Sample records for purkinje cell clusters

  1. Palisade pattern of mormyrid Purkinje cells: a correlated light and electron microscopic study.

    PubMed

    Meek, J; Nieuwenhuys, R

    1991-04-01

    The present study is devoted to a detailed analysis of the structural and synaptic organization of mormyrid Purkinje cells in order to evaluate the possible functional significance of their dendritic palisade pattern. For this purpose, the properties of Golgi-impregnated as well as unimpregnated Purkinje cells in lobe C1 and C3 of the cerebellum of Gnathonemus petersii were light and electron microscopically analyzed, quantified, reconstructed, and mutually compared. Special attention was paid to the degree of regularity of their dendritic trees, their relations with Bergmann glia, and the distribution and numerical properties of their synaptic connections with parallel fibers, stellate cells, "climbing" fibers, and Purkinje axonal boutons. The highest degree of palisade specialization was encountered in lobe C1, where Purkinje cells have on average 50 palisade dendrites with a very regular distribution in a sagittal plane. Their spine density decreases from superficial to deep (from 14 to 6 per micron dendritic length), a gradient correlated with a decreasing parallel fiber density but an increasing parallel fiber diameter. Each Purkinje cell makes on average 75,000 synaptic contacts with parallel fibers, some of which are rather coarse (0.45 microns), and provided with numerous short collaterals. Climbing fibers do not climb, since their synaptic contacts are restricted to the ganglionic layer (i.e., the layer of Purkinje and eurydendroid projection cells), where they make about 130 synaptic contacts per cell with 2 or 3 clusters of thorns on the proximal dendrites. These clusters contain also a type of "shunting" elements that make desmosome-like junctions with both the climbing fiber boutons and the necks of the thorns. The axons of Purkinje cells in lobe C1 make small terminal arborizations, with about 20 boutons, that may be substantially (up to 500 microns) displaced rostrally or caudally with respect to the soma. Purkinje axonal boutons were observed to

  2. Inverse Stochastic Resonance in Cerebellar Purkinje Cells

    PubMed Central

    Häusser, Michael; Gutkin, Boris S.; Roth, Arnd

    2016-01-01

    Purkinje neurons play an important role in cerebellar computation since their axons are the only projection from the cerebellar cortex to deeper cerebellar structures. They have complex internal dynamics, which allow them to fire spontaneously, display bistability, and also to be involved in network phenomena such as high frequency oscillations and travelling waves. Purkinje cells exhibit type II excitability, which can be revealed by a discontinuity in their f-I curves. We show that this excitability mechanism allows Purkinje cells to be efficiently inhibited by noise of a particular variance, a phenomenon known as inverse stochastic resonance (ISR). While ISR has been described in theoretical models of single neurons, here we provide the first experimental evidence for this effect. We find that an adaptive exponential integrate-and-fire model fitted to the basic Purkinje cell characteristics using a modified dynamic IV method displays ISR and bistability between the resting state and a repetitive activity limit cycle. ISR allows the Purkinje cell to operate in different functional regimes: the all-or-none toggle or the linear filter mode, depending on the variance of the synaptic input. We propose that synaptic noise allows Purkinje cells to quickly switch between these functional regimes. Using mutual information analysis, we demonstrate that ISR can lead to a locally optimal information transfer between the input and output spike train of the Purkinje cell. These results provide the first experimental evidence for ISR and suggest a functional role for ISR in cerebellar information processing. PMID:27541958

  3. BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells.

    PubMed

    Hirono, Moritoshi; Ogawa, Yasuhiro; Misono, Kaori; Zollinger, Daniel R; Trimmer, James S; Rasband, Matthew N; Misonou, Hiroaki

    2015-05-06

    In myelinated axons, K(+) channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na(+) channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K(+) channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K(+) channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni(2+) elicited a similar effect on APs, indicating the involvement of Ni(2+)-sensitive Ca(2+) channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex. Copyright © 2015 the authors 0270-6474/15/357082-13$15.00/0.

  4. Possible role of pineal allopregnanolone in Purkinje cell survival

    PubMed Central

    Haraguchi, Shogo; Hara, Sakurako; Ubuka, Takayoshi; Mita, Masatoshi; Tsutsui, Kazuyoshi

    2012-01-01

    It is believed that neurosteroids are produced in the brain and other nervous systems. Here, we show that allopregnanolone (ALLO), a neurosteroid, is exceedingly produced in the pineal gland compared with the brain and that pineal ALLO acts on the Purkinje cell, a principal cerebellar neuron, to prevent apoptosis in the juvenile quail. We first demonstrated that the pineal gland is a major organ of neurosteroidogenesis. A series of experiments using molecular and biochemical techniques has further demonstrated that the pineal gland produces a variety of neurosteroids de novo from cholesterol in the juvenile quail. Importantly, ALLO was far more actively produced in the pineal gland than in the brain. Pinealectomy (Px) decreased ALLO concentration in the cerebellum and induced apoptosis of Purkinje cells, whereas administration of ALLO to Px quail chicks prevented apoptosis of Purkinje cells. We further found that Px significantly increased the number of Purkinje cells that expressed active caspase-3, a key protease in apoptotic pathway, and daily injection of ALLO to Px quail chicks decreased the number of Purkinje cells expressing active caspase-3. These results indicate that the neuroprotective effect of pineal ALLO is associated with the decrease in caspase-3 activity during the early stage of neuronal development. We thus provide evidence that the pineal gland is an important neurosteroidogenic organ and that pineal ALLO may be involved in Purkinje cell survival during development. This is an important function of the pineal gland in the formation of neuronal circuits in the developing cerebellum. PMID:23213208

  5. Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.

    PubMed

    Kang, Guoxin; Giovannone, Steven F; Liu, Nian; Liu, Fang-Yu; Zhang, Jie; Priori, Silvia G; Fishman, Glenn I

    2010-08-20

    The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R

  6. Purkinje Cell Activity in the Cerebellar Anterior Lobe after Rabbit Eyeblink Conditioning

    ERIC Educational Resources Information Center

    Green, John T.; Steinmetz, Joseph E.

    2005-01-01

    The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus.…

  7. Climbing fibers mediate vestibular modulation of both "complex" and "simple spikes" in Purkinje cells.

    PubMed

    Barmack, N H; Yakhnitsa, V

    2015-10-01

    Climbing and mossy fibers comprise two distinct afferent paths to the cerebellum. Climbing fibers directly evoke a large multispiked action potential in Purkinje cells termed a "complex spike" (CS). By logical exclusion, the other class of Purkinje cell action potential, termed "simple spike" (SS), has often been attributed to activity conveyed by mossy fibers and relayed to Purkinje cells through granule cells. Here, we investigate the relative importance of climbing and mossy fiber pathways in modulating neuronal activity by recording extracellularly from Purkinje cells, as well as from mossy fiber terminals and interneurons in folia 8-10. Sinusoidal roll-tilt vestibular stimulation vigorously modulates the discharge of climbing and mossy fiber afferents, Purkinje cells, and interneurons in folia 9-10 in anesthetized mice. Roll-tilt onto the side ipsilateral to the recording site increases the discharge of both climbing fibers (CSs) and mossy fibers. However, the discharges of SSs decrease during ipsilateral roll-tilt. Unilateral microlesions of the beta nucleus (β-nucleus) of the inferior olive blocks vestibular modulation of both CSs and SSs in contralateral Purkinje cells. The blockage of SSs occurs even though primary and secondary vestibular mossy fibers remain intact. When mossy fiber afferents are damaged by a unilateral labyrinthectomy (UL), vestibular modulation of SSs in Purkinje cells ipsilateral to the UL remains intact. Two inhibitory interneurons, Golgi and stellate cells, could potentially contribute to climbing fiber-induced modulation of SSs. However, during sinusoidal roll-tilt, only stellate cells discharge appropriately out of phase with the discharge of SSs. Golgi cells discharge in phase with SSs. When the vestibularly modulated discharge is blocked by a microlesion of the inferior olive, the modulated discharge of CSs and SSs is also blocked. When the vestibular mossy fiber pathway is destroyed, vestibular modulation of ipsilateral CSs and

  8. In vivo analysis of Purkinje cell firing properties during postnatal mouse development

    PubMed Central

    Arancillo, Marife; White, Joshua J.; Lin, Tao; Stay, Trace L.

    2014-01-01

    Purkinje cell activity is essential for controlling motor behavior. During motor behavior Purkinje cells fire two types of action potentials: simple spikes that are generated intrinsically and complex spikes that are induced by climbing fiber inputs. Although the functions of these spikes are becoming clear, how they are established is still poorly understood. Here, we used in vivo electrophysiology approaches conducted in anesthetized and awake mice to record Purkinje cell activity starting from the second postnatal week of development through to adulthood. We found that the rate of complex spike firing increases sharply at 3 wk of age whereas the rate of simple spike firing gradually increases until 4 wk of age. We also found that compared with adult, the pattern of simple spike firing during development is more irregular as the cells tend to fire in bursts that are interrupted by long pauses. The regularity in simple spike firing only reached maturity at 4 wk of age. In contrast, the adult complex spike pattern was already evident by the second week of life, remaining consistent across all ages. Analyses of Purkinje cells in alert behaving mice suggested that the adult patterns are attained more than a week after the completion of key morphogenetic processes such as migration, lamination, and foliation. Purkinje cell activity is therefore dynamically sculpted throughout postnatal development, traversing several critical events that are required for circuit formation. Overall, we show that simple spike and complex spike firing develop with unique developmental trajectories. PMID:25355961

  9. Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake

    PubMed Central

    Wang, Fushun; Xu, Qiwu; Wang, Weishan; Takano, Takahiro; Nedergaard, Maiken

    2012-01-01

    Recent studies have shown that cerebellar Bergmann glia display coordinated Ca2+ transients in live mice. However, the functional significance of Bergmann glial Ca2+ signaling remains poorly understood. Using transgenic mice that allow selective stimulation of glial cells, we report here that cytosolic Ca2+ regulates uptake of K+ by Bergmann glia, thus providing a powerful mechanism for control of Purkinje cell-membrane potential. The decline in extracellular K+ evoked by agonist-induced Ca2+ in Bergmann glia transiently increased spike activity of Purkinje cells in cerebellar slices as well as in live anesthetized mice. Thus, Bergmann glia play a previously unappreciated role in controlling the membrane potential and thereby the activity of adjacent Purkinje cells. PMID:22547829

  10. Functional compatibility between Purkinje cell axon branches and their target neurons in the cerebellum.

    PubMed

    Yang, Zhilai; Chen, Na; Ge, Rongjing; Qian, Hao; Wang, Jin-Hui

    2017-09-22

    A neuron sprouts an axon, and its branches to innervate many target neurons that are divergent in their functions. In order to efficiently regulate the diversified cells, the axon branches should differentiate functionally to be compatible with their target neurons, i.e., a function compatibility between presynaptic and postsynaptic partners. We have examined this hypothesis by using electrophysiological method in the cerebellum, in which the main axon of Purkinje cell projected to deep nucleus cells and the recurrent axons innervated the adjacent Purkinje cells. The fidelity of spike propagation is superior in the recurrent branches than the main axon. The capabilities of encoding spikes and processing GABAergic inputs are advanced in Purkinje cells versus deep nucleus cells. The functional differences among Purkinje's axonal branches and their postsynaptic neurons are preset by the variable dynamics of their voltage-gated sodium channels. In addition, activity strengths between presynaptic and postsynaptic partners are proportionally correlated, i.e., active axonal branches innervate active target neurons, or vice versa. The physiological impact of the functional compatibility is to make the neurons in their circuits to be activated appropriately. In conclusion, each cerebellar Purkinje cell sprouts the differentiated axon branches to be compatible with the diversified target cells in their functions, in order to construct the homeostatic and efficient units for their coordinated activity in neural circuits.

  11. Functional compatibility between Purkinje cell axon branches and their target neurons in the cerebellum

    PubMed Central

    Qian, Hao; Wang, Jin-Hui

    2017-01-01

    A neuron sprouts an axon, and its branches to innervate many target neurons that are divergent in their functions. In order to efficiently regulate the diversified cells, the axon branches should differentiate functionally to be compatible with their target neurons, i.e., a function compatibility between presynaptic and postsynaptic partners. We have examined this hypothesis by using electrophysiological method in the cerebellum, in which the main axon of Purkinje cell projected to deep nucleus cells and the recurrent axons innervated the adjacent Purkinje cells. The fidelity of spike propagation is superior in the recurrent branches than the main axon. The capabilities of encoding spikes and processing GABAergic inputs are advanced in Purkinje cells versus deep nucleus cells. The functional differences among Purkinje's axonal branches and their postsynaptic neurons are preset by the variable dynamics of their voltage-gated sodium channels. In addition, activity strengths between presynaptic and postsynaptic partners are proportionally correlated, i.e., active axonal branches innervate active target neurons, or vice versa. The physiological impact of the functional compatibility is to make the neurons in their circuits to be activated appropriately. In conclusion, each cerebellar Purkinje cell sprouts the differentiated axon branches to be compatible with the diversified target cells in their functions, in order to construct the homeostatic and efficient units for their coordinated activity in neural circuits. PMID:29069799

  12. Current source density correlates of cerebellar Golgi and Purkinje cell responses to tactile input

    PubMed Central

    Tahon, Koen; Wijnants, Mike; De Schutter, Erik

    2011-01-01

    The overall circuitry of the cerebellar cortex has been known for over a century, but the function of many synaptic connections remains poorly characterized in vivo. We used a one-dimensional multielectrode probe to estimate the current source density (CSD) of Crus IIa in response to perioral tactile stimuli in anesthetized rats and to correlate current sinks and sources to changes in the spike rate of corecorded Golgi and Purkinje cells. The punctate stimuli evoked two distinct early waves of excitation (at <10 and ∼20 ms) associated with current sinks in the granular layer. The second wave was putatively of corticopontine origin, and its associated sink was located higher in the granular layer than the first trigeminal sink. The distinctive patterns of granular-layer sinks correlated with the spike responses of corecorded Golgi cells. In general, Golgi cell spike responses could be linearly reconstructed from the CSD profile. A dip in simple-spike activity of coregistered Purkinje cells correlated with a current source deep in the molecular layer, probably generated by basket cell synapses, interspersed between sparse early sinks presumably generated by synapses from granule cells. The late (>30 ms) enhancement of simple-spike activity in Purkinje cells was characterized by the absence of simultaneous sinks in the granular layer and by the suppression of corecorded Golgi cell activity, pointing at inhibition of Golgi cells by Purkinje axon collaterals as a likely mechanism of late Purkinje cell excitation. PMID:21228303

  13. Regulation of spinogenesis in mature Purkinje cells via mGluR/PKC-mediated phosphorylation of CaMKIIβ

    PubMed Central

    Sugawara, Takeyuki; Hisatsune, Chihiro; Miyamoto, Hiroyuki; Ogawa, Naoko; Mikoshiba, Katsuhiko

    2017-01-01

    Dendritic spines of Purkinje cells form excitatory synapses with parallel fiber terminals, which are the primary sites for cerebellar synaptic plasticity. Nevertheless, how density and morphology of these spines are properly maintained in mature Purkinje cells is not well understood. Here we show an activity-dependent mechanism that represses excessive spine development in mature Purkinje cells. We found that CaMKIIβ promotes spine formation and elongation in Purkinje cells through its F-actin bundling activity. Importantly, activation of group I mGluR, but not AMPAR, triggers PKC-mediated phosphorylation of CaMKIIβ, which results in dissociation of the CaMKIIβ/F-actin complex. Defective function of the PKC-mediated CaMKIIβ phosphorylation promotes excess F-actin bundling and leads to abnormally numerous and elongated spines in mature IP3R1-deficient Purkinje cells. Thus, our data suggest that phosphorylation of CaMKIIβ through the mGluR/IP3R1/PKC signaling pathway represses excessive spine formation and elongation in mature Purkinje cells. PMID:28607044

  14. A New Approach for Determining Phase Response Curves Reveals that Purkinje Cells Can Act as Perfect Integrators

    PubMed Central

    Roth, Arnd; Häusser, Michael

    2010-01-01

    Cerebellar Purkinje cells display complex intrinsic dynamics. They fire spontaneously, exhibit bistability, and via mutual network interactions are involved in the generation of high frequency oscillations and travelling waves of activity. To probe the dynamical properties of Purkinje cells we measured their phase response curves (PRCs). PRCs quantify the change in spike phase caused by a stimulus as a function of its temporal position within the interspike interval, and are widely used to predict neuronal responses to more complex stimulus patterns. Significant variability in the interspike interval during spontaneous firing can lead to PRCs with a low signal-to-noise ratio, requiring averaging over thousands of trials. We show using electrophysiological experiments and simulations that the PRC calculated in the traditional way by sampling the interspike interval with brief current pulses is biased. We introduce a corrected approach for calculating PRCs which eliminates this bias. Using our new approach, we show that Purkinje cell PRCs change qualitatively depending on the firing frequency of the cell. At high firing rates, Purkinje cells exhibit single-peaked, or monophasic PRCs. Surprisingly, at low firing rates, Purkinje cell PRCs are largely independent of phase, resembling PRCs of ideal non-leaky integrate-and-fire neurons. These results indicate that Purkinje cells can act as perfect integrators at low firing rates, and that the integration mode of Purkinje cells depends on their firing rate. PMID:20442875

  15. Effect of Early Neonatal Exposure to Deltamethrin on the Purkinje Cell Number in Rat Cerebellum

    PubMed Central

    Asari, Mohd Asnizam; Abdullah, Mohammad Shukri; Abdullah, Suryati

    2008-01-01

    Deltamethrin is a widely used insecticide belonging to the class of pyrethroid. Although the neurotoxicity of pyrethroids including deltamethrin is well established, it is still unclear whether exposure to deltamethrin during neonatal period has any deleterious effect on the survival of the Purkinje cells in the cerebellum. In the study, we investigated the total number of Purkinje cells in experimental rats exposed to deltamethrin using a stereological method, the fractionator. Deltamethrin in a dose of 1 mg/kg/day (corresponds to 20% of LD50 ) was administered through oral gavage to male pups from 2nd to 5th postnatal day (PND). At PND 21 the animals were sacrificed and their cerebelli were removed. The cerebelli were systematically sampled using the fractionator method and stained with cresyl fast violet. The number of the Purkinje cells was counted for each cerebellum. The results showed that there was no significant difference in the total number of Purkinje cells in the deltamethrin-treated group as compared to the control animals. This suggests that deltamethrin exposure at the current dosage during the neonatal period do not have any significant effect on the survival of the Purkinje cells in the cerebellum. PMID:22570585

  16. The Vps33a gene regulates behavior and cerebellar Purkinje cell number

    PubMed Central

    Chintala, Sreenivasulu; Novak, Edward K.; Spernyak, Joseph A.; Mazurchuk, Richard; Torres, German; Patel, Suchith; Busch, Kristie; Meeder, Beth A.; Horowitz, Judith M.; Vaughan, Mary M.; Swank, Richard T.

    2015-01-01

    A mutation in the Vps33a gene causes Hermansky–Pudlak Syndrome (HPS)-like-symptoms in the buff (bf) mouse mutant. The encoded product, Vps33a, is a member of the Sec1 and Class C multi-protein complex that regulates vesicle trafficking to specialized lysosome-related organelles. As Sec1 signaling pathways have been implicated in pre-synaptic function, we examined brain size, cerebellar cell number and the behavioral phenotype of bf mutants. Standardized behavioral tests (SHIRPA protocols) demonstrated significant motor deficits (e.g., grip strength, righting reflex and touch escape) in bf mutants, worsening with age. Histological examination of brain revealed significant Purkinje cell loss that was confirmed with staining for calbindin, a calcium binding protein enriched in Purkinje cells. This pathologic finding was progressive, as older bf mutants (13–14 months) showed a greater attrition of neurons, with their cerebella appearing to be particularly reduced (~30%) in size relative to those of age-matched-control cohorts. These studies suggest that loss of Purkinje neurons is the most obvious neurological atrophy in the bf mutant, a structural change that generates motor coordination deficits and impaired postural phenotypes. It is conceivable therefore that death of cerebellar cells may alsobea clinical feature of HPS patients, a pathological event which has not been reported in the literature. In general, the bf mutant may be a potentially new and useful model for understanding Purkinje cell development and function. PMID:19254700

  17. PCPP-260, PURKINJE CELL-SPECIFIC CYCLE AMP-REGULATED MEMBRANE PHOSPHOPROTEIN OF (M SUB R) 260,000

    EPA Science Inventory

    The present study reports the existence of Purkinje cell-specific phosphoprotein, Mr260,000 (PCPP-260), a neuronal membrance phosphoprotein, in cerebellar Purkinje cells. PCPP-260, which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has an apparaent molecular mass ...

  18. Contribution of cerebellar intracortical inhibition to Purkinje cell response during vestibulo-ocular reflex of alert rabbits.

    PubMed Central

    Miyashita, Y; Nagao, S

    1984-01-01

    Ionophoretic application of bicuculline, an antagonist of gamma-aminobutyric acid (GABA), was used to examine the contribution of intracortical inhibition to vestibular responses of Purkinje cells in the cerebellar flocculus of alert rabbits. Purkinje cells were sampled extracellularly (with triple-barrelled micropipettes) from the floccular area where electrical stimulation through the micro-electrode evoked abduction of the ipsilateral eye, indicating its close functional relationship to the horizontal vestibulo-ocular reflex. These cells exhibited frequency modulation of simple spike discharges in-phase or out-phase with sinusoidal head rotation (0.5 cycles/s, 5 degrees peak-to-peak) in the horizontal plane. Bicuculline was ejected ionophoretically through one barrel with a 20-60 nA current. The pharmacological effectiveness of the ejected bicuculline was confirmed for each Purkinje cell by its blocking action upon the depressant action of GABA applied ionophoretically through another barrel. Bicuculline usually shifted the simple spike modulation in the in-phase direction: it reduced the amplitude of out-phase modulation in three cells, converted out-phase modulation to the in-phase type in four cells, and increased in-phase modulation in five cells. In three other cells, however, bicuculline shifted the modulation in the out-phase direction. Because bicuculline application usually increased the resting discharge level of a Purkinje cell, ionophoretic application of DL-homocysteate was used in ten Purkinje cells to control for the effect of a generalized increase in excitability. In contrast to bicuculline, DL-homocysteate generally induced a slight increase of the simple spike modulation regardless of the phase relationship. Since frequency modulation of the simple spike discharges of flocculus Purkinje cells is presumed to contribute to the control of vestibulo-ocular reflexes, these results point to an important functional role of intracortical post

  19. A novel approach to non-biased systematic random sampling: a stereologic estimate of Purkinje cells in the human cerebellum.

    PubMed

    Agashiwala, Rajiv M; Louis, Elan D; Hof, Patrick R; Perl, Daniel P

    2008-10-21

    Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well.

  20. A novel approach to non-biased systematic random sampling: A stereologic estimate of Purkinje cells in the human cerebellum

    PubMed Central

    Agashiwala, Rajiv M.; Louis, Elan D.; Hof, Patrick R.; Perl, Daniel P.

    2010-01-01

    Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well. PMID:18725208

  1. Increased protein kinase C gamma activity induces Purkinje cell pathology in a mouse model of spinocerebellar ataxia 14.

    PubMed

    Ji, Jingmin; Hassler, Melanie L; Shimobayashi, Etsuko; Paka, Nagendher; Streit, Raphael; Kapfhammer, Josef P

    2014-10-01

    Spinocerebellar ataxias (SCAs) are hereditary diseases leading to Purkinje cell degeneration and cerebellar dysfunction. Most forms of SCA are caused by expansion of CAG repeats similar to other polyglutamine disorders such as Huntington's disease. In contrast, in the autosomal dominant SCA-14 the disease is caused by mutations in the protein kinase C gamma (PKCγ) gene which is a well characterized signaling molecule in cerebellar Purkinje cells. The study of SCA-14, therefore, offers the unique opportunity to reveal the molecular and pathological mechanism eventually leading to Purkinje cell dysfunction and degeneration. We have created a mouse model of SCA-14 in which PKCγ protein with a mutation found in SCA-14 is specifically expressed in cerebellar Purkinje cells. We find that in mice expressing the mutated PKCγ protein the morphology of Purkinje cells in cerebellar slice cultures is drastically altered and mimics closely the morphology seen after pharmacological PKC activation. Similar morphological abnormalities were seen in localized areas of the cerebellum of juvenile transgenic mice in vivo. In adult transgenic mice there is evidence for some localized loss of Purkinje cells but there is no overall cerebellar atrophy. Transgenic mice show a mild cerebellar ataxia revealed by testing on the rotarod and on the walking beam. Our findings provide evidence for both an increased PKCγ activity in Purkinje cells in vivo and for pathological changes typical for cerebellar disease thus linking the increased and dysregulated activity of PKCγ tightly to the development of cerebellar disease in SCA-14 and possibly also in other forms of SCA. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging.

    PubMed

    Fattoretti, P; Bertoni-Freddari, C; Caselli, U; Paoloni, R; Meier-Ruge, W

    1998-03-16

    The perikaryal Purkinje cell mitochondria positive to the copper ferrocyanide histochemical reaction for succinic dehydrogenase (SDH) have been investigated by means of semiautomatic morphometric methods in rats of 3, 12 and 24 months of age. The number of organelles/microm3 of Purkinje cell cytoplasm (Numeric density: Nv), the average mitochondrial volume (V) and the mitochondrial volume fraction (Volume density: Vv) were the ultrastructural parameters taken into account. Nv was significantly higher at 12 than at 3 and 24 months of age. V was significantly decreased at 12 and 24 months of age, but no difference was envisaged between adult and old rats. Vv was significantly decreased in old animals vs. the other age groups. In young and old rats, the percentage of organelles larger than 0.32 microm3 was 13.5 and 11%, respectively, while these enlarged mitochondria accounted for less than 1% in the adult group. Since SDH activity is of critical importance when energy demand is high, the marked decrease of Vv supports an impaired capacity of the old Purkinje cells to match actual energy supply at sustained transmission of the nervous impulse. However, the high percentage of enlarged organelles found in old rats may witness a morphofunctional compensatory response.

  3. Cadm1-Expressing Synapses on Purkinje Cell Dendrites Are Involved in Mouse Ultrasonic Vocalization Activity

    PubMed Central

    Fujita, Eriko; Tanabe, Yuko; Imhof, Beat A.; Momoi, Mariko Y.; Momoi, Takashi

    2012-01-01

    Foxp2(R552H) knock-in (KI) mouse pups with a mutation related to human speech–language disorders exhibit poor development of cerebellar Purkinje cells and impaired ultrasonic vocalization (USV), a communication tool for mother-offspring interactions. Thus, human speech and mouse USV appear to have a Foxp2-mediated common molecular basis in the cerebellum. Mutations in the gene encoding the synaptic adhesion molecule CADM1 (RA175/Necl2/SynCAM1/Cadm1) have been identified in people with autism spectrum disorder (ASD) who have impaired speech and language. In the present study, we show that both Cadm1-deficient knockout (KO) pups and Foxp2(R552H) KI pups exhibit impaired USV and smaller cerebellums. Cadm1 was preferentially localized to the apical–distal portion of the dendritic arbor of Purkinje cells in the molecular layer of wild-type pups, and VGluT1 level decreased in the cerebellum of Cadm1 KO mice. In addition, we detected reduced immunoreactivity of Cadm1 and VGluT1 on the poorly developed dendritic arbor of Purkinje cells in the Foxp2(R552H) KI pups. However, Cadm1 mRNA expression was not altered in the Foxp2(R552H) KI pups. These results suggest that although the Foxp2 transcription factor does not target Cadm1, Cadm1 at the synapses of Purkinje cells and parallel fibers is necessary for USV function. The loss of Cadm1-expressing synapses on the dendrites of Purkinje cells may be associated with the USV impairment that Cadm1 KO and Foxp2(R552H) KI mice exhibit. PMID:22272290

  4. Administration of a non-NMDA antagonist, GYKI 52466, increases excitotoxic Purkinje cell degeneration caused by ibogaine.

    PubMed

    O'Hearn, E; Molliver, M E

    2004-01-01

    Ibogaine is a tremorigenic hallucinogen that has been proposed for clinical use in treating addiction. We previously reported that ibogaine, administered systemically, produces degeneration of a subset of Purkinje cells in the cerebellum, primarily within the vermis. Ablation of the inferior olive affords protection against ibogaine-induced neurotoxicity leading to the interpretation that ibogaine itself is not directly toxic to Purkinje cells. We postulated that ibogaine produces sustained excitation of inferior olivary neurons that leads to excessive glutamate release at climbing fiber terminals, causing subsequent excitotoxic injury to Purkinje cells. The neuronal degeneration induced by ibogaine provides an animal model for studying excitotoxic injury in order to analyze the contribution of glutamate receptors to this injury and to evaluate neuroprotective strategies. Since non-N-methyl-D-aspartate (NMDA) receptors mediate Purkinje cell excitation by climbing fibers, we hypothesized that 1-4-aminophenyl-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466), which antagonizes non-NMDA receptors, may have a neuroprotective effect by blocking glutamatergic excitation at climbing fiber synapses. To test this hypothesis, rats were administered systemic ibogaine plus GYKI-52466 and the degree of neuronal injury was analyzed in cerebellar sections. The results indicate that the AMPA antagonist GYKI-52466 (10 mg/kg i.p. x 3) does not protect against Purkinje cell injury at the doses used. Rather, co-administration of GYKI-52466 with ibogaine produces increased toxicity evidenced by more extensive Purkinje cell degeneration. Several hypotheses that may underlie this result are discussed. Although the reason for the increased toxicity found in this study is not fully explained, the present results show that a non-NMDA antagonist can produce increased excitotoxic injury under some conditions. Therefore, caution should be exercised before employing glutamate

  5. Temporal expression and mitochondrial localization of a Foxp2 isoform lacking the forkhead domain in developing Purkinje cells.

    PubMed

    Tanabe, Yuko; Fujiwara, Yuji; Matsuzaki, Ayumi; Fujita, Eriko; Kasahara, Tadashi; Yuasa, Shigeki; Momoi, Takashi

    2012-07-01

    FOXP2, a forkhead box-containing transcription factor, forms homo- or hetero-dimers with FOXP family members and localizes to the nucleus, while FOXP2(R553H), which contains a mutation related to speech/language disorders, features reduced DNA binding activity and both cytoplasmic and nuclear localization. In addition to being a loss-of-function mutation, it is possible that FOXP2(R553H) also may act as a gain-of-function mutation to inhibit the functions of FOXP2 isoforms including FOXP2Ex10+ lacking forkhead domain. Foxp2(R552H) knock-in mouse pups exhibit impaired ultrasonic vocalization and poor dendritic development in Purkinje cells. However, expressions of Foxp2 isoforms in the developing Purkinje are unclear. The appearance of 'apical cytoplasmic swelling' (mitochondria-rich regions that are the source of budding processes) correlates with dendritic development of Purkinje cells. In the present study, we focused on Foxp2 isoforms localizing to the apical cytoplasmic swelling and identified two isoforms lacking forkhead domain: Foxp2Ex12+ and Foxp2Ex15. They partly localized to the membrane fraction that includes mitochondria. Foxp2Ex12+ mainly localized to the apical cytoplasmic swelling in early developing Purkinje cells at the stellate stage (P2-P4). Mitochondrial localization of Foxp2Ex12+ in Purkinje cells was confirmed by immune-electron microscopic analysis. Foxp2Ex12+ may play a role in dendritic development in Purkinje cells. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  6. Red sorrel (Hibiscus Sabdariffa) prevents the ethanol-induced deficits of Purkinje cells in the cerebellum.

    PubMed

    Suryanti, S; Partadiredja, G; Atthobari, J

    2015-01-01

    The present study is aimed at investigating the possible protective effects of H. sabdariffa on ethanol-elicited deficits of motor coordination and estimated total number of the Purkinje cells of the cerebellums of adolescent male Wistar rats. Forty male Wistar rats aged 21 days were divided into five groups. Na/wtr group was given water orally and injected with normal saline intra peritoneally (ip). Eth/wtr group was given water orally and ethanol (ip). Another three experimental groups (Eth/Hsab) were given different dosages of H. sabdariffa and ethanol (ip). All groups were treated intermittently for the total period of treatment of two weeks. The motor coordination of rats was tested prior and subsequent to the treatments. The rats were euthanized, and their cerebellums were examined. The total number of Purkinje cells was estimated using physical fractionator method. Upon revolving drum test, the number of falls of rats increased following ethanol treatment. There was no significant difference between the total number of falls prior and subsequent to treatment in all Eth/Hsab groups. The estimated total number of Purkinje cells in Eth/Hsab groups was higher than in Eth/wtr group. H. sabdariffa may prevent the ethanol-induced deficits of motor coordination and estimated total number of Purkinje cells of the cerebellums in adolescent rats (Tab. 3, Fig. 1, Ref. 42).

  7. Discharge properties of Purkinje cells in the oculomotor vermis during visually guided saccades in the macaque monkey.

    PubMed

    Ohtsuka, K; Noda, H

    1995-11-01

    1. We previously described discharge properties of cerebellar output cells in the fastigial nucleus during ipsilateral and contralateral saccades. Fastigial cells exhibited unique responses depending on the direction of saccades and were involved in execution of accurate targeting saccades. Purkinje cells in the oculomotor vermis (lobules VIc and VII) are thought to modulate these discharges of fastigial cells. In this study we reexamine discharge properties of Purkinje cells on the basis of this hypothesis. 2. Initially we physiologically identified the right and left sides of the oculomotor vermis. Saccade-related discharges of 79 Purkinje cells were recorded from both sides of the vermis during visually guided saccades toward the sides ipsilateral and contralateral to the recording side in two trained macaque monkeys. To clarify the correlation of Purkinje cell discharge with burst activities in the fastigial nucleus during saccadic eye movements, we analyzed our data by employing methods used in the study of fastigial neurons. 3. Among the 79 cells, 56 (71%) showed burst discharges during saccades (saccadic burst cells). Of the 56 cells, 29 exhibited a peak of burst discharges in both the contralateral and ipsilateral directions (bidirectional cells). The remaining 27 saccadic burst cells showed a peak of burst discharges during either contralateral or ipsilateral saccades (unidirectional cells). Among the 79 cells, 14 (18%) exhibited a pause of discharges during contralateral saccades (pause cells). Among the 79 cells, 9 (11%) showed burst discharge during contralateral saccades followed by tonic discharge that was correlated with eye position (burst tonic cells). 4. The timing of bursts in bidirectional cells with respect to saccade onset was dependent on the direction of saccade. During ipsilateral saccades, Purkinje cells exhibited a long lead burst that built up gradually, peaked near the onset of the saccade, and terminated sharply near midsaccade. The

  8. Chick cerebellar Purkinje cells express omega-conotoxin GVIA-sensitive rather than funnel-web spider toxin-sensitive calcium channels.

    PubMed

    Angulo, M C; Parra, P; Dieudonné, S

    1998-03-01

    Voltage-gated calcium channels form a complex family of distinct molecular entities which participate in multiple neuronal functions. In cerebellar Purkinje cells these channels contribute to the characteristic electrophysiological pattern of complex spikes, first described in birds and later in mammals. A specific calcium channel, the P-type channel, has been shown to mediate the majority of the voltage-gated calcium flux in mammalian Purkinje cells. P-type channels play an essential role in synaptic transmission of mammalian cerebellum. It is unclear whether the P-type calcium channel is present in birds. Studies in chick synaptosomal preparations show that the pharmacological profile of calcium channels is complex and suggest a minimal expression of the P-type channel in avian central nervous system. In the present work, we studied voltage-gated calcium channels in dissociated chick cerebellar Purkinje cells to examine the presence of different calcium channel types. Purkinje cells were used because, in mammals, they express predominantly P-type channels and because the morphology of these cells is thought to be phylogenetically conserved. We found that omega-conotoxin GVIA (omega-CgTx GVIA), a specific antagonist of N-type calcium channel, rather than the synthetic funnel-web spider toxin (sFTX), a P-type channel antagonist, blocks the majority of the barium current flowing through calcium channels in chick Purkinje neurons.

  9. Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

    PubMed

    Verheijen, Bert M; Gentier, Romina J G; Hermes, Denise J H P; van Leeuwen, Fred W; Hopkins, David A

    2017-06-01

    The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B +1 (UBB +1 ), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB +1 -expressing transgenic mice display widespread labeling for UBB +1 in brain and exhibit behavioral deficits. Here, we show that UBB +1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB +1 -expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

  10. Sensorimotor-correlated discharge recorded from ensembles of cerebellar Purkinje cells varies across the estrous cycle of the rat.

    PubMed

    Smith, S S

    1995-09-01

    1. In the present study, locomotor-correlated activity of cerebellar Purkinje cells, recorded using arrays of microwires chronically implanted in adult female rats, was examined across estrous-cycle-associated fluctuations in endogenous sex steroids. Ongoing studies from this laboratory have shown that systemic and local administration of the sex steroid 17 beta-estradiol (E2) augments excitatory responses of cerebellar Purkinje cells to iontophoretically applied glutamate, recorded in vivo from anesthetized female rats. In addition, this steroid potentiated discharge correlated with limb movement. For the present study, extracellular single-unit activity was recorded from as many as 5-11 Purkinje cells simultaneously during treadmill locomotion paradigms. Motor modulation of activity was recorded across three to five consecutive estrous cycles from behaviorally identified cohorts of neurons to test the hypothesis that fluctuations in endogenous sex steroids alter motor modulation of Purkinje cell discharge. 2. Locomotor-associated discharge correlated with treadmill locomotion was increased by a mean of 47% on proestrus, when E2 levels are elevated, relative to diestrus 1. These changes in discharge rate during treadmill locomotion were of significantly greater magnitude than corresponding cyclic alterations in discharge during stationary periods. 3. Correlations with the circadian cycle were also significant, because peak levels of locomotor-associated discharge on the night of behavioral estrus, following elevations in circulating E2, were on average 67% greater than corresponding discharge recorded during the light (proestrus). 4. Alterations in the step cycle were also observed across the estrous cycle: significant decreases in the duration of the flexion phase (by 265 ms, P < 0.05) were noted on estrus compared with diestrus. 5. When recorded on estrus, Purkinje cell discharge correlated with the stance or flexion phase of the step cycle was greater in

  11. Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

    PubMed

    Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo

    2010-06-01

    Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

  12. HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition

    PubMed Central

    Rinaldi, Arianna; Defterali, Cagla; Mialot, Antoine; Garden, Derek L F; Beraneck, Mathieu; Nolan, Matthew F

    2013-01-01

    Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type-specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo-ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour. PMID:24000178

  13. Effects of subchronic inhalation of vaporized plastic cement on exploratory behavior and Purkinje cell differentiation in the rat.

    PubMed

    Pascual, R; Salgado, C; Viancos, L; Figueroa, H R

    1996-12-06

    In the present study, the effects of preweaning cement vapor inhalation on exploratory behavior and cerebellar Purkinje cell differentiation were assessed. Sprague-Dawley albino rats were daily exposed to glue vapors between postnatal d 2 and 21. At postnatal d 22, all animals were submitted to the open-field test in order to evaluate their exploratory behavior. Then they were sacrificed, their brains dissected out, and cerebella stained according to the Golgi-Cox-Sholl procedure. Purkinje cells sampled from parasagittal sections of the cerebellar vermis were drawn under camera lucida and their dendritic domain was determined. The collected data indicate that glue solvent inhalation impairs both Purkinje cell differentiation and locomotor exploratory behavior.

  14. The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes.

    PubMed

    Burroughs, Amelia; Wise, Andrew K; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y; Lang, Eric J; Apps, Richard; Cerminara, Nadia L

    2017-01-01

    Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes. Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets. The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear. We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number. This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake cats. In addition

  15. The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes

    PubMed Central

    Burroughs, Amelia; Wise, Andrew K.; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y.; Lang, Eric J.

    2016-01-01

    Key points Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes.Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets.The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear.We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number.This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Abstract Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake

  16. Acute adaptation of the vestibuloocular reflex: signal processing by floccular and ventral parafloccular Purkinje cells.

    PubMed

    Hirata, Y; Highstein, S M

    2001-05-01

    The gain of the vertical vestibuloocular reflex (VVOR), defined as eye velocity/head velocity was adapted in squirrel monkeys by employing visual-vestibular mismatch stimuli. VVOR gain, measured in the dark, could be trained to values between 0.4 and 1.5. Single-unit activity of vertical zone Purkinje cells was recorded from the flocculus and ventral paraflocculus in alert squirrel monkeys before and during the gain change training. Our goal was to evaluate the site(s) of learning of the gain change. To aid in the evaluation, a model of the vertical optokinetic reflex (VOKR) and VVOR was constructed consisting of floccular and nonfloccular systems divided into subsystems based on the known anatomy and input and output parameters. Three kinds of input to floccular Purkinje cells via mossy fibers were explicitly described, namely vestibular, visual (retinal slip), and efference copy of eye movement. The characteristics of each subsystem (gain and phase) were identified at different VOR gains by reconstructing single-unit activity of Purkinje cells during VOKR and VVOR with multiple linear regression models consisting of sensory input and motor output signals. Model adequacy was checked by evaluating the residual following the regressions and by predicting Purkinje cells' activity during visual-vestibular mismatch paradigms. As a result, parallel changes in identified characteristics with VVOR adaptation were found in the prefloccular/floccular subsystem that conveys vestibular signals and in the nonfloccular subsystem that conveys vestibular signals, while no change was found in other subsystems, namely prefloccular/floccular subsystems conveying efference copy or visual signals, nonfloccular subsystem conveying visual signals, and postfloccular subsystem transforming Purkinje cell activity to eye movements. The result suggests multiple sites for VVOR motor learning including both flocculus and nonflocculus pathways. The gain change in the nonfloccular vestibular

  17. Multiple subclasses of Purkinje cells in the primate floccular complex provide similar signals to guide learning in the vestibulo-ocular reflex

    NASA Technical Reports Server (NTRS)

    Raymond, J. L.; Lisberger, S. G.

    1997-01-01

    The neural "learning rules" governing the induction of plasticity in the cerebellum were analyzed by recording the patterns of neural activity in awake, behaving animals during stimuli that induce a form of cerebellum-dependent learning. We recorded the simple- and complex-spike responses of a broad sample of Purkinje cells in the floccular complex during a number of stimulus conditions that induce motor learning in the vestibulo-ocular reflex (VOR). Each subclass of Purkinje cells carried essentially the same information about required changes in the gain of the VOR. The correlation of simple-spike activity in Purkinje cells with activity in vestibular pathways could guide learning during low-frequency but not high-frequency stimuli. Climbing fiber activity could guide learning during all stimuli tested but only if compared with the activity present approximately 100 msec earlier in either vestibular pathways or Purkinje cells.

  18. Resveratrol Protects Purkinje Neurons and Restores Muscle Activity in Rat Model of Cerebellar Ataxia.

    PubMed

    Ghorbani, Zeynab; Farahani, Reza Mastery; Aliaghaei, Abbas; Khodagholi, Fariba; Meftahi, Gholam Houssein; Danyali, Samira; Abdollahifar, Mohammad Amin; Daftari, Mahtab; Boroujeni, Mahdi Eskandarian; Sadeghi, Yousef

    2018-05-01

    Cerebellar ataxia (CA) is regarded as a miscellaneous cluster of brain disorders related to the cerebellum. Resveratrol is a naturally occurring polyphenolic compound. Previous reports suggest that resveratrol confers neuroprotection in various animal models of brain damage. Indeed, we considered it invaluable to investigate whether a treatment with resveratrol has a therapeutic role against CA induced by 3-acetylpyridine (3-AP) in rats. In addition, no investigation has examined neuroprotective effect of resveratrol in rat model of CA. Initially, 3-AP administration generated CA rat models followed by intraperitoneal injection with resveratrol. Then, motor performance and muscle electromyography (EMG) activity were assessed. Moreover, the anti-apoptotic role of resveratrol in CA and its relationship to protection of Purkinje cells were explored. According to what we have found, resveratrol administration improved the muscle activity and movement coordination in 3-AP-lesioned rats. Also under resveratrol treatment, the total number of the Purkinje neurons increased whereas a reduction in apoptotic bodies was observed. In conclusion, post-treatment with resveratrol evidently ameliorated motor performance as well as muscle activity accompanied by a protection of Purkinje cells in ataxic rats.

  19. Apoptosis of Purkinje and granular cells of the cerebellum following chronic ethanol intake.

    PubMed

    Oliveira, Suelen A; Chuffa, Luiz Gustavo A; Fioruci-Fontanelli, Beatriz Aparecida; Lizarte Neto, Fermino Sanches; Novais, Paulo Cezar; Tirapelli, Luiz Fernando; Oishi, Jorge Camargo; Takase, Luiz Fernando; Stefanini, Maira Aparecida; Martinez, Marcelo; Martinez, Francisco Eduardo

    2014-12-01

    Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10% (<2 g ethanol/kg body weight/day). At 120 days old, rats were anesthetized and decapitated, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), caspase-3, X-linked inhibitor of apoptosis protein (XIAP), and insulin-like growth factor 1-receptor (IGF-1R); real-time PCR (RT-PCR) to determine caspase-3, XIAP, and IGF-1R gene expression; and transmission electron microscopy (TEM). We identified fragmentation of DNA and an increase in caspase-3 protein and XIAP in Purkinje cells, whereas granule cells exhibited increased caspase-3 and XIAP. IGF-1R expression was unchanged. There was no significant difference in gene expression of caspase-3, XIAP, and IGF-1R. There were an increase in lipid droplets, a reduction in the cellular cytoplasm in electron-dense nuclei, and changes in the myelin sheath in the cerebellar cortex. In conclusion, our data demonstrated that ethanol induced apoptosis in the Purkinje and granule cells of the cerebellum of adult UChA rats.

  20. Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

    PubMed Central

    Fu, Chenglai; Xu, Jing; Li, Ruo-Jing; Crawford, Joshua A.; Khan, A. Basit; Ma, Ting Martin; Cha, Jiyoung Y.; Snowman, Adele M.; Pletnikov, Mikhail V.

    2015-01-01

    The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function. PMID:26245967

  1. Chronic treadmill exercise in rats delicately alters the Purkinje cell structure to improve motor performance and toxin resistance in the cerebellum.

    PubMed

    Huang, Tung-Yi; Lin, Lung-Sheng; Cho, Keng-Chi; Chen, Shean-Jen; Kuo, Yu-Min; Yu, Lung; Wu, Fong-Sen; Chuang, Jih-Ing; Chen, Hsiun-Ing; Jen, Chauying J

    2012-09-01

    Although exercise usually improves motor performance, the underlying cellular changes in the cerebellum remain to be elucidated. This study aimed to investigate whether and how chronic treadmill exercise in young rats induced Purkinje cell changes to improve motor performance and rendered the cerebellum less vulnerable to toxin insults. After 1-wk familiarization of treadmill running, 6-wk-old male Wistar rats were divided into exercise and sedentary groups. The exercise group was then subjected to 8 wk of exercise training at moderate intensity. The rotarod test was carried out to evaluate motor performance. Purkinje cells in cerebellar slices were visualized by lucifer yellow labeling in single neurons and by calbindin immunostaining in groups of neurons. Compared with sedentary control rats, exercised rats not only performed better in the rotarod task, but also showed finer Purkinje cell structure (higher dendritic volume and spine density with the same dendritic field). The exercise-improved cerebellar functions were further evaluated by monitoring the long-lasting effects of intraventricular application of OX7-saporin. In the sedentary group, OX7-saporin treatment retarded the rotarod performance and induced ∼60% Purkinje cell loss in 3 wk. As a comparison, the exercise group showed much milder injuries in the cerebellum by the same toxin treatment. In conclusion, exercise training in young rats increased the dendritic density of Purkinje cells, which might play an important role in improving the motor performance. Furthermore, as Purkinje cells in the exercise group were relatively toxin resistant, the exercised rats showed good motor performance, even under toxin-treated conditions.

  2. Changes in Purkinje cell simple spike encoding of reach kinematics during adaption to a mechanical perturbation.

    PubMed

    Hewitt, Angela L; Popa, Laurentiu S; Ebner, Timothy J

    2015-01-21

    The cerebellum is essential in motor learning. At the cellular level, changes occur in both the simple spike and complex spike firing of Purkinje cells. Because simple spike discharge reflects the main output of the cerebellar cortex, changes in simple spike firing likely reflect the contribution of the cerebellum to the adapted behavior. Therefore, we investigated in Rhesus monkeys how the representation of arm kinematics in Purkinje cell simple spike discharge changed during adaptation to mechanical perturbations of reach movements. Monkeys rapidly adapted to a novel assistive or resistive perturbation along the direction of the reach. Adaptation consisted of matching the amplitude and timing of the perturbation to minimize its effect on the reach. In a majority of Purkinje cells, simple spike firing recorded before and during adaptation demonstrated significant changes in position, velocity, and acceleration sensitivity. The timing of the simple spike representations change within individual cells, including shifts in predictive versus feedback signals. At the population level, feedback-based encoding of position increases early in learning and velocity decreases. Both timing changes reverse later in learning. The complex spike discharge was only weakly modulated by the perturbations, demonstrating that the changes in simple spike firing can be independent of climbing fiber input. In summary, we observed extensive alterations in individual Purkinje cell encoding of reach kinematics, although the movements were nearly identical in the baseline and adapted states. Therefore, adaption to mechanical perturbation of a reaching movement is accompanied by widespread modifications in the simple spike encoding. Copyright © 2015 the authors 0270-6474/15/351106-19$15.00/0.

  3. Predictive and Feedback Performance Errors are Signaled in the Simple Spike Discharge of Individual Purkinje Cells

    PubMed Central

    Popa, Laurentiu S.; Hewitt, Angela L.; Ebner, Timothy J.

    2012-01-01

    The cerebellum has been implicated in processing motor errors required for online control of movement and motor learning. The dominant view is that Purkinje cell complex spike discharge signals motor errors. This study investigated whether errors are encoded in the simple spike discharge of Purkinje cells in monkeys trained to manually track a pseudo-randomly moving target. Four task error signals were evaluated based on cursor movement relative to target movement. Linear regression analyses based on firing residuals ensured that the modulation with a specific error parameter was independent of the other error parameters and kinematics. The results demonstrate that simple spike firing in lobules IV–VI is significantly correlated with position, distance and directional errors. Independent of the error signals, the same Purkinje cells encode kinematics. The strongest error modulation occurs at feedback timing. However, in 72% of cells at least one of the R2 temporal profiles resulting from regressing firing with individual errors exhibit two peak R2 values. For these bimodal profiles, the first peak is at a negative τ (lead) and a second peak at a positive τ (lag), implying that Purkinje cells encode both prediction and feedback about an error. For the majority of the bimodal profiles, the signs of the regression coefficients or preferred directions reverse at the times of the peaks. The sign reversal results in opposing simple spike modulation for the predictive and feedback components. Dual error representations may provide the signals needed to generate sensory prediction errors used to update a forward internal model. PMID:23115173

  4. Changes in Purkinje Cell Simple Spike Encoding of Reach Kinematics during Adaption to a Mechanical Perturbation

    PubMed Central

    Hewitt, Angela L.; Popa, Laurentiu S.

    2015-01-01

    The cerebellum is essential in motor learning. At the cellular level, changes occur in both the simple spike and complex spike firing of Purkinje cells. Because simple spike discharge reflects the main output of the cerebellar cortex, changes in simple spike firing likely reflect the contribution of the cerebellum to the adapted behavior. Therefore, we investigated in Rhesus monkeys how the representation of arm kinematics in Purkinje cell simple spike discharge changed during adaptation to mechanical perturbations of reach movements. Monkeys rapidly adapted to a novel assistive or resistive perturbation along the direction of the reach. Adaptation consisted of matching the amplitude and timing of the perturbation to minimize its effect on the reach. In a majority of Purkinje cells, simple spike firing recorded before and during adaptation demonstrated significant changes in position, velocity, and acceleration sensitivity. The timing of the simple spike representations change within individual cells, including shifts in predictive versus feedback signals. At the population level, feedback-based encoding of position increases early in learning and velocity decreases. Both timing changes reverse later in learning. The complex spike discharge was only weakly modulated by the perturbations, demonstrating that the changes in simple spike firing can be independent of climbing fiber input. In summary, we observed extensive alterations in individual Purkinje cell encoding of reach kinematics, although the movements were nearly identical in the baseline and adapted states. Therefore, adaption to mechanical perturbation of a reaching movement is accompanied by widespread modifications in the simple spike encoding. PMID:25609626

  5. Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models

    PubMed Central

    Yokoi, Fumiaki; Dang, Mai T.; Yang, Guang; Li, JinDong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

    2011-01-01

    Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ε-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally-inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ε-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ε-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally-inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ε-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ε-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. PMID:22040906

  6. TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres.

    PubMed

    Hof, Thomas; Sallé, Laurent; Coulbault, Laurent; Richer, Romain; Alexandre, Joachim; Rouet, René; Manrique, Alain; Guinamard, Romain

    2016-01-15

    The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells. Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate

  7. Lrp5/6 are required for cerebellar development and for suppressing TH expression in Purkinje cells via β-catenin.

    PubMed

    Huang, Ying; Zhang, Qiong; Song, Ning-Ning; Zhang, Lei; Sun, Yu-Ling; Hu, Ling; Chen, Jia-Ying; Zhu, Weidong; Li, Jue; Ding, Yu-Qiang

    2016-01-15

    The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons. We report here that the low-density lipoprotein receptors (Lrp) 5 and 6, Wnt co-receptors, are required for the development of the cerebellum and for suppressing ectopic TH expression in Purkinje cells. Simultaneous inactivation of Lrp 5 and 6 by Nestin-Cre results in defective lamination and foliation of the cerebellum during postnatal development. Surprisingly, TH is ectopically expressed by Purkinje cells, although they still keep its other neurochemical characteristics. These phenotypes are also observed in the cerebellum of GFAP-Cre;β-catenin(flox/flox) mice, and AAV2-Cre-mediated gene deletion leads to ectopic TH expression in Purkinje cells of β-catenin(flox/flox) mice as well. Our results revealed a new role of the canonical Lrp5/6-β-catenin pathway in regulating the morphogenesis of the cerebellum during postnatal development.

  8. Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models.

    PubMed

    Yokoi, Fumiaki; Dang, Mai T; Yang, Guang; Li, Jindong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

    2012-02-01

    Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ɛ-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ɛ-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ɛ-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ɛ-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ɛ-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments.

    PubMed

    Fukatsu, Kazumi; Bannai, Hiroko; Inoue, Takafumi; Mikoshiba, Katsuhiko

    2010-09-01

    Inositol 1,4,5-trisphosphate receptor type 1 (IP(3) R1) is an intracellular Ca(2+) release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3) R1 on the endoplasmic reticulum membrane and the distribution of IP(3) R1 in neurons are thought to be important for the spatial regulation of Ca(2+) release. In this study, we analyzed the lateral diffusion of IP(3) R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3) R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 μm(2) /s, and the diffusion of IP(3) R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3) R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca(2+) transient in Purkinje cells, decreased the diffusion of IP(3) R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3) R1 in spines. © 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.

  10. Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and Purkinje fibers.

    PubMed

    Nayak, Alok Ranjan; Panfilov, A V; Pandit, Rahul

    2017-02-01

    We present systematic numerical studies of the possible effects of the coupling of human endocardial and Purkinje cells at cellular and two-dimensional tissue levels. We find that the autorhythmic-activity frequency of the Purkinje cell in a composite decreases with an increase in the coupling strength; this can even eliminate the autorhythmicity. We observe a delay between the beginning of the action potentials of endocardial and Purkinje cells in a composite; such a delay increases as we decrease the diffusive coupling, and eventually a failure of transmission occurs. An increase in the diffusive coupling decreases the slope of the action-potential-duration-restitution curve of an endocardial cell in a composite. By using a minimal model for the Purkinje network, in which we have a two-dimensional, bilayer tissue, with a layer of Purkinje cells on top of a layer of endocardial cells, we can stabilize spiral-wave turbulence; however, for a sparse distribution of Purkinje-ventricular junctions, at which these two layers are coupled, we can also obtain additional focal activity and many complex transient regimes. We also present additional effects resulting from the coupling of Purkinje and endocardial layers and discuss the relation of our results to the studies performed in anatomically accurate models of the Purkinje network.

  11. Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and Purkinje fibers

    NASA Astrophysics Data System (ADS)

    Nayak, Alok Ranjan; Panfilov, A. V.; Pandit, Rahul

    2017-02-01

    We present systematic numerical studies of the possible effects of the coupling of human endocardial and Purkinje cells at cellular and two-dimensional tissue levels. We find that the autorhythmic-activity frequency of the Purkinje cell in a composite decreases with an increase in the coupling strength; this can even eliminate the autorhythmicity. We observe a delay between the beginning of the action potentials of endocardial and Purkinje cells in a composite; such a delay increases as we decrease the diffusive coupling, and eventually a failure of transmission occurs. An increase in the diffusive coupling decreases the slope of the action-potential-duration-restitution curve of an endocardial cell in a composite. By using a minimal model for the Purkinje network, in which we have a two-dimensional, bilayer tissue, with a layer of Purkinje cells on top of a layer of endocardial cells, we can stabilize spiral-wave turbulence; however, for a sparse distribution of Purkinje-ventricular junctions, at which these two layers are coupled, we can also obtain additional focal activity and many complex transient regimes. We also present additional effects resulting from the coupling of Purkinje and endocardial layers and discuss the relation of our results to the studies performed in anatomically accurate models of the Purkinje network.

  12. Crista Supraventricularis Purkinje Network and Its Relation to Intraseptal Purkinje Network.

    PubMed

    De Almeida, Marcos C; Araujo, Mayssa; Duque, Mathias; Vilhena, Virginia

    2017-10-01

    Using transparent specimens with a dual color injection, microscopy, and computer tomography, this report shows that the right and left ventricular subendocardial Purkinje networks are connected by an extensive septal network in the bovine heart. The septal network is present along the entire septum except at a free zone below ventricular valves. Being the only communication of the basal right septum with the right free wall, the supraventricular crest is an enigmatic but not, by any means, hidden muscular structure. It is one of the last structures to be activated in human heart. It is shown here that the supraventricular crest Purkinje network connects the anterosuperior right ventricular basal free wall Purkinje network to anterior right ventricular basal septal Purkinje network. It is suggested that the stimulus initiated at middle left ventricular endocardium will activate the supraventricular crest. The intraseptal connection found between the basal left ventricular subendocardial septal Purkinje network and the right ventricular basal septal Purkinje network is, probably, the pathway for the stimulus. An anatomic basis is provided to explain why the inflow tract contracts earlier than the outflow tract in the right ventricle systole. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1793-1801, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Modulated discharge of Purkinje and stellate cells persists after unilateral loss of vestibular primary afferent mossy fibers in mice

    PubMed Central

    Yakhnitsa, V.

    2013-01-01

    Cerebellar Purkinje cells are excited by two afferent pathways: climbing and mossy fibers. Climbing fibers evoke large “complex spikes” (CSs) that discharge at low frequencies. Mossy fibers synapse on granule cells whose parallel fibers excite Purkinje cells and may contribute to the genesis of “simple spikes” (SSs). Both afferent systems convey vestibular information to folia 9c–10. After making a unilateral labyrinthectomy (UL) in mice, we tested how the discharge of CSs and SSs was changed by the loss of primary vestibular afferent mossy fibers during sinusoidal roll tilt. We recorded from cells identified by juxtacellular neurobiotin labeling. The UL preferentially reduced vestibular modulation of CSs and SSs in folia 8–10 contralateral to the UL. The effects of a UL on Purkinje cell discharge were similar in folia 9c–10, to which vestibular primary afferents project, and in folia 8–9a, to which they do not project, suggesting that vestibular primary afferent mossy fibers were not responsible for the UL-induced alteration of SS discharge. UL also induced reduced vestibular modulation of stellate cell discharge contralateral to the UL. We attribute the decreased modulation to reduced vestibular modulation of climbing fibers. In summary, climbing fibers modulate CSs directly and SSs indirectly through activation of stellate cells. Whereas vestibular primary afferent mossy fibers cannot account for the modulated discharge of SSs or stellate cells, the nonspecific excitation of Purkinje cells by parallel fibers may set an operating point about which the discharges of SSs are sculpted by climbing fibers. PMID:23966673

  14. Purkinje cell heterotopy with cerebellar hypoplasia in two free-living American kestrels (Falco sparverius)

    USDA-ARS?s Scientific Manuscript database

    Two wild fledgling kestrels exhibited lack of motor coordination, postural reaction deficits, and abnormal propioception. At necropsy, the cerebellum and brainstem were markedly underdeveloped. Microscopically, there was Purkinje cells heterotopy, abnormal circuitry, and hypoplasia with defective fo...

  15. Structural study of Purkinje cell axonal torpedoes in essential tremor.

    PubMed

    Louis, Elan D; Yi, Hong; Erickson-Davis, Cordelia; Vonsattel, Jean-Paul G; Faust, Phyllis L

    2009-02-06

    Essential tremor (ET) is one of the most common neurological diseases. A basic understanding of its neuropathology is now emerging. Aside from Purkinje cell loss, a prominent finding is an abundance of torpedoes (rounded swellings of Purkinje cell axons). Such swellings often result from the mis-accumulation of cell constituents. Identifying the basic nature of these accumulations is an important step in understanding the underlying disease process. Torpedoes, only recently identified in ET, have not yet been characterized ultrastructurally. Light and electron microscopy were used to characterize the structural constituents of torpedoes in ET. Formalin-fixed cerebellar cortical tissue from four prospectively collected ET brains was sectioned and immunostained with a monoclonal phosphorylated neurofilament antibody (SMI-31, Covance, Emeryville, CA). Using additional sections from three ET brains, torpedoes were assessed using electron microscopy. Immunoreactivity for phosphorylated neurofilament protein revealed clear labeling of torpedoes in each case. Torpedoes were strongly immunoreactive; in many instances, two or more torpedoes were noted in close proximity to one another. On electron microscopy, torpedoes were packed with randomly arranged 10-12nm neurofilaments. Mitochondria and smooth endoplasmic reticulum were abundant as well, particularly at the periphery of the torpedo. We demonstrated that the torpedoes in ET represent the mis-accumulation of disorganized neurofilaments and other organelles. It is not known where in the pathogenic cascade these accumulations occur (i.e., whether these accumulations are the primary event or a secondary/downstream event) and this deserves further study.

  16. High dosage of monosodium glutamate causes deficits of the motor coordination and the number of cerebellar Purkinje cells of rats.

    PubMed

    Prastiwi, D; Djunaidi, A; Partadiredja, G

    2015-11-01

    Monosodium glutamate (MSG) has been widely used throughout the world as a flavoring agent of food. However, MSG at certain dosages is also thought to cause damage to many organs, including cerebellum. This study aimed at investigating the effects of different doses of MSG on the motor coordination and the number of Purkinje cells of the cerebellum of Wistar rats. A total of 24 male rats aged 4 to 5 weeks were divided into four groups, namely, control (C), T2.5, T3, and T3.5 groups, which received intraperitoneal injection of 0.9% sodium chloride solution, 2.5 mg/g body weight (bw) of MSG, 3.0 mg/g bw of MSG, and 3.5 mg/g bw of MSG, respectively, for 10 consecutive days. The motor coordination of the rats was examined prior and subsequent to the treatment. The number of cerebellar Purkinje cells was estimated using physical fractionator method. It has been found that the administration of MSG at a dosage of 3.5 mg/g bw, but not at lower dosages, caused a significant decrease of motor coordination and the estimated total number of Purkinje cells of rats. There was also a significant correlation between motor coordination and the total number of Purkinje cells. © The Author(s) 2015.

  17. Dissociation of spatial navigation and visual guidance performance in Purkinje cell degeneration (pcd) mutant mice.

    PubMed

    Goodlett, C R; Hamre, K M; West, J R

    1992-04-10

    Spatial learning in rodents requires normal functioning of hippocampal and cortical structures. Recent data suggest that the cerebellum may also be essential. Neurological mutant mice with dysgenesis of the cerebellum provide useful models to examine the effects of abnormal cerebellar function. Mice with one such mutation, Purkinje cell degeneration (pcd), in which Purkinje cells degenerate between the third and fourth postnatal weeks, were evaluated for performance of spatial navigation learning and visual guidance learning in the Morris maze swim-escape task. Unaffected littermates and C57BL/6J mice served as controls. Separate groups of pcd and control mice were tested at 30, 50 and 110 days of age. At all ages, pcd mice had severe deficits in distal-cue (spatial) navigation, failing to decrease path lengths over training and failing to express appropriate spatial biases on probe trials. On the proximal-cue (visual guidance) task, whenever performance differences between groups did occur, they were limited to the initial trials. The ability of the pcd mice to perform the proximal-cue but not the distal-cue task indicates that the massive spatial navigation deficit was not due simply to motor dysfunction. Histological evaluations confirmed that the pcd mutation resulted in Purkinje cell loss without significant depletion of cells in the hippocampal formation. These data provide further evidence that the cerebellum is vital for the expression of behavior directed by spatial cognitive processes.

  18. Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy

    PubMed Central

    Kros, Lieke; Lindeman, Sander; Eelkman Rooda, Oscar H. J.; Murugesan, Pavithra; Bina, Lorenzo; Bosman, Laurens W. J.; De Zeeuw, Chris I.; Hoebeek, Freek E.

    2017-01-01

    Absence epilepsy is characterized by the occurrence of generalized spike and wave discharges (GSWDs) in electrocorticographical (ECoG) recordings representing oscillatory activity in thalamocortical networks. The oscillatory nature of GSWDs has been shown to be reflected in the simple spike activity of cerebellar Purkinje cells and in the activity of their target neurons in the cerebellar nuclei, but it is unclear to what extent complex spike activity is implicated in generalized epilepsy. Purkinje cell complex spike firing is elicited by climbing fiber activation and reflects action potential firing in the inferior olive. Here, we investigated to what extent modulation of complex spike firing is reflected in the temporal patterns of seizures. Extracellular single-unit recordings in awake, head-restrained homozygous tottering mice, which suffer from a mutation in the voltage-gated CaV2.1 calcium channel, revealed that a substantial proportion of Purkinje cells (26%) showed increased complex spike activity and rhythmicity during GSWDs. Moreover, Purkinje cells, recorded either electrophysiologically or by using Ca2+-imaging, showed a significant increase in complex spike synchronicity for both adjacent and remote Purkinje cells during ictal events. These seizure-related changes in firing frequency, rhythmicity and synchronicity were most prominent in the lateral cerebellum, a region known to receive cerebral input via the inferior olive. These data indicate profound and widespread changes in olivary firing that are most likely induced by seizure-related activity changes in the thalamocortical network, thereby highlighting the possibility that olivary neurons can compensate for pathological brain-state changes by dampening oscillations. PMID:29163057

  19. Responses of Purkinje cells in the oculomotor vermis of monkeys during smooth pursuit eye movements and saccades: comparison with floccular complex.

    PubMed

    Raghavan, Ramanujan T; Lisberger, Stephen G

    2017-08-01

    We recorded the responses of Purkinje cells in the oculomotor vermis during smooth pursuit and saccadic eye movements. Our goal was to characterize the responses in the vermis using approaches that would allow direct comparisons with responses of Purkinje cells in another cerebellar area for pursuit, the floccular complex. Simple-spike firing of vermis Purkinje cells is direction selective during both pursuit and saccades, but the preferred directions are sufficiently independent so that downstream circuits could decode signals to drive pursuit and saccades separately. Complex spikes also were direction selective during pursuit, and almost all Purkinje cells showed a peak in the probability of complex spikes during the initiation of pursuit in at least one direction. Unlike the floccular complex, the preferred directions for simple spikes and complex spikes were not opposite. The kinematics of smooth eye movement described the simple-spike responses of vermis Purkinje cells well. Sensitivities were similar to those in the floccular complex for eye position and considerably lower for eye velocity and acceleration. The kinematic relations were quite different for saccades vs. pursuit, supporting the idea that the contributions from the vermis to each kind of movement could contribute independently in downstream areas. Finally, neither the complex-spike nor the simple-spike responses of vermis Purkinje cells were appropriate to support direction learning in pursuit. Complex spikes were not triggered reliably by an instructive change in target direction; simple-spike responses showed very small amounts of learning. We conclude that the vermis plays a different role in pursuit eye movements compared with the floccular complex. NEW & NOTEWORTHY The midline oculomotor cerebellum plays a different role in smooth pursuit eye movements compared with the lateral, floccular complex and appears to be much less involved in direction learning in pursuit. The output from the

  20. Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

    PubMed

    Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

    2015-01-01

    Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

  1. Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    PubMed Central

    Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

    2015-01-01

    Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

  2. Regular Patterns in Cerebellar Purkinje Cell Simple Spike Trains

    PubMed Central

    Shin, Soon-Lim; Hoebeek, Freek E.; Schonewille, Martijn; De Zeeuw, Chris I.; Aertsen, Ad; De Schutter, Erik

    2007-01-01

    Background Cerebellar Purkinje cells (PC) in vivo are commonly reported to generate irregular spike trains, documented by high coefficients of variation of interspike-intervals (ISI). In strong contrast, they fire very regularly in the in vitro slice preparation. We studied the nature of this difference in firing properties by focusing on short-term variability and its dependence on behavioral state. Methodology/Principal Findings Using an analysis based on CV2 values, we could isolate precise regular spiking patterns, lasting up to hundreds of milliseconds, in PC simple spike trains recorded in both anesthetized and awake rodents. Regular spike patterns, defined by low variability of successive ISIs, comprised over half of the spikes, showed a wide range of mean ISIs, and were affected by behavioral state and tactile stimulation. Interestingly, regular patterns often coincided in nearby Purkinje cells without precise synchronization of individual spikes. Regular patterns exclusively appeared during the up state of the PC membrane potential, while single ISIs occurred both during up and down states. Possible functional consequences of regular spike patterns were investigated by modeling the synaptic conductance in neurons of the deep cerebellar nuclei (DCN). Simulations showed that these regular patterns caused epochs of relatively constant synaptic conductance in DCN neurons. Conclusions/Significance Our findings indicate that the apparent irregularity in cerebellar PC simple spike trains in vivo is most likely caused by mixing of different regular spike patterns, separated by single long intervals, over time. We propose that PCs may signal information, at least in part, in regular spike patterns to downstream DCN neurons. PMID:17534435

  3. [The effect of modulators of SK channels on simple spike firing frequency in the discharge of the cerebellar Purkinje cells in laboratory mice].

    PubMed

    Egorova, P A; Karelina, T V; Vlasova, O L; Antonov, S M; Besprozvanny, I B

    2014-01-01

    The effect of CyPPA, a positive modulator of small conductance calcium-activated potassium channels of type 3 and 2 (SK3/SK2), and of NS309, an activator of intermediate and small conductance calcium-activated potassium channels (IK/SK), on the activity of cerebellar Purkinje cells was studied in 2-month-old male mice. The use of 1 mM of CyPPA has led to a decrease of simple spike firing frequency in the discharge of Purkinje cells by 25%, on average, during 1 h after application. At the same time, application of 100 μM of NS309 has promoted a decrease in simple spike firing frequency by 47 %, on average, during 1 h after the beginning of the action. The obtained results confirm the hypothesis that SK channels participate in regulation of simple spike firing frequency in the discharge of Purkinje cells and are responsible for restriction of signal frequency. The effect of NS309 on simple spike firing frequency was more pronounced; therefore, the IK/SK channels may be suggested to play the cardinal role in regulation of spike activity of Purkinje cells. Since increasing simple spike frequency in the discharge of Purkinje cells is observed at many disturbances of motor activity, in particular, at spinocerebellar ataxia, it can be suggested that the studied compounds or substances of similar action are of interest as potential medicinal agents.

  4. Differential association of GABAB receptors with their effector ion channels in Purkinje cells.

    PubMed

    Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Cózar, Javier; Kleindienst, David; de la Ossa, Luis; Bettler, Bernhard; Wickman, Kevin; Watanabe, Masahiko; Shigemoto, Ryuichi; Fukazawa, Yugo

    2018-04-01

    Metabotropic GABA B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K + (GIRK/Kir3) channel and the voltage-dependent Ca 2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA B receptors co-assembled with GIRK and Ca V 2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA B1 and Ca V 2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA B1 and GIRK2 or Ca V 2.1 channels was detected, inter-cluster distance for GABA B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA B1 and Ca V 2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA B receptors are associated with GIRK and Ca V 2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA B receptors and their effector ion channels in the cerebellar network.

  5. Reminiscing about Jan Evangelista Purkinje: a pioneer of modern experimental physiology.

    PubMed

    Cavero, Icilio; Guillon, Jean-Michel; Holzgrefe, Henry H

    2017-12-01

    This article reminisces about the life and key scientific achievements of Jan Evangelista Purkinje (1787-1869), a versatile 19th century Czech pioneer of modern experimental physiology. In 1804, after completing senior high school, Purkinje joined the Piarist monk order, but, after a 3-yr novitiate, he gave up the religious calling "to deal more freely with science." In 1818, he earned a Medical Doctor degree from Prague University by defending a dissertation on intraocular phenomena observed in oneself. In 1823, Purkinje became a Physiology and Pathology professor at the Prussian Medical University in Breslau, where he innovated the traditional teaching methods of physiology. Purkinje's contributions to physiology were manifold: accurate descriptions of various visual phenomena (e.g., Purkinje-Sanson images, Purkinje phenomenon), discovery of the terminal network of the cardiac conduction system (Purkinje fibers), identification of cerebellar neuronal bodies (Purkinje cells), formulation of the vertigo law (Purkinje's law), discovery of criteria to classify human fingerprints, etc. In 1850, Purkinje accepted and held until his death the Physiology chair at Prague Medical Faculty. During this period, he succeeded in introducing the Czech idiom (in addition to long-established German and Latin) as a Medical Faculty teaching language. Additionally, as a zealous Czech patriot, he actively contributed to the naissance and consolidation of a national Czech identity conscience. Purkinje was a trend-setting scientist who, throughout his career, worked to pave the way for the renovation of physiology from a speculative discipline, ancilla of anatomy, into a factual, autonomous science committed to the discovery of mechanisms governing in-life functions. Copyright © 2017 the American Physiological Society.

  6. Sensory processing and corollary discharge effects in posterior caudal lobe Purkinje cells in a weakly electric mormyrid fish.

    PubMed

    Alviña, Karina; Sawtell, Nathaniel B

    2014-07-15

    Although it has been suggested that the cerebellum functions to predict the sensory consequences of motor commands, how such predictions are implemented in cerebellar circuitry remains largely unknown. A detailed and relatively complete account of predictive mechanisms has emerged from studies of cerebellum-like sensory structures in fish, suggesting that comparisons of the cerebellum and cerebellum-like structures may be useful. Here we characterize electrophysiological response properties of Purkinje cells in a region of the cerebellum proper of weakly electric mormyrid fish, the posterior caudal lobe (LCp), which receives the same mossy fiber inputs and projects to the same target structures as the electrosensory lobe (ELL), a well-studied cerebellum-like structure. We describe patterns of simple spike and climbing fiber activation in LCp Purkinje cells in response to motor corollary discharge, electrosensory, and proprioceptive inputs and provide evidence for two functionally distinct Purkinje cell subtypes within LCp. Protocols that induce rapid associative plasticity in ELL fail to induce plasticity in LCp, suggesting differences in the adaptive functions of the two structures. Similarities and differences between LCp and ELL are discussed in light of these results. Copyright © 2014 the American Physiological Society.

  7. Tissue Plasminogen Activator Induction in Purkinje Neurons After Cerebellar Motor Learning

    NASA Astrophysics Data System (ADS)

    Seeds, Nicholas W.; Williams, Brian L.; Bickford, Paula C.

    1995-12-01

    The cerebellar cortex is implicated in the learning of complex motor skills. This learning may require synaptic remodeling of Purkinje cell inputs. An extracellular serine protease, tissue plasminogen activator (tPA), is involved in remodeling various nonneural tissues and is associated with developing and regenerating neurons. In situ hybridization showed that expression of tPA messenger RNA was increased in the Purkinje neurons of rats within an hour of their being trained for a complex motor task. Antibody to tPA also showed the induction of tPA protein associated with cerebellar Purkinje cells. Thus, the induction of tPA during motor learning may play a role in activity-dependent synaptic plasticity.

  8. Step-related discharges of Purkinje cells in the paravermal cortex of the cerebellar anterior lobe in the cat.

    PubMed Central

    Edgley, S A; Lidierth, M

    1988-01-01

    1. Extracellular recordings were made of the simple spike discharges of Purkinje cells in the lateral part of the paravermal cortex of lobule V in the cerebellum of awake cats. The cells were located within the c2 and c3 zones of Oscarsson (1979). 2. The peripheral receptive fields in which light mechanical stimuli could evoke simple spikes were examined in 252 Purkinje cells. Ninety-two per cent were activated by stimulation of the ipsilateral forelimb and 52% of 113 tested cells also discharged simple spikes in response to stimulation of the contralateral forelimb. The receptive fields were concentrated on the distal parts of the limbs: 67% of the 139 cells which were examined in most detail responded to stimulation of the paw or wrist of the ipsilateral forelimb. 3. In 135 of the Purkinje cells, the discharges were recorded during locomotion. Simple spikes were discharged at a mean rate of 54.3 +/- 27.8 impulses/s (S.D., n = 135) during steady walking on a belt moving at 0.5-0.7 m/s. The discharges of each cell were rhythmically modulated in time with the movements of stepping and although the timings of the discharges were highly variable between cells, activity in the population was greatest at the times of transition between the stance and swing phases in the ipsilateral forelimb and least during mid-stance. 4. As a population Purkinje cells with simple spike receptive fields on the distal parts of the forelimb(s) exhibited two activity maxima. These occurred during early stance and during the transition from stance to swing in the ipsilateral forelimb. Cells with receptive fields on the proximal parts of the limb achieved an activity maximum during late swing, and their average discharge rate fell at the time of onset of the swing phase in the ipsilateral forelimb instead of rising as was the case for the distal group. 5. The present results are compared with those from cells located more medially in the paravermal cortex. It is shown that medially located

  9. Effect of glutamate, aspartate and related derivatives on cerebellar Purkinje cell dendrites in the rat: an in vitro study

    PubMed Central

    Crepel, F.; Dhanjal, S. S.; Sears, T. A.

    1982-01-01

    1. The responses of Purkinje cells to short duration (pulse) ionophoretic applications of L-aspartate (L-asp), L-glutamate (L-glu), N-methyl DL-aspartate (NMDLA) and quisqualic acid in their dendritic fields were studied in vitro on sagittal slices of lobules IX and X of the adult rat cerebellum. 2. Pulse application of L-asp or L-glu evoked transient and dose-dependent increases in the firing rate of the simple spikes recorded extracellularly as single units. When the ionophoretic electrode was positioned in the dendritic field of the Purkinje cells, the lowest thresholds for L-glu and L-asp mediated excitations of the cells were as low as 25 and 35 pC respectively, with a latency for maximal responses as brief as 7 ms. 3. In intracellular recordings these excitatory responses consisted of depolarizations of up to 18 mV in amplitude and with depolarizing slopes up to 0·52 mV/ms. They were generally unaccompanied by changes in cell input resistance in contrast to the marked decrease which occurred in response to steady applications of large doses of L-asp and L-glu. 4. The spatial distribution of the excitatory sites confirmed that the dendritic sensitivity to L-glu was greater than that of the soma and showed that the same was true for L-asp. In 34% of cells the sensitivity for L-asp declined markedly in the upper region of the molecular layer, whereas it remained high for L-glu; no such differential sensitivity was detected in the remaining 66% of cells. 5. Inhibitory responses, antagonized by 10-5 M-bicuculline in the bath, were also induced in Purkinje cells by L-glu and L-asp when the ionophoretic electrode was withdrawn from the excitatory sites by as little as 8 μm and up to 40 μm upward or downward along the track of parallel fibres or positioned as far as 250 μm laterally. 6. Whenever it was applied in the molecular layer, the pulse application of NMDLA elicited no excitatory response in Purkinje cells recorded extra or intracellularly. However, slow

  10. Sumoylation of FOXP2 Regulates Motor Function and Vocal Communication Through Purkinje Cell Development.

    PubMed

    Usui, Noriyoshi; Co, Marissa; Harper, Matthew; Rieger, Michael A; Dougherty, Joseph D; Konopka, Genevieve

    2017-02-01

    Mutations in the gene encoding the transcription factor forkhead box P2 (FOXP2) result in brain developmental abnormalities, including reduced gray matter in both human patients and rodent models and speech and language deficits. However, neither the region-specific function of FOXP2 in the brain, in particular the cerebellum, nor the effects of any posttranslational modifications of FOXP2 in the brain and disorders have been explored. We characterized sumoylation of FOXP2 biochemically and analyzed the region-specific function and sumoylation of FOXP2 in the developing mouse cerebellum. Using in utero electroporation to manipulate the sumoylation state of FOXP2 as well as Foxp2 expression levels in Purkinje cells of the cerebellum in vivo, we reduced Foxp2 expression approximately 40% in the mouse cerebellum. Such a reduction approximates the haploinsufficiency observed in human patients who demonstrate speech and language impairments. We identified sumoylation of FOXP2 at K674 (K673 in mice) in the cerebellum of neonates. In vitro co-immunoprecipitation and in vivo colocalization experiments suggest that PIAS3 acts as the small ubiquitin-like modifier E3 ligase for FOXP2 sumoylation. This sumoylation modifies transcriptional regulation by FOXP2. We demonstrated that FOXP2 sumoylation is required for regulation of cerebellar motor function and vocal communication, likely through dendritic outgrowth and arborization of Purkinje cells in the mouse cerebellum. Sumoylation of FOXP2 in neonatal mouse cerebellum regulates Purkinje cell development and motor functions and vocal communication, demonstrating evidence for sumoylation in regulating mammalian behaviors. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  11. MICROLESIONS OF THE INFERIOR OLIVE REDUCE VESTIBULAR MODULATION OF PURKINJE CELL COMPLEX AND SIMPLE SPIKES IN MOUSE CEREBELLUM

    PubMed Central

    Barmack, N.H.; Yakhnitsa, V.

    2011-01-01

    Cerebellar Purkinje cells have two distinct action potentials: Complex spikes (CSs) are evoked by single climbing fibers that originate from the contralateral inferior olive. Simple spikes (SSs) are often ascribed to mossy fiber---granule cell---parallel fiber inputs to Purkinje cells. Although generally accepted, this view lacks experimental support. Vestibular stimulation independently activates primary afferent mossy fibers and tertiary afferent climbing fibers that project to theuvula-nodulus (folia 8-10). CSs and SSs normally discharge antiphasically during sinusoidal roll-tilt. When CSs increase, SSs decrease. We tested the relative independence of these pathways in mice by making electrolytic microlesions of the two inferior olivary nuclei from which vestibular climbing fibers originate; the β-nucleus and dorsomedial cell column (DMCC). This reduced vestibular climbing fiber signaling to the contralateral folia 8-10, while leaving intact vestibular primary and secondary afferent mossy fibers. We recorded from Purkinje cells and interneurons in folia 8-10, identified by juxtacellular labeling with neurobiotin. Microlesions of the inferior olive increased the spontaneous discharge of SSs in contralateral folia 8-10, but blocked their modulation during vestibular stimulation. The vestibularly-evoked discharge of excitatory cerebellar interneurons (granule cells and unipolar brush cells) was not modified by olivary microlesions. The modulated discharge of stellate cells, but not Golgi cells was reduced by olivary microlesions. We conclude that vestibular modulation of CSs and SSs depends on intact climbing fibers. The absence of vestibularly-modulated SSs following olivary microlesions reflects the loss of climbing fiber-evoked stellate cell discharge. PMID:21734274

  12. Voltage signals of individual Purkinje cell dendrites in rat cerebellar slices.

    PubMed

    Borst, A; Heck, D; Thomann, M

    1997-11-28

    For investigating neuronal information processing at the cellular level, a technique which visualizes the voltage distribution within single neurons in situ would be extremely useful. Voltage-sensitive dyes are, in principle, capable of reporting membrane potential [Cohen, L.B. and Salzberg, B.M., Rev. Physiol. Biochem. Pharmacol., 83 (1978) 35-88; Grinvald, A., Lieke, E.E., Frostig, R.D. and Hildesheim, R., J. Neurosci., 14 (1994) 2545-2568; Kleinfeld, D., Delaney, K.R., Fee, M.S., Flores, J.A., Tank, D.W. and Gelperin, A., J. Neurophysiol., 72 (1994) 1402-1419]. However, their application to single cells internally is technically difficult [Antic, S. and Zecevic, D., J. Neurosci., 15 (1995) 1392-1405; Grinvald, A., Salzberg, B.M., Lev-Ram, V. and Hildesheim, R., Biophys. J., 51 (1987) 643-651; Kogan, A., Ross, W.N., Zecevic, D. and Lasser-Ross, N., Brain Res., 700 (1995) 235-239; Zecevic, D., Nature, 381 (1996) 322-325]. An alternative strategy consists in applying the dye from the outside to all cells in the tissue, while manipulating a single cell by current injection [Krauthamer, V. and Ross, W.N., J. Neurosci., 4 (1984) 673-682; Ross, W.N. and Krauthamer, V., J. Neurosci., 4 (1984) 659-672]. Here, we modify this technique to further enhance spatial at the cost of temporal resolution [Borst, A., Z. Naturforsch., 50 (1995) 435-438]. Applied to rat cerebellar slices we demonstrate that the potential spread in individual Purkinje cells can be imaged up to even fine dendritic branches. The acquired optical signals suggest that steadily hyperpolarized Purkinje cells are electrically compact. When permanently depolarized, the somatic input resistance is significantly diminished, yet the spatial voltage drop along the dendrites remains unchanged. As demonstrated by compartmental modeling, this hints to a concentration of outward rectifying currents at the soma of the cells.

  13. Representation of limb kinematics in Purkinje cell simple spike discharge is conserved across multiple tasks

    PubMed Central

    Hewitt, Angela L.; Popa, Laurentiu S.; Pasalar, Siavash; Hendrix, Claudia M.

    2011-01-01

    Encoding of movement kinematics in Purkinje cell simple spike discharge has important implications for hypotheses of cerebellar cortical function. Several outstanding questions remain regarding representation of these kinematic signals. It is uncertain whether kinematic encoding occurs in unpredictable, feedback-dependent tasks or kinematic signals are conserved across tasks. Additionally, there is a need to understand the signals encoded in the instantaneous discharge of single cells without averaging across trials or time. To address these questions, this study recorded Purkinje cell firing in monkeys trained to perform a manual random tracking task in addition to circular tracking and center-out reach. Random tracking provides for extensive coverage of kinematic workspaces. Direction and speed errors are significantly greater during random than circular tracking. Cross-correlation analyses comparing hand and target velocity profiles show that hand velocity lags target velocity during random tracking. Correlations between simple spike firing from 120 Purkinje cells and hand position, velocity, and speed were evaluated with linear regression models including a time constant, τ, as a measure of the firing lead/lag relative to the kinematic parameters. Across the population, velocity accounts for the majority of simple spike firing variability (63 ± 30% of Radj2), followed by position (28 ± 24% of Radj2) and speed (11 ± 19% of Radj2). Simple spike firing often leads hand kinematics. Comparison of regression models based on averaged vs. nonaveraged firing and kinematics reveals lower Radj2 values for nonaveraged data; however, regression coefficients and τ values are highly similar. Finally, for most cells, model coefficients generated from random tracking accurately estimate simple spike firing in either circular tracking or center-out reach. These findings imply that the cerebellum controls movement kinematics, consistent with a forward internal model that

  14. Representation of limb kinematics in Purkinje cell simple spike discharge is conserved across multiple tasks.

    PubMed

    Hewitt, Angela L; Popa, Laurentiu S; Pasalar, Siavash; Hendrix, Claudia M; Ebner, Timothy J

    2011-11-01

    Encoding of movement kinematics in Purkinje cell simple spike discharge has important implications for hypotheses of cerebellar cortical function. Several outstanding questions remain regarding representation of these kinematic signals. It is uncertain whether kinematic encoding occurs in unpredictable, feedback-dependent tasks or kinematic signals are conserved across tasks. Additionally, there is a need to understand the signals encoded in the instantaneous discharge of single cells without averaging across trials or time. To address these questions, this study recorded Purkinje cell firing in monkeys trained to perform a manual random tracking task in addition to circular tracking and center-out reach. Random tracking provides for extensive coverage of kinematic workspaces. Direction and speed errors are significantly greater during random than circular tracking. Cross-correlation analyses comparing hand and target velocity profiles show that hand velocity lags target velocity during random tracking. Correlations between simple spike firing from 120 Purkinje cells and hand position, velocity, and speed were evaluated with linear regression models including a time constant, τ, as a measure of the firing lead/lag relative to the kinematic parameters. Across the population, velocity accounts for the majority of simple spike firing variability (63 ± 30% of R(adj)(2)), followed by position (28 ± 24% of R(adj)(2)) and speed (11 ± 19% of R(adj)(2)). Simple spike firing often leads hand kinematics. Comparison of regression models based on averaged vs. nonaveraged firing and kinematics reveals lower R(adj)(2) values for nonaveraged data; however, regression coefficients and τ values are highly similar. Finally, for most cells, model coefficients generated from random tracking accurately estimate simple spike firing in either circular tracking or center-out reach. These findings imply that the cerebellum controls movement kinematics, consistent with a forward

  15. Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

    PubMed

    Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

    2012-05-01

    Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

    PubMed Central

    Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

    2012-01-01

    Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. PMID:22391119

  17. Dendritic excitability modulates dendritic information processing in a purkinje cell model.

    PubMed

    Coop, Allan D; Cornelis, Hugo; Santamaria, Fidel

    2010-01-01

    Using an electrophysiological compartmental model of a Purkinje cell we quantified the contribution of individual active dendritic currents to processing of synaptic activity from granule cells. We used mutual information as a measure to quantify the information from the total excitatory input current (I(Glu)) encoded in each dendritic current. In this context, each active current was considered an information channel. Our analyses showed that most of the information was encoded by the calcium (I(CaP)) and calcium activated potassium (I(Kc)) currents. Mutual information between I(Glu) and I(CaP) and I(Kc) was sensitive to different levels of excitatory and inhibitory synaptic activity that, at the same time, resulted in the same firing rate at the soma. Since dendritic excitability could be a mechanism to regulate information processing in neurons we quantified the changes in mutual information between I(Glu) and all Purkinje cell currents as a function of the density of dendritic Ca (g(CaP)) and Kca (g(Kc)) conductances. We extended our analysis to determine the window of temporal integration of I(Glu) by I(CaP) and I(Kc) as a function of channel density and synaptic activity. The window of information integration has a stronger dependence on increasing values of g(Kc) than on g(CaP), but at high levels of synaptic stimulation information integration is reduced to a few milliseconds. Overall, our results show that different dendritic conductances differentially encode synaptic activity and that dendritic excitability and the level of synaptic activity regulate the flow of information in dendrites.

  18. Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.

    PubMed

    Chung, Chan; Elrick, Matthew J; Dell'Orco, James M; Qin, Zhaohui S; Kalyana-Sundaram, Shanker; Chinnaiyan, Arul M; Shakkottai, Vikram G; Lieberman, Andrew P

    2016-05-01

    Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.

  19. The Importance of Purkinje Activation in Long Duration Ventricular Fibrillation

    PubMed Central

    Huang, Jian; Dosdall, Derek J.; Cheng, Kang‐An; Li, Li; Rogers, Jack M.; Ideker, Raymond E.

    2014-01-01

    Background The mechanisms that maintain long duration ventricular fibrillation (LDVF) are unclear. The difference in distribution of the Purkinje system in dogs and pigs was explored to determine if Purkinje activation propagates to stimulate working myocardium (WM) during LDVF and WM pacing. Methods and Results In‐vivo extracellular recordings were made from 1044 intramural plunge and epicardial plaque electrodes in 6 pig and 6 dog hearts. Sinus activation propagated sequentially from the endocardium to the epicardium in dogs but not pigs. During epicardial pacing, activation propagated along the endocardium and traversed the LV wall almost parallel to the epicardium in dogs, but in pigs propagated away from the pacing site approximately perpendicular to the epicardium. After 1 minute of VF, activation rate near the endocardium was significantly faster than near the epicardium in dogs (P<0.01) but not pigs (P>0.05). From 2 to 10 minutes of LDVF, recordings exhibiting Purkinje activations were near the endocardium in dogs (P<0.01) but were scattered transmurally in pigs, and the WM activation rate in recordings in which Purkinje activations were present was significantly faster than the WM activation rate in recordings in which Purkinje activations were absent (P<0.01). In 10 isolated perfused dog hearts, the LV endocardium was exposed and 2 microelectrodes were inserted into Purkinje and adjacent myocardial cells. After 5 minutes of LDVF, mean Purkinje activation rate was significantly faster than mean WM activation rate (P<0.01). Conclusion These extracellular and intracellular findings about activation support the hypothesis that Purkinje activation propagates to stimulate WM during sinus rhythm, pacing, and LDVF. PMID:24584738

  20. Cross talk between metabotropic and ionotropic glutamate receptor-mediated signaling in parallel fiber-induced inositol 1,4,5-trisphosphate production in cerebellar Purkinje cells.

    PubMed

    Okubo, Yohei; Kakizawa, Sho; Hirose, Kenzo; Iino, Masamitsu

    2004-10-27

    In many excitatory glutamatergic synapses, both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are closely distributed on the postsynaptic membrane. However, the functional significance of the close distribution of the two types of glutamate receptors has not been fully clarified. In this study, we examined the functional interaction between iGluR and mGluR at parallel fiber (PF)--> Purkinje cell synapses in the generation of inositol 1,4,5-trisphosphate (IP3), a key second messenger that regulates many important cellular functions. We visualized local IP3 dynamics in Purkinje cells using the green fluorescent protein-tagged pleckstrin homology domain (GFP-PHD) as a fluorescent IP3 probe. Purkinje cells were transduced with Sindbis virus encoding GFP-PHD and imaged with a two-photon laser scanning microscope. Translocation of GFP-PHD from the plasma membrane to the cytoplasm attributable to an increase in IP3 concentration was observed on PF stimulation in fine dendrites of Purkinje cells. Surprisingly, this PF-induced IP3 production was blocked not only by the group I mGluR antagonist but also by the AMPA receptor (AMPAR) antagonist. The PF-induced IP3 production was blocked by either the inhibition of G-protein activation by GDP-betaS or intracellular Ca2+ buffering by BAPTA. These results show that IP3 production is mediated cooperatively by group I mGluR and AMPAR through G-protein activation and Ca2+ influx at PF--> Purkinje cell synapses, identifying the robust cross talk between iGluR and mGluR for the generation of IP3 signals.

  1. AMPA receptor activation controls type I metabotropic glutamate receptor signalling via a tyrosine kinase at parallel fibre-Purkinje cell synapses.

    PubMed

    Auger, Céline; Ogden, David

    2010-08-15

    Metabotropic glutamate receptors type 1 (mGluR1s) and ionotropic AMPA receptors (AMPARs) are colocalized at parallel fibre (PF) to Purkinje cell synapses of the cerebellum. Single stimulation of PFs activates fast AMPAR excitatory postsynaptic currents, whereas the activation of mGluR1s requires burst stimulation. mGluR1s signal through several pathways in Purkinje cells and the most prominent is the activation of a slow EPSC (sEPSC). To separate the two synaptic currents, studies of the sEPSC have commonly been performed in the presence of AMPA/KA receptor antagonists. We show here in rat cerebellar slices that inhibition of the fast EPSC by AMPAR antagonists strongly and selectively potentiates the mGluR1 sEPSC, showing a negative regulation of mGluR1 by AMPAR. This effect is observed with low concentrations of NBQX (300 nM to 1 microM), with the selective AMPAR antagonist GYKI 53655 and also with gamma-DGG, a low affinity glutamate receptor antagonist. When photorelease of glutamate from MNI-glutamate was used to study the postsynaptic responses in isolation, AMPAR inhibition produced a similar potentiation of the mGluR1 sEPSC, showing that the interaction is postsynaptic. Finally, perfusion of the postsynaptic cell with PP1, an inhibitor of src-family tyrosine kinase, increased the amplitude of the mGluR1 sEPSC and occluded the effect of AMPAR inhibition. Thus, at PF to Purkinje cell synapses, AMPAR activation inhibits the mGluR1 sEPSC via activation of a src-family tyrosine kinase. Consequently mGluR1 signalling will be more sensitive to spillover of glutamate than to local synaptic release. Furthermore, it will be enhanced at silent PF synapses which are the majority in Purkinje cells.

  2. Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata

    PubMed Central

    Benton, Mark D.; Lewis, Amanda H.; Bant, Jason S.

    2013-01-01

    Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17–21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (β4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell

  3. Train stimulation of parallel fibre to Purkinje cell inputs reveals two populations of synaptic responses with different receptor signatures

    PubMed Central

    Devi, Suma Priya Sudarsana; Howe, James R.

    2016-01-01

    Key points Purkinje cells of the cerebellum receive ∼180,000 parallel fibre synapses, which have often been viewed as a homogeneous synaptic population and studied using single action potentials.Many parallel fibre synapses might be silent, however, and granule cells in vivo fire in bursts. Here, we used trains of stimuli to study parallel fibre inputs to Purkinje cells in rat cerebellar slices.Analysis of train EPSCs revealed two synaptic components, phase 1 and 2. Phase 1 is initially large and saturates rapidly, whereas phase 2 is initially small and facilitates throughout the train. The two components have a heterogeneous distribution at dendritic sites and different pharmacological profiles.The differential sensitivity of phase 1 and phase 2 to inhibition by pentobarbital and NBQX mirrors the differential sensitivity of AMPA receptors associated with the transmembrane AMPA receptor regulatory protein, γ‐2, gating in the low‐ and high‐open probability modes, respectively. Abstract Cerebellar granule cells fire in bursts, and their parallel fibre axons (PFs) form ∼180,000 excitatory synapses onto the dendritic tree of a Purkinje cell. As many as 85% of these synapses have been proposed to be silent, but most are labelled for AMPA receptors. Here, we studied PF to Purkinje cell synapses using trains of 100 Hz stimulation in rat cerebellar slices. The PF train EPSC consisted of two components that were present in variable proportions at different dendritic sites: one, with large initial EPSC amplitude, saturated after three stimuli and dominated the early phase of the train EPSC; and the other, with small initial amplitude, increased steadily throughout the train of 10 stimuli and dominated the late phase of the train EPSC. The two phases also displayed different pharmacological profiles. Phase 2 was less sensitive to inhibition by NBQX but more sensitive to block by pentobarbital than phase 1. Comparison of synaptic results with fast glutamate

  4. Intracellular calcium dynamics permit a Purkinje neuron model to perform toggle and gain computations upon its inputs

    PubMed Central

    Forrest, Michael D.

    2014-01-01

    Without synaptic input, Purkinje neurons can spontaneously fire in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence. Climbing fiber input (CF) switches Purkinje neurons out of the trimodal firing pattern and toggles them between a tonic firing and a quiescent state, while setting the gain of their response to Parallel Fiber (PF) input. The basis to this transition is unclear. We investigate it using a biophysical Purkinje cell model under conditions of CF and PF input. The model can replicate these toggle and gain functions, dependent upon a novel account of intracellular calcium dynamics that we hypothesize to be applicable in real Purkinje cells. PMID:25191262

  5. Chronic pharmacological blockade of the Na+ /Ca2+ exchanger modulates the growth and development of the Purkinje cell dendritic arbor in mouse cerebellar slice cultures.

    PubMed

    Sherkhane, Pradeep; Kapfhammer, Josef P

    2017-09-01

    The Na + /Ca 2+ exchanger (NCX) is a bidirectional plasma membrane antiporter involved in Ca 2+ homeostasis in eukaryotes. NCX has three isoforms, NCX1-3, and all of them are expressed in the cerebellum. Immunostaining on cerebellar slice cultures indicates that NCX is widely expressed in the cerebellum, including expression in Purkinje cells. The pharmacological blockade of the forward mode of NCX (Ca 2+ efflux mode) by bepridil moderately inhibited growth and development of Purkinje cell dendritic arbor in cerebellar slice cultures. However, the blockade of the reverse mode (Ca 2+ influx mode) by KB-R7943 severely reduced the dendritic arbor and induced a morphological change with thickened distal dendrites. The effect of KB-R7943 on dendritic growth was unrelated to the activity of voltage-gated calcium channels and was also apparent in the absence of bioelectrical activity indicating that it was mediated by NCX expressed in Purkinje cells. We have used additional NCX inhibitors including CB-DMB, ORM-10103, SEA0400, YM-244769, and SN-6 which have higher specificity for NCX isoforms and target either the forward, reverse, or both modes. These inhibitors caused a strong dendritic reduction similar to that seen with KB-R7943, but did not elicit thickening of distal dendrites. Our findings indicate that disturbance of the NCX-dependent calcium transport in Purkinje cells induces a reduction of dendritic arbor, which is presumably caused by changes in the calcium handling, and underline the importance of the calcium equilibrium for the dendritic development in cerebellar Purkinje cells. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  6. A Simple Mathematical Model Inspired by the Purkinje Cells: From Delayed Travelling Waves to Fractional Diffusion.

    PubMed

    Dipierro, Serena; Valdinoci, Enrico

    2018-07-01

    Recently, several experiments have demonstrated the existence of fractional diffusion in the neuronal transmission occurring in the Purkinje cells, whose malfunctioning is known to be related to the lack of voluntary coordination and the appearance of tremors. Also, a classical mathematical feature is that (fractional) parabolic equations possess smoothing effects, in contrast with the case of hyperbolic equations, which typically exhibit shocks and discontinuities. In this paper, we show how a simple toy-model of a highly ramified structure, somehow inspired by that of the Purkinje cells, may produce a fractional diffusion via the superposition of travelling waves that solve a hyperbolic equation. This could suggest that the high ramification of the Purkinje cells might have provided an evolutionary advantage of "smoothing" the transmission of signals and avoiding shock propagations (at the price of slowing a bit such transmission). Although an experimental confirmation of the possibility of such evolutionary advantage goes well beyond the goals of this paper, we think that it is intriguing, as a mathematical counterpart, to consider the time fractional diffusion as arising from the superposition of delayed travelling waves in highly ramified transmission media. The case of a travelling concave parabola with sufficiently small curvature is explicitly computed. The new link that we propose between time fractional diffusion and hyperbolic equation also provides a novelty with respect to the usual paradigm relating time fractional diffusion with parabolic equations in the limit. This paper is written in such a way as to be of interest to both biologists and mathematician alike. In order to accomplish this aim, both complete explanations of the objects considered and detailed lists of references are provided.

  7. l-Serine and glycine serve as major astroglia-derived trophic factors for cerebellar Purkinje neurons

    PubMed Central

    Furuya, Shigeki; Tabata, Toshihide; Mitoma, Junya; Yamada, Keiko; Yamasaki, Miwako; Makino, Asami; Yamamoto, Toshifumi; Watanabe, Masahiko; Kano, Masanobu; Hirabayashi, Yoshio

    2000-01-01

    Glial cells support the survival and development of central neurons through the supply of trophic factors. Here we demonstrate that l-serine (l-Ser) and glycine (Gly) also are glia-derived trophic factors. These amino acids are released by astroglial cells and promote the survival, dendritogenesis, and electrophysiological development of cultured cerebellar Purkinje neurons. Although l-Ser and Gly are generally classified as nonessential amino acids, 3-phosphoglycerate dehydrogenase (3PGDH), a key enzyme for their biosynthesis, is not expressed in Purkinje neurons. By contrast, the Bergman glia, a native astroglia in the cerebellar cortex, highly expresses 3PGDH. These data suggest that l-Ser and Gly mediate the trophic actions of glial cells on Purkinje neurons. PMID:11016963

  8. Long-Term Predictive and Feedback Encoding of Motor Signals in the Simple Spike Discharge of Purkinje Cells

    PubMed Central

    Popa, Laurentiu S.; Streng, Martha L.

    2017-01-01

    Abstract Most hypotheses of cerebellar function emphasize a role in real-time control of movements. However, the cerebellum’s use of current information to adjust future movements and its involvement in sequencing, working memory, and attention argues for predicting and maintaining information over extended time windows. The present study examines the time course of Purkinje cell discharge modulation in the monkey (Macaca mulatta) during manual, pseudo-random tracking. Analysis of the simple spike firing from 183 Purkinje cells during tracking reveals modulation up to 2 s before and after kinematics and position error. Modulation significance was assessed against trial shuffled firing, which decoupled simple spike activity from behavior and abolished long-range encoding while preserving data statistics. Position, velocity, and position errors have the most frequent and strongest long-range feedforward and feedback modulations, with less common, weaker long-term correlations for speed and radial error. Position, velocity, and position errors can be decoded from the population simple spike firing with considerable accuracy for even the longest predictive (-2000 to -1500 ms) and feedback (1500 to 2000 ms) epochs. Separate analysis of the simple spike firing in the initial hold period preceding tracking shows similar long-range feedforward encoding of the upcoming movement and in the final hold period feedback encoding of the just completed movement, respectively. Complex spike analysis reveals little long-term modulation with behavior. We conclude that Purkinje cell simple spike discharge includes short- and long-range representations of both upcoming and preceding behavior that could underlie cerebellar involvement in error correction, working memory, and sequencing. PMID:28413823

  9. Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells.

    PubMed

    Engbers, Jordan D T; Anderson, Dustin; Asmara, Hadhimulya; Rehak, Renata; Mehaffey, W Hamish; Hameed, Shahid; McKay, Bruce E; Kruskic, Mirna; Zamponi, Gerald W; Turner, Ray W

    2012-02-14

    Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.

  10. Ataxia with loss of Purkinje cells in a mouse model for Refsum disease

    PubMed Central

    Ferdinandusse, Sacha; Zomer, Anna W. M.; Komen, Jasper C.; van den Brink, Christina E.; Thanos, Melissa; Hamers, Frank P. T.; Wanders, Ronald J. A.; van der Saag, Paul T.; Poll-The, Bwee Tien; Brites, Pedro

    2008-01-01

    Refsum disease is caused by a deficiency of phytanoyl-CoA hydroxylase (PHYH), the first enzyme of the peroxisomal α-oxidation system, resulting in the accumulation of the branched-chain fatty acid phytanic acid. The main clinical symptoms are polyneuropathy, cerebellar ataxia, and retinitis pigmentosa. To study the pathogenesis of Refsum disease, we generated and characterized a Phyh knockout mouse. We studied the pathological effects of phytanic acid accumulation in Phyh−/− mice fed a diet supplemented with phytol, the precursor of phytanic acid. Phytanic acid accumulation caused a reduction in body weight, hepatic steatosis, and testicular atrophy with loss of spermatogonia. Phenotype assessment using the SHIRPA protocol and subsequent automated gait analysis using the CatWalk system revealed unsteady gait with strongly reduced paw print area for both fore- and hindpaws and reduced base of support for the hindpaws. Histochemical analyses in the CNS showed astrocytosis and up-regulation of calcium-binding proteins. In addition, a loss of Purkinje cells in the cerebellum was observed. No demyelination was present in the CNS. Motor nerve conduction velocity measurements revealed a peripheral neuropathy. Our results show that, in the mouse, high phytanic acid levels cause a peripheral neuropathy and ataxia with loss of Purkinje cells. These findings provide important insights in the pathophysiology of Refsum disease. PMID:19004801

  11. Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

    NASA Astrophysics Data System (ADS)

    He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

    2015-07-01

    Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

  12. Morphological study of the atrioventricular conduction system and Purkinje fibers in yak.

    PubMed

    Duan, Deyong; Yu, Sijiu; Cui, Yan; Li, Chaoxu

    2017-07-01

    We studied the morphology of the atrioventricular conduction system (AVCS) and Purkinje fibers of the yak. Light and transmission electron microscopy were used to study the histological features of AVCS. The distributional characteristics of the His-bundle, the left bundle branch (LBB), right bundle branch (RBB), and Purkinje fiber network of yak hearts were examined using gross dissection, ink injection, and ABS casting. The results showed that the atrioventricular node (AVN) of yak located in the right side of interatrial septum and had a flattened ovoid shape. The AVN of yak is composed of the slender, interweaving cells formed almost entirely of the transitional cells (T-cells). The His-bundle extended from the AVN, and split into left LBB and RBB at the crest of the interventricular septum. The LBB descended along the left side of interventricular septum. At approximately the upper 1/3 of the interventricular septum, the LBB typically divided into three branches. The RBB ran under the endocardium of the right side of interventricular septum, and extended to the base of septal papillary muscle, passed into the moderator band, crossed the right ventricular cavity to reach the base of anterior papillary muscle, and divided into four fascicles under the subendocardial layer. The Purkinje fibers in the ventricle formed a complex spatial network. The distributional and cellular component characteristics of the AVCS and Purkinje fibers ensured normal cardiac function. © 2017 Wiley Periodicals, Inc.

  13. Frequency-dependent reliability of spike propagation is function of axonal voltage-gated sodium channels in cerebellar Purkinje cells.

    PubMed

    Yang, Zhilai; Wang, Jin-Hui

    2013-12-01

    The spike propagation on nerve axons, like synaptic transmission, is essential to ensure neuronal communication. The secure propagation of sequential spikes toward axonal terminals has been challenged in the neurons with a high firing rate, such as cerebellar Purkinje cells. The shortfall of spike propagation makes some digital spikes disappearing at axonal terminals, such that the elucidation of the mechanisms underlying spike propagation reliability is crucial to find the strategy of preventing loss of neuronal codes. As the spike propagation failure is influenced by the membrane potentials, this process is likely caused by altering the functional status of voltage-gated sodium channels (VGSC). We examined this hypothesis in Purkinje cells by using pair-recordings at their somata and axonal blebs in cerebellar slices. The reliability of spike propagation was deteriorated by elevating spike frequency. The frequency-dependent reliability of spike propagation was attenuated by inactivating VGSCs and improved by removing their inactivation. Thus, the functional status of axonal VGSCs influences the reliability of spike propagation.

  14. Purkinje image eyetracking: A market survey

    NASA Technical Reports Server (NTRS)

    Christy, L. F.

    1979-01-01

    The Purkinje image eyetracking system was analyzed to determine the marketability of the system. The eyetracking system is a synthesis of two separate instruments, the optometer that measures the refractive power of the eye and the dual Purkinje image eyetracker that measures the direction of the visual axis.

  15. Role of cytosolic calcium diffusion in cardiac purkinje cells.

    PubMed

    Limbu, Bijay; Shah, Kushal; Deo, Makarand

    2016-08-01

    The Cardiac Purkinje cells (PCs) exhibit distinct calcium (Ca2+) homeostasis than that in ventricular myocytes (VMs). Due to lack of t-tubules in PCs, the Ca2+ ions entering the cell have to diffuse through the cytoplasm to reach the sarcoplasmic reticulum (SR) before triggering Ca2+-induced-Ca2+-release (CICR). In recent experimental studies PCs have been shown to be more susceptible to action potential (AP) abnormalities than the VMs, however the exact mechanisms are poorly understood. In this study, we utilize morphologically realistic detailed biophysical mathematical model of a murine PC to systematically examine the role intracellular Ca2+ diffusion in the APs of PCs. A biphasic spatiotemporal Ca2+ diffusion process, as observed experimentally, was implemented in the model which includes radial Ca2+ wavelets and cell wide longitudinal Ca2+ diffusion wave (CWW). The AP morphology, specifically plateau, is affected due to changes in intracellular Ca2+ dynamics. When Ca2+ concentration in sarcolemmal region is elevated, it activated inward sodium Ca2+ exchanger (NCX) current resulting into prolongation of the plateau at faster diffusion rates. Our results demonstrate that the cytosolic Ca2+ diffusion waves play a significant role in shaping APs of PCs and could provide mechanistic insights into the increased arrhythmogeneity of PCs.

  16. Essential role of axonal VGSC inactivation in time-dependent deceleration and unreliability of spike propagation at cerebellar Purkinje cells

    PubMed Central

    2014-01-01

    Background The output of the neuronal digital spikes is fulfilled by axonal propagation and synaptic transmission to influence postsynaptic cells. Similar to synaptic transmission, spike propagation on the axon is not secure, especially in cerebellar Purkinje cells whose spiking rate is high. The characteristics, mechanisms and physiological impacts of propagation deceleration and infidelity remain elusive. The spike propagation is presumably initiated by local currents that raise membrane potential to the threshold of activating voltage-gated sodium channels (VGSC). Results We have investigated the natures of spike propagation and the role of VGSCs in this process by recording spikes simultaneously on the somata and axonal terminals of Purkinje cells in cerebellar slices. The velocity and fidelity of spike propagation decreased during long-lasting spikes, to which the velocity change was more sensitive than fidelity change. These time-dependent deceleration and infidelity of spike propagation were improved by facilitating axonal VGSC reactivation, and worsen by intensifying VGSC inactivation. Conclusion Our studies indicate that the functional status of axonal VGSCs is essential to influencing the velocity and fidelity of spike propagation. PMID:24382121

  17. Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons.

    PubMed

    Eitan, Erez; Braverman, Carmel; Tichon, Ailone; Gitler, Daniel; Hutchison, Emmette R; Mattson, Mark P; Priel, Esther

    2016-08-01

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

  18. Purkinje cells signal hand shape and grasp force during reach-to-grasp in the monkey.

    PubMed

    Mason, Carolyn R; Hendrix, Claudia M; Ebner, Timothy J

    2006-01-01

    The cerebellar cortex and nuclei play important roles in the learning, planning, and execution of reach-to-grasp and prehensile movements. However, few studies have investigated the signals carried by cerebellar neurons during reach-to-grasp, particularly signals relating to target object properties, hand shape, and grasp force. In this study, the simple spike discharge of 77 Purkinje cells was recorded as two rhesus monkeys reached and grasped 16 objects. The objects varied systematically in volume, shape, and orientation and each was grasped at five different force levels. Linear multiple regression analyses showed the simple spike discharge was significantly modulated in relation to objects and force levels. Object related modulation occurred preferentially during reach or early in the grasp and was linearly related to grasp aperture. The simple spike discharge was positively correlated with grasp force during both the reach and the grasp. There was no significant interaction between object and grasp force modulation, supporting previous kinematic findings that grasp kinematics and force are signaled independently. Singular value decomposition (SVD) was used to quantify the temporal patterns in the simple spike discharge. Most cells had a predominant discharge pattern that remained relatively constant across object grasp dimensions and force levels. A single predominant simple spike discharge pattern that spans reach and grasp and accounts for most of the variation (>60%) is consistent with the concept that the cerebellum is involved with synergies underlying prehension. Therefore Purkinje cells are involved with the signaling of prehension, providing independent signals for hand shaping and grasp force.

  19. Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

    PubMed Central

    Preston, Chet; Wang, Louis; Yi, Jae Kyo; Lin, Chih-Li; Sun, Wei; Spyropoulos, Demetri D.; Rhee, Soyoung; Li, Mingsong; Zhou, Jie; Ge, Shaoyu; Zhang, Guofeng; Snider, Ashley J.; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

    2015-01-01

    Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration. PMID:26474409

  20. Development of "Pinceaux" formations and dendritic translocation of climbing fibers during the acquisition of the balance between glutamatergic and gamma-aminobutyric acidergic inputs in developing Purkinje cells.

    PubMed

    Sotelo, Constantino

    2008-01-10

    The acquisition of the dynamic balance between excitation and inhibition in developing Purkinje cells, necessary for their proper function, is analyzed. Newborn (P0) mouse cerebellum contains glutamatergic (VGLUT2-IR) and gamma-aminobutyric acid (GABA)-ergic (VIAAT-IR) axons. The former prevail and belong to climbing fibers, whereas the latter neither colabel with calbindin-expressing fibers nor belong to axons of the cortical GABAergic interneurons. During the first postnatal week, VIAAT-IR axons in the Purkinje cell neighborhood remains very low, and the first synapses with basket fibers are formed at P7, when climbing fibers have already established dense pericellular nets. The descending basket fibers reach the Purkinje cell axon initial segment by P9, immediately establishing axoaxonic synapses. The pinceaux appear as primitive vortex-like arrangements by P12, and by P20 interbasket fiber septate-like junctions, typical of fully mature pinceaux, are still missing. The climbing fiber's somatodendritic translocation occurs later than expected, after the regression of the multiple innervation, and follows the ascending collaterals of the basket axons, which are apparently the optimal substrate for the proper subcellular targeting of the climbing fibers. These results emphasize that chemical transmission in the axon initial segment precedes the electrical inhibition generated by field effects. In addition, GABAergic Purkinje cells, as opposed to glutamatergic projection neurons in other cortical structures, do not begin to receive their excitation to inhibition balance until the end of the first postnatal week, despite the early presence of potentially functional GABAergic axons that possess the required vesicular transport system. (c) 2007 Wiley-Liss, Inc.

  1. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    PubMed Central

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  2. Bursting patterns and mixed-mode oscillations in reduced Purkinje model

    NASA Astrophysics Data System (ADS)

    Zhan, Feibiao; Liu, Shenquan; Wang, Jing; Lu, Bo

    2018-02-01

    Bursting discharge is a ubiquitous behavior in neurons, and abundant bursting patterns imply many physiological information. There exists a closely potential link between bifurcation phenomenon and the number of spikes per burst as well as mixed-mode oscillations (MMOs). In this paper, we have mainly explored the dynamical behavior of the reduced Purkinje cell and the existence of MMOs. First, we adopted the codimension-one bifurcation to illustrate the generation mechanism of bursting in the reduced Purkinje cell model via slow-fast dynamics analysis and demonstrate the process of spike-adding. Furthermore, we have computed the first Lyapunov coefficient of Hopf bifurcation to determine whether it is subcritical or supercritical and depicted the diagrams of inter-spike intervals (ISIs) to examine the chaos. Moreover, the bifurcation diagram near the cusp point is obtained by making the codimension-two bifurcation analysis for the fast subsystem. Finally, we have a discussion on mixed-mode oscillations and it is further investigated using the characteristic index that is Devil’s staircase.

  3. Micropatterning of neural stem cells and Purkinje neurons using a polydimethylsiloxane (PDMS) stencil.

    PubMed

    Choi, Jin Ho; Lee, Hyun; Jin, Hee Kyung; Bae, Jae-sung; Kim, Gyu Man

    2012-12-07

    A new fabrication method of a polydimethylsiloxane (PDMS) stencil embedded microwell plate is proposed and applied to a localized culture of Purkinje neurons (PNs) and neural stem cells (NSCs). A microwell plate combines a PDMS stencil and well plate. The PDMS stencil was fabricated by spin casting from an SU-8 master mold. Gas blowing using nitrogen was adopted to perforate the stencil membrane. An acrylic well plate compartment mold was fabricated using computer numerical control (CNC) machining. By PDMS casting using a stencil placed on an acrylic mold, microwell plates were fabricated without punching or the use of a plasma bonding process. By using the stencil as a physical mask for the cell culture, PNs and NSCs were successfully cultured into micropatterns. The microwell plate could be applied to the localizing and culturing of a cell. The micropatterned NSCs were differentiated into neurons, astrocytes, and oligodendrocytes. The results showed that cells could be cultured and differentiated into micropatterns in a precisely controlled manner in any shape and in specific sizes for bioscience study and bioengineering applications.

  4. Distribution of zebrin-immunoreactive Purkinje cell terminals in the cerebellar and vestibular nuclei of birds.

    PubMed

    Wylie, Douglas R; Pakan, Janelle M P; Huynh, Hang; Graham, David J; Iwaniuk, Andrew N

    2012-05-01

    Zebrin II (aldolase C) is expressed in a subset of Purkinje cells in the mammalian and avian cerebella such that there is a characteristic parasagittal organization of zebrin-immunopositive stripes alternating with zebrin-immunonegative stripes. Zebrin is expressed not only in the soma and dendrites of Purkinje cells but also in their axonal terminals. Here we describe the distribution of zebrin immunoreactivity in both the vestibular and the cerebellar nuclei of pigeons (Columba livia) and hummingbirds (Calypte anna, Selasphorus rufus). In the medial cerebellar nucleus, zebrin-positive labeling was particularly heavy in the “shell,” whereas the “core” was zebrin negative. In the lateral cerebellar nucleus, labeling was not as heavy, but a positive shell and negative core were also observed. In the vestibular nuclear complex, zebrin-positive terminal labeling was heavy in the dorsolateral vestibular nucleus and the lateral margin of the superior vestibular nucleus. The central and medial regions of the superior nucleus were generally zebrin negative. Labeling was moderate to heavy in the medial vestibular nucleus, particulary the rostral half of the parvocellular subnucleus. A moderate amount of zebrin-positive labeling was present in the descending vestibular nucleus: this was heaviest laterally, and the central region was generally zebrin negative. Zebrin-positive terminals were also observed in the the cerebellovestibular process, prepositus hypoglossi, and lateral tangential nucleus. We discuss our findings in light of similar studies in rats and with respect to the corticonuclear projections to the cerebellar nuclei and the functional connections of the vestibulocerebellum with the vestibular nuclei. Copyright © 2011 Wiley Periodicals, Inc.

  5. Miglustat Improves Purkinje Cell Survival and Alters Microglial Phenotype in Feline Niemann-Pick Disease Type C

    PubMed Central

    Stein, Veronika M.; Crooks, Alexandra; Ding, Wenge; Prociuk, Maria; O’Donnell, Patricia; Bryan, Caroline; Sikora, Tracey; Dingemanse, Jasper; Vanier, Marie T.; Walkley, Steven U.; Vite, Charles H.

    2012-01-01

    Niemann-Pick disease type C (NPC disease) is an incurable cellular lipid trafficking disorder characterized by neurodegeneration and intralysosomal accumulation of cholesterol and glycosphingolipids. Treatment with miglustat, a small imino sugar that reversibly inhibits glucosylceramide synthase, which is necessary for glycosphingolipid synthesis, has been shown to benefit patients with NPC disease. The mechanism(s) and extent of brain cellular changes underlying this benefit are not understood. To investigate the basis of the efficacy of miglustat, cats with disease homologous to the juvenile-onset form of human NPC disease received daily miglustat orally beginning at 3 weeks of age. The plasma half-life of miglustat was 6.6 ± 1.1 hours, with a tmax, Cmax, and area under the plasma concentration-time curve of 1.7 ± 0.6 hours, 20.3 ± 4.6 μg/ml, and 104.1 ± 16.6 μg hours/ml, respectively. Miglustat delayed the onset of neurological signs and increased the lifespan of treated cats, and was associated with decreased GM2 ganglioside accumulation in the cerebellum and improved Purkinje cell survival. Ex vivo examination of microglia from the brains of treated cats revealed normalization of CD1c and class II major histocompatibility complex expression, as well as generation of reactive oxygen species. Together, these results suggest that prolonged Purkinje cell survival, reduced glycosphingolipid accumulation, and/or the modulation of microglial immunophenotype and function contribute to miglustat-induced neurological improvement in treated cats. PMID:22487861

  6. Decimetre Waves and Cerebellar Cortex Key Element - Purkinje Cells

    NASA Astrophysics Data System (ADS)

    Maharramov, Akif A.

    2007-04-01

    Acute experiments have been carried out on both decerebrated and anaesthetized adult cats exposed to Decimetre Range Microwaves (DRM) (λ=65 cm, duration of exposition -10 min., at least) by the help of a 2 cm radius contact applicator located on the temple part of the head with cerebellum in the centre of projection of irradiation conducted from portable physiotherapeutic apparatus ``Romashka''. Extracelullar registration of Purkinje Cells (PC) impulse activities have been led by glass microelectrodes. Statistical and computational analyses of PC activities have been realized by the help of histograms - the characteristic distribution of the number of interimpulse intervals (II) between electrical discharges of a neuron on the II durations, drawn up by the help of a computer. The results obtained revealed the reaction of PC to DRM as a succession of starting to react of PC electrophysiological parameters, beginning from decreasing of known ``Inhibitory Pause'' duration, and further, at first, ``Simple Spikes'' then ``Complex Spikes'' frequencies' increase, and furthermore, durations' increase in ``Big Interimpulse Intervals'', parameter, introduced for the first time by us, in this way, showing the ``evolutionary'' nature of Electromagnetic Fields and Living object interactions.

  7. Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Collin, Ludovic; Doretto, Sandrine; Department of Psychiatry and Human Behavior, University of California Irvine, 3226 Gillespie Neuroscience Research Facility, Irvine CA 92697

    2007-08-01

    Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiationmore » and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.« less

  8. Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells

    PubMed Central

    Fujita, Eriko; Tanabe, Yuko; Shiota, Akira; Ueda, Masatsugu; Suwa, Kiyotaka; Momoi, Mariko Y.; Momoi, Takashi

    2008-01-01

    Previous studies have demonstrated that mutation in the forkhead domain of the forkhead box P2 (FOXP2) protein (R553H) causes speech-language disorders. To further analyze FOXP2 function in speech learning, we generated a knockin (KI) mouse for Foxp2 (R552H) [Foxp2 (R552H)-KI], corresponding to the human FOXP2 (R553H) mutation, by homologous recombination. Homozygous Foxp2 (R552H)-KI mice showed reduced weight, immature development of the cerebellum with incompletely folded folia, Purkinje cells with poor dendritic arbors and less synaptophysin immunoreactivity, and achieved crisis stage for survival 3 weeks after birth. At postnatal day 10, these mice also showed severe ultrasonic vocalization (USV) and motor impairment, whereas the heterozygous Foxp2 (R552H)-KI mice exhibited modest impairments. Similar to the wild-type protein, Foxp2 (R552H) localized in the nuclei of the Purkinje cells and the thalamus, striatum, cortex, and hippocampus (CA1) neurons of the homozygous Foxp2 (R552H)-KI mice (postnatal day 10), and some of the neurons showed nuclear aggregates of Foxp2 (R552H). In addition to the immature development of the cerebellum, Foxp2 (R552H) nuclear aggregates may further compromise the function of the Purkinje cells and cerebral neurons of the homozygous mice, resulting in their death. In contrast, heterozygous Foxp2 (R552H)-KI mice, which showed modest impairment of USVs with different USV qualities and which did not exhibit nuclear aggregates, should provide insights into the common molecular mechanisms between the mouse USV and human speech learning and the relationship between the USV and motor neural systems. PMID:18287060

  9. Axonal propagation of simple and complex spikes in cerebellar Purkinje neurons.

    PubMed

    Khaliq, Zayd M; Raman, Indira M

    2005-01-12

    In cerebellar Purkinje neurons, the reliability of propagation of high-frequency simple spikes and spikelets of complex spikes is likely to regulate inhibition of Purkinje target neurons. To test the extent to which a one-to-one correspondence exists between somatic and axonal spikes, we made dual somatic and axonal recordings from Purkinje neurons in mouse cerebellar slices. Somatic action potentials were recorded with a whole-cell pipette, and the corresponding axonal signals were recorded extracellularly with a loose-patch pipette. Propagation of spontaneous and evoked simple spikes was highly reliable. At somatic firing rates of approximately 200 spikes/sec, <10% of spikes failed to propagate, with failures becoming more frequent only at maximal somatic firing rates (approximately 260 spikes/sec). Complex spikes were elicited by climbing fiber stimulation, and their somatic waveforms were modulated by tonic current injection, as well as by paired stimulation to depress the underlying EPSCs. Across conditions, the mean number of propagating action potentials remained just above two spikes per climbing fiber stimulation, but the instantaneous frequency of the propagating spikes changed, from approximately 375 Hz during somatic hyperpolarizations that silenced spontaneous firing to approximately 150 Hz during spontaneous activity. The probability of propagation of individual spikelets could be described quantitatively as a saturating function of spikelet amplitude, rate of rise, or preceding interspike interval. The results suggest that ion channels of Purkinje axons are adapted to produce extremely short refractory periods and that brief bursts of forward-propagating action potentials generated by complex spikes may contribute transiently to inhibition of postsynaptic neurons.

  10. The origin of downbeat nystagmus: an asymmetry in the distribution of on-directions of vertical gaze-velocity Purkinje cells.

    PubMed

    Marti, Sarah; Straumann, Dominik; Glasauer, Stefan

    2005-04-01

    Various hypotheses on the origin of cerebellar downbeat nystagmus (DBN) have been presented; the exact pathomechanism, however, is still not known. Based on previous anatomical and electrophysiological studies, we propose that an asymmetry in the distribution of on-directions of vertical gaze-velocity Purkinje cells leads to spontaneous upward ocular drift in cerebellar disease, and therefore, to DBN. Our hypothesis is supported by a computational model for vertical eye movements.

  11. Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures.

    PubMed

    Campeau, Jody L; Wu, Gengshu; Bell, John R; Rasmussen, Jay; Sim, Valerie L

    2013-01-01

    Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

  12. Signals and circuits in the purkinje neuron.

    PubMed

    Abrams, Zéev R; Zhang, Xiang

    2011-01-01

    Purkinje neurons (PN) in the cerebellum have over 100,000 inputs organized in an orthogonal geometry, and a single output channel. As the sole output of the cerebellar cortex layer, their complex firing pattern has been associated with motor control and learning. As such they have been extensively modeled and measured using tools ranging from electrophysiology and neuroanatomy, to dynamic systems and artificial intelligence methods. However, there is an alternative approach to analyze and describe the neuronal output of these cells using concepts from electrical engineering, particularly signal processing and digital/analog circuits. By viewing the PN as an unknown circuit to be reverse-engineered, we can use the tools that provide the foundations of today's integrated circuits and communication systems to analyze the Purkinje system at the circuit level. We use Fourier transforms to analyze and isolate the inherent frequency modes in the PN and define three unique frequency ranges associated with the cells' output. Comparing the PN to a signal generator that can be externally modulated adds an entire level of complexity to the functional role of these neurons both in terms of data analysis and information processing, relying on Fourier analysis methods in place of statistical ones. We also re-describe some of the recent literature in the field, using the nomenclature of signal processing. Furthermore, by comparing the experimental data of the past decade with basic electronic circuitry, we can resolve the outstanding controversy in the field, by recognizing that the PN can act as a multivibrator circuit.

  13. Behavior related pauses in simple spike activity of mouse Purkinje cells are linked to spike rate modulation

    PubMed Central

    Cao, Ying; Maran, Selva K.; Dhamala, Mukesh; Jaeger, Dieter; Heck, Detlef H.

    2012-01-01

    Purkinje cells (PCs) in the mammalian cerebellum express high frequency spontaneous activity with average spike rates between 30 and 200 Hz. Cerebellar nuclear (CN) neurons receive converging input from many PCs resulting in a continuous barrage of inhibitory inputs. It has been hypothesized that pauses in PC activity trigger increases in CN spiking activity. A prediction derived from this hypothesis is that pauses in PC simple spike activity represent relevant behavioral or sensory events. Here we asked whether pauses in the simple spike activity of PCs related to either fluid licking or respiration, play a special role in representing information about behavior. Both behaviors are widely represented in cerebellar PC simple spike activity. We recorded PC activity in the vermis and lobus simplex of head fixed mice while monitoring licking and respiratory behavior. Using cross correlation and Granger causality analysis we examined whether short ISIs had a different temporal relation to behavior than long ISIs or pauses. Behavior related simple spike pauses occurred during low-rate simple spike activity in both licking and breathing related PCs. Granger causality analysis revealed causal relationships between simple spike pauses and behavior. However, the same results were obtained from an analysis of surrogate spike trains with gamma ISI distributions constructed to match rate modulations of behavior related Purkinje cells. Our results therefore suggest that the occurrence of pauses in simple spike activity does not represent additional information about behavioral or sensory events that goes beyond the simple spike rate modulations. PMID:22723707

  14. Distinct responses of Purkinje neurons and roles of simple spikes during associative motor learning in larval zebrafish

    PubMed Central

    Harmon, Thomas C; Magaram, Uri; McLean, David L; Raman, Indira M

    2017-01-01

    To study cerebellar activity during learning, we made whole-cell recordings from larval zebrafish Purkinje cells while monitoring fictive swimming during associative conditioning. Fish learned to swim in response to visual stimulation preceding tactile stimulation of the tail. Learning was abolished by cerebellar ablation. All Purkinje cells showed task-related activity. Based on how many complex spikes emerged during learned swimming, they were classified as multiple, single, or zero complex spike (MCS, SCS, ZCS) cells. With learning, MCS and ZCS cells developed increased climbing fiber (MCS) or parallel fiber (ZCS) input during visual stimulation; SCS cells fired complex spikes associated with learned swimming episodes. The categories correlated with location. Optogenetically suppressing simple spikes only during visual stimulation demonstrated that simple spikes are required for acquisition and early stages of expression of learned responses, but not their maintenance, consistent with a transient, instructive role for simple spikes during cerebellar learning in larval zebrafish. DOI: http://dx.doi.org/10.7554/eLife.22537.001 PMID:28541889

  15. Heterogeneity of Purkinje cell simple spike-complex spike interactions: zebrin- and non-zebrin-related variations.

    PubMed

    Tang, Tianyu; Xiao, Jianqiang; Suh, Colleen Y; Burroughs, Amelia; Cerminara, Nadia L; Jia, Linjia; Marshall, Sarah P; Wise, Andrew K; Apps, Richard; Sugihara, Izumi; Lang, Eric J

    2017-08-01

    Cerebellar Purkinje cells (PCs) generate two types of action potentials, simple and complex spikes. Although they are generated by distinct mechanisms, interactions between the two spike types exist. Zebrin staining produces alternating positive and negative stripes of PCs across most of the cerebellar cortex. Thus, here we compared simple spike-complex spike interactions both within and across zebrin populations. Simple spike activity undergoes a complex modulation preceding and following a complex spike. The amplitudes of the pre- and post-complex spike modulation phases were correlated across PCs. On average, the modulation was larger for PCs in zebrin positive regions. Correlations between aspects of the complex spike waveform and simple spike activity were found, some of which varied between zebrin positive and negative PCs. The implications of the results are discussed with regard to hypotheses that complex spikes are triggered by rises in simple spike activity for either motor learning or homeostatic functions. Purkinje cells (PCs) generate two types of action potentials, called simple and complex spikes (SSs and CSs). We first investigated the CS-associated modulation of SS activity and its relationship to the zebrin status of the PC. The modulation pattern consisted of a pre-CS rise in SS activity, and then, following the CS, a pause, a rebound, and finally a late inhibition of SS activity for both zebrin positive (Z+) and negative (Z-) cells, though the amplitudes of the phases were larger in Z+ cells. Moreover, the amplitudes of the pre-CS rise with the late inhibitory phase of the modulation were correlated across PCs. In contrast, correlations between modulation phases across CSs of individual PCs were generally weak. Next, the relationship between CS spikelets and SS activity was investigated. The number of spikelets/CS correlated with the average SS firing rate only for Z+ cells. In contrast, correlations across CSs between spikelet numbers and the

  16. Evidence that GABA ρ subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

    PubMed Central

    Harvey, Victoria L; Duguid, Ian C; Krasel, Cornelius; Stephens, Gary J

    2006-01-01

    Ionotropic γ-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for ρ subunit-containing GABAC over other GABA receptors. Exogenous application of the GABAC-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABAC antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABAA receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABAA/GABAC pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone–Purkinje cell (IN–PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that ρ subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABAA α1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that ρ subunits can form complexes

  17. Glial S100B Positive Vacuoles In Purkinje Cells: Earliest Morphological Abnormality In SCA1 Transgenic Mice

    PubMed Central

    VIG, Parminder J.S.; LOPEZ, Maripar E.; WEI, Jinrong; D’SOUZA, David R.; SUBRAMONY, SH; HENEGAR, Jeffrey; FRATKIN, Jonathan D.

    2007-01-01

    Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeat within the disease protein, ataxin-1. The overexpression of mutant ataxin-1 in SCA1 transgenic mice results in the formation of cytoplasmic vacuoles in Purkinje neurons (PKN) of the cerebellum. PKN are closely associated with neighboring Bergmann glia. To elucidate the role of Bergmann glia in SCA1 pathogenesis, cerebellar tissue from 7 days to 6 wks old SCA1 transgenic and wildtype mice were used. We observed that Bergmann glial S100B protein is localized to the cytoplasmic vacuoles in SCA1 PKN. These S100B positive cytoplasmic vacuoles began appearing much before the onset of behavioral abnormalities, and were negative for other glial and PKN marker proteins. Electron micrographs revealed that vacuoles have a double membrane. In the vacuoles, S100B colocalized with receptors of advanced glycation end-products (RAGE), and S100B co-immunoprecipated with cerebellar RAGE. In SCA1 PKN cultures, exogenous S100B protein interacted with the PKN membranes and was internalized. These data suggest that glial S100B though extrinsic to PKN is sequestered into cytoplasmic vacuoles in SCA1 mice at early postnatal ages. Further, S100B may be binding to RAGE on Purkinje cell membranes before these membranes are internalized. PMID:18176630

  18. [Hering, Vintschgau and the problem of Purkinje's succession].

    PubMed

    Sablik, K

    1989-01-01

    The problem of Jan Evangelista Purkinje's succession will be presented according to the results of archival research. The Ministery of Cult and Education in Vienna, and especially Karl Rokitansky, who was the adviser for medical education, in 1867 created a new professorship and Institute for Physiology, beside Purkinje and his Institute. Maximilian Vintschgau was to assist the world-famous 80 years old Purkinje but was not permitted to teach the whole field of physiology and to examine students. The fact that the professors of the Prague Medical Faculty in 1868 started to remove the restrictions for Vintschgau with the argument of academic freedom and in 1869 tried to keep the second institute for the future, is not yet mentioned in the literature. Discussions about the problems of the Czech language and its use in physiological lectures were scarcely mentioned by the Ministery: if one day there should be a Czech-speaking lecturer, the problem would be solved. Unfortunately Purkinje had no genuine pupil in Prague, and after his death, Vintschgau was provisional director of the Institute for half a year. In this situation Rokitansky decided that there should only be one institute for physiology in Prague. The Medical Faculty wanted to have Hermann Helmholtz to succeed Purkinje, but Helmholtz refused to come. Ewald Hering, who was nominated in the second place by the Faculty, accepted the call. Vintschgau had only rank four, third was Conrad Eckhard from Giessen. The Ministery in Vienna, however, made a special decision: The Medical Faculty of Innsbruck was founded in 1869, and there was not professor for physiology at the beginning of 1870. The candidates of the Insbruck Medical Faculty were neglected in favour of Vintschgau, who was considered to be a trustworthy Austrian patriot. Hering and Vintschgau became professors on March 6, 1870, and Hering started his work in Prague in a new institute in the "Wenzelsbad".

  19. Muscarinic acetylcholine receptor activation blocks long-term potentiation at cerebellar parallel fiber-Purkinje cell synapses via cannabinoid signaling.

    PubMed

    Rinaldo, Lorenzo; Hansel, Christian

    2013-07-02

    Muscarinic acetylcholine receptors (mAChRs) are known to modulate synaptic plasticity in various brain areas. A signaling pathway triggered by mAChR activation is the production and release of endocannabinoids that bind to type 1 cannabinoid receptors (CB1R) located on synaptic terminals. Using whole-cell patch-clamp recordings from rat cerebellar slices, we have demonstrated that the muscarinic agonist oxotremorine-m (oxo-m) blocks the induction of presynaptic long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell synapses in a CB1R-dependent manner. Under control conditions, LTP was induced by delivering 120 PF stimuli at 8 Hz. In contrast, no LTP was observed when oxo-m was present during tetanization. PF-LTP was restored when the CB1R antagonist N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) was coapplied with oxo-m. Furthermore, the suppressive effect of oxo-m on PF-LTP was abrogated by the GDP analog GDP-β-S (applied intracellularly), the phospholipase C inhibitor U-73122, and the diacylglycerol lipase inhibitor tetrahydrolipstatin (THL), suggesting that cannabinoid synthesis results from the activation of Gq-coupled mAChRs present on Purkinje cells. The oxo-m-mediated suppression of LTP was also prevented in the presence of the M3 receptor antagonist DAU 5884, and was absent in M1/M3 receptor double-KO mice, identifying M3 receptors as primary oxo-m targets. Our findings allow for the possibility that cholinergic signaling in the cerebellum--which may result from long-term depression (LTD)-related disinhibition of cholinergic neurons in the vestibular nuclei--suppresses presynaptic LTP to prevent an up-regulation of transmitter release that opposes the reduction of postsynaptic responsiveness. This modulatory capacity of mAChR signaling could promote the functional penetrance of LTD.

  20. Treadmill performance of mice with cerebellar lesions: 1. Purkinje cell degeneration mutant mice.

    PubMed

    Le Marec, N; Lalonde, R

    1998-02-01

    The purpose of this study was to evaluate the sensorimotor skills of a spontaneous mouse mutant, Purkinje cell degeneration (PCD), marked by selective cerebellar cortical atrophy on a treadmill activated at 1 of 2 speeds and at 1 of 3 slopes, requiring forward movements to avoid footshocks. There was no difference in latencies before falling from the belt between PCD mutants and controls during acquisition. However, PCD mutants were impaired on the fast treadmill during retention, implicating the cerebellum in the memory of a motor skill. During acquisition of the slow treadmill task at the 2 lowest slopes of inclination, PCD mutants spent more time walking than controls, an indication of a decreased ability of coordinating whole body movements. The same pattern of higher walking time on the slow treadmill in PCD mutants was evident during retention. These results indicate that the cerebellar cortex is involved in the acquisition and the retention of a task requiring equilibrium.

  1. Systematic Regional Variations in Purkinje Cell Spiking Patterns

    PubMed Central

    Xiao, Jianqiang; Cerminara, Nadia L.; Kotsurovskyy, Yuriy; Aoki, Hanako; Burroughs, Amelia; Wise, Andrew K.; Luo, Yuanjun; Marshall, Sarah P.; Sugihara, Izumi; Apps, Richard; Lang, Eric J.

    2014-01-01

    In contrast to the uniform anatomy of the cerebellar cortex, molecular and physiological studies indicate that significant differences exist between cortical regions, suggesting that the spiking activity of Purkinje cells (PCs) in different regions could also show distinct characteristics. To investigate this possibility we obtained extracellular recordings from PCs in different zebrin bands in crus IIa and vermis lobules VIII and IX in anesthetized rats in order to compare PC firing characteristics between zebrin positive (Z+) and negative (Z−) bands. In addition, we analyzed recordings from PCs in the A2 and C1 zones of several lobules in the posterior lobe, which largely contain Z+ and Z− PCs, respectively. In both datasets significant differences in simple spike (SS) activity were observed between cortical regions. Specifically, Z− and C1 PCs had higher SS firing rates than Z+ and A2 PCs, respectively. The irregularity of SS firing (as assessed by measures of interspike interval distribution) was greater in Z+ bands in both absolute and relative terms. The results regarding systematic variations in complex spike (CS) activity were less consistent, suggesting that while real differences can exist, they may be sensitive to other factors than the cortical location of the PC. However, differences in the interactions between SSs and CSs, including the post-CS pause in SSs and post-pause modulation of SSs, were also consistently observed between bands. Similar, though less strong trends were observed in the zonal recordings. These systematic variations in spontaneous firing characteristics of PCs between zebrin bands in vivo, raises the possibility that fundamental differences in information encoding exist between cerebellar cortical regions. PMID:25144311

  2. Cerebellar Nuclear Neurons Use Time and Rate Coding to Transmit Purkinje Neuron Pauses.

    PubMed

    Sudhakar, Shyam Kumar; Torben-Nielsen, Benjamin; De Schutter, Erik

    2015-12-01

    Neurons of the cerebellar nuclei convey the final output of the cerebellum to their targets in various parts of the brain. Within the cerebellum their direct upstream connections originate from inhibitory Purkinje neurons. Purkinje neurons have a complex firing pattern of regular spikes interrupted by intermittent pauses of variable length. How can the cerebellar nucleus process this complex input pattern? In this modeling study, we investigate different forms of Purkinje neuron simple spike pause synchrony and its influence on candidate coding strategies in the cerebellar nuclei. That is, we investigate how different alignments of synchronous pauses in synthetic Purkinje neuron spike trains affect either time-locking or rate-changes in the downstream nuclei. We find that Purkinje neuron synchrony is mainly represented by changes in the firing rate of cerebellar nuclei neurons. Pause beginning synchronization produced a unique effect on nuclei neuron firing, while the effect of pause ending and pause overlapping synchronization could not be distinguished from each other. Pause beginning synchronization produced better time-locking of nuclear neurons for short length pauses. We also characterize the effect of pause length and spike jitter on the nuclear neuron firing. Additionally, we find that the rate of rebound responses in nuclear neurons after a synchronous pause is controlled by the firing rate of Purkinje neurons preceding it.

  3. Cerebellar Purkinje Cells Generate Highly Correlated Spontaneous Slow-Rate Fluctuations.

    PubMed

    Cao, Ying; Liu, Yu; Jaeger, Dieter; Heck, Detlef H

    2017-01-01

    Cerebellar Purkinje cells (PC) fire action potentials at high, sustained rates. Changes in spike rate that last a few tens of milliseconds encode sensory and behavioral events. Here we investigated spontaneous fluctuations of PC simple spike rate at a slow time scale of the order of 1 s. Simultaneous recordings from pairs of PCs that were aligned either along the sagittal or transversal axis of the cerebellar cortex revealed that simple spike rate fluctuations at the 1 s time scale were highly correlated. Each pair of PCs had either a predominantly positive or negative slow-rate correlation, with negative correlations observed only in PC pairs aligned along the transversal axis. Slow-rate correlations were independent of faster rate changes that were correlated with fluid licking behavior. Simultaneous recordings from PCs and cerebellar nuclear (CN) neurons showed that slow-rate fluctuations in PC and CN activity were also highly correlated, but their correlations continually alternated between periods of positive and negative correlation. The functional significance of this new aspect of cerebellar spike activity remains to be determined. Correlated slow-rate fluctuations seem too slow to be involved in the real-time control of ongoing behavior. However, slow-rate fluctuations of PCs converging on the same CN neuron are likely to modulate the excitability of the CN neuron, thus introduce a possible slow modulation of cerebellar output activity.

  4. Treadmill exercise ameliorates symptoms of attention deficit/hyperactivity disorder through reducing Purkinje cell loss and astrocytic reaction in spontaneous hypertensive rats

    PubMed Central

    Yun, Hyo-Soon; Park, Mi-Sook; Ji, Eun-Sang; Kim, Tae-Woon; Ko, Il-Gyu; Kim, Hyun-Bae; Kim, Hong

    2014-01-01

    Attention deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder of cognition. We investigated the effects of treadmill exercise on Purkinje cell and astrocytic reaction in the cerebellum of the ADHD rat. Adult male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKYR) weighing 210± 10 g were used. The animals were randomly divided into four groups (n= 15): control group, ADHD group, ADHD and methylphenidate (MPH)-treated group, ADHD and treadmill exercise group. The rats in the MPH-treated group as a positive control received 1 mg/kg MPH orally once a day for 28 consecutive days. The rats in the treadmill exercise group were made to run on a treadmill for 30 min once a day for 28 days. Motor coordination and balance were determined by vertical pole test. Immunohistochemistry for the expression of calbindinD-28 and glial fibrillary acidic protein (GFAP) in the cerebellar vermis and Western blot for GFAP, Bax, and Bcl-2 were conducted. In the present results, ADHD significantly decreased balance and the number of calbindin-positive cells, while GFAP expression and Bax/Bcl-2 ratio in the cerebellum were significantly increased in the ADHD group compared to the control group (P< 0.05, respectively). In contrast, treadmill exercise and MPH alleviated the ADHD-induced the decrease of balance and the number of calbindine-positive cells, and the increase of GFAP expression and Bax/Bcl-2 ratio in the cerebellum (P< 0.05, respectively). Therefore, the present results suggested that treadmill exercise might exert ameliorating effect on ADHD through reduction of Purkinje cell loss and astrocytic reaction in the cerebellum. PMID:24678501

  5. Comparison of intraocular lens decentration and tilt measurements using 2 Purkinje meter systems.

    PubMed

    Maedel, Sophie; Hirnschall, Nino; Bayer, Natascha; Markovic, Sabine; Tabernero, Juan; Artal, Pablo; Schaeffel, Frank; Findl, Oliver

    2017-05-01

    To evaluate the difference in intraocular lens tilt and decentration measurements with 2 Purkinje meters. Vienna Institute for Research in Ocular Surgery, Hanusch Hospital, Vienna, Austria. Prospective evaluation of diagnostic test. This single-center study included pseudophakic patients in 2 substudies in which 3 consecutive measurements were performed with 2 Purkinje meters (Spanish and German). In substudy 1, an inexperienced examiner performed all measurements after a short learning period. In substudy 2, all measurements were taken by experienced examiners under direct supervision of the inventors of the devices. Substudy 1 included 53 pseudophakic eyes in which all 53 scans were successful with the Spanish device; however, only 35 measurements (66%) were successful with the German Purkinje meter. The mean tilt measured with the Spanish Purkinje meter was 4.35 degrees ± 2.50 (SD) and 9.20 ± 6.96 degrees with the German Purkinje meter. The mean decentration was 0.44 ± 0.19 mm and 0.74 ± 0.91 mm, (P = .44), respectively. In substudy 2 (29 pseudophakic eyes), the number of successful scans was 29 (100%) and 18 (62%) for the Spanish meter and German Purkinje meter, respectively. The mean horizontal and vertical tilt difference vector between the 2 systems was 4.89 ± 3.24 degrees and 7.57 ± 3.82 degrees, respectively. Concerning clinical feasibility, the Spanish Purkinje meter had a greater percentage of successful scans than the German device. In addition, this device measured significantly higher tilt values than the Spanish Purkinje meter. Copyright © 2017 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  6. Electrical properties associated with wide intercellular clefts in rabbit Purkinje fibres.

    PubMed Central

    Colatsky, T J; Tsien, R W

    1979-01-01

    1. Rabbit Purkinje fibres were studied using micro-electrode recordings of electrical activity or a two-micro-electrode voltage clamp. Previous morphological work had suggested that these preparations offer structural advantages for the analysis of ionic permeability mechanisms. 2. Viable preparations could be obtained consistently by exposure to a K glutamate Tyrode solution during excision and recovery. In NaCl Tyrode solution, the action potential showed a large overshoot and fully developed plateau, but no pacemaker depolarization at negative potentials. 3. The passive electrical properties were consistent with morphological evidence for the accessibility of cleft membranes within the cell bundle. Electrotonic responses to intracellular current steps showed the behaviour expected for a simple leaky capacitative cable. Capacitative current transients under voltage clamp were changed very little by an eightfold reduction in the external solution conductivity. 4. Slow current changes attributable to K depletion were small compared to those found in other cardiac preparations. The amount of depletion was close to that predicted by a cleft model which assumed free K diffusion in 1 micron clefts. 5. Step depolarizations over the plateau range of potentials evoked a slow inward current which was resistant to tetrodotoxin but blocked by D600. 6. Strong depolarizations to potentials near 0 mV elicited a transient outward current and a slowly activating late outward current. Both components resembled currents found in sheep or calf Purkinje fibres. 7. These experiments support previous interpretations of slow plateau currents in terms of genuine permeability changes. The rabbit Purkinje fibre may allow various ionic channels to be studied with relatively little interference from radial non-uniformities in membrane potential or ion concentration. Images Fig. 7 PMID:469754

  7. An Inotropic Action Caused by Muscarinic Receptor Subtype 3 in Canine Cardiac Purkinje Fibers

    PubMed Central

    Urushidani, Tetsuro; Tachibana, Shigehiro

    2013-01-01

    Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers. PMID:24260719

  8. A spiking network model of cerebellar Purkinje cells and molecular layer interneurons exhibiting irregular firing

    PubMed Central

    Lennon, William; Hecht-Nielsen, Robert; Yamazaki, Tadashi

    2014-01-01

    While the anatomy of the cerebellar microcircuit is well-studied, how it implements cerebellar function is not understood. A number of models have been proposed to describe this mechanism but few emphasize the role of the vast network Purkinje cells (PKJs) form with the molecular layer interneurons (MLIs)—the stellate and basket cells. We propose a model of the MLI-PKJ network composed of simple spiking neurons incorporating the major anatomical and physiological features. In computer simulations, the model reproduces the irregular firing patterns observed in PKJs and MLIs in vitro and a shift toward faster, more regular firing patterns when inhibitory synaptic currents are blocked. In the model, the time between PKJ spikes is shown to be proportional to the amount of feedforward inhibition from an MLI on average. The two key elements of the model are: (1) spontaneously active PKJs and MLIs due to an endogenous depolarizing current, and (2) adherence to known anatomical connectivity along a parasagittal strip of cerebellar cortex. We propose this model to extend previous spiking network models of the cerebellum and for further computational investigation into the role of irregular firing and MLIs in cerebellar learning and function. PMID:25520646

  9. Numerical approximation of the electromechanical coupling in the left ventricle with inclusion of the Purkinje network.

    PubMed

    Landajuela, Mikel; Vergara, Christian; Gerbi, Antonello; Dedé, Luca; Formaggia, Luca; Quarteroni, Alfio

    2018-03-25

    In this work, we consider the numerical approximation of the electromechanical coupling in the left ventricle with inclusion of the Purkinje network. The mathematical model couples the 3D elastodynamics and bidomain equations for the electrophysiology in the myocardium with the 1D monodomain equation in the Purkinje network. For the numerical solution of the coupled problem, we consider a fixed-point iterative algorithm that enables a partitioned solution of the myocardium and Purkinje network problems. Different levels of myocardium-Purkinje network splitting are considered and analyzed. The results are compared with those obtained using standard strategies proposed in the literature to trigger the electrical activation. Finally, we present a numerical study that, although performed in an idealized computational domain, features all the physiological issues that characterize a heartbeat simulation, including the initiation of the signal in the Purkinje network and the systolic and diastolic phases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Aberrant connections between climbing fibres and Purkinje cells induce alterations in the timing of an instrumental response in the rat.

    PubMed

    Gaytán-Tocavén, Lorena; López-Vázquez, Miguel Ángel; Guevara, Miguel Ángel; Olvera-Cortés, María Esther

    2017-09-01

    Cerebellar participation in timing and sensory-motor sequences has been supported by several experimental and clinical studies. A relevant role of the cerebellum in timing of conditioned responses in the range of milliseconds has been demonstrated, but less is known regarding the role of the cerebellum in supra-second timing of operant responses. A dissociated role of the cerebellum and striatum in timing in the millisecond and second range had been reported, respectively. The climbing fibre-Purkinje cell synapse is crucial in timing models; thus, the aberrant connection between these cellular elements is a suitable model for evaluating the contribution of the cerebellum in timing in the supra-second range. The aberrant connection between climbing fibres and Purkinje cells was induced by administration of the antagonist of NMDA receptors MK-801 to Sprague-Dawley rats at postnatal days 7-14. The timing of an operant response with two fixed intervals (5 and 8 s) and egocentric sequential learning was evaluated in 60-day-old adult rats. The aberrant connections caused a reduced accuracy in the timing of the instrumental response that was more evident in the 8-s interval and a reduced number of successive correct responses (responses emitted in the correct second without any other response between them) in the 8-s interval. In addition, an inability to incorporate new information in a sequence previously learned in egocentric-based sequence learning was apparent in rats with aberrant CF-PC synapses. These results support a relevant role for the cerebellum in the fine-tuning of the timing of operant responses in the supra-second range.

  11. Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

    PubMed

    Weber, Thomas; Namikawa, Kazuhiko; Winter, Barbara; Müller-Brown, Karina; Kühn, Ralf; Wurst, Wolfgang; Köster, Reinhard W

    2016-11-15

    The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTAC TM ) further expands the repertoire of genetic tools for conditional interrogation of cellular functions. © 2016. Published by The Company of Biologists Ltd.

  12. Regional Alterations in Purkinje Cell Density in Patients with Autism

    PubMed Central

    Skefos, Jerry; Cummings, Christopher; Enzer, Katelyn; Holiday, Jarrod; Weed, Katrina; Levy, Ezra; Yuce, Tarik; Kemper, Thomas; Bauman, Margaret

    2014-01-01

    Neuropathological studies, using a variety of techniques, have reported a decrease in Purkinje cell (PC) density in the cerebellum in autism. We have used a systematic sampling technique that significantly reduces experimenter bias and variance to estimate PC densities in the postmortem brains of eight clinically well-documented individuals with autism, and eight age- and gender-matched controls. Four cerebellar regions were analyzed: a sensorimotor area comprised of hemispheric lobules IV–VI, crus I & II of the posterior lobe, and lobule X of the flocculonodular lobe. Overall PC density was thus estimated using data from all three cerebellar lobes and was found to be lower in the cases with autism as compared to controls, an effect that was most prominent in crus I and II (p<0.05). Lobule X demonstrated a trend towards lower PC density in only the males with autism (p = 0.05). Brain weight, a correlate of tissue volume, was found to significantly contribute to the lower lobule X PC density observed in males with autism, but not to the finding of lower PC density in crus I & II. Therefore, lower crus I & II PC density in autism is more likely due to a lower number of PCs. The PC density in lobule X was found to correlate with the ADI-R measure of the patient's use of social eye contact (R2 = −0.75, p = 0.012). These findings support the hypothesis that abnormal PC density may contribute to selected clinical features of the autism phenotype. PMID:24586223

  13. Maturation of Cerebellar Purkinje Cell Population Activity during Postnatal Refinement of Climbing Fiber Network.

    PubMed

    Good, Jean-Marc; Mahoney, Michael; Miyazaki, Taisuke; Tanaka, Kenji F; Sakimura, Kenji; Watanabe, Masahiko; Kitamura, Kazuo; Kano, Masanobu

    2017-11-21

    Neural circuits undergo massive refinements during postnatal development. In the developing cerebellum, the climbing fiber (CF) to Purkinje cell (PC) network is drastically reshaped by eliminating early-formed redundant CF to PC synapses. To investigate the impact of CF network refinement on PC population activity during postnatal development, we monitored spontaneous CF responses in neighboring PCs and the activity of populations of nearby CF terminals using in vivo two-photon calcium imaging. Population activity is highly synchronized in newborn mice, and the degree of synchrony gradually declines during the first postnatal week in PCs and, to a lesser extent, in CF terminals. Knockout mice lacking P/Q-type voltage-gated calcium channel or glutamate receptor δ2, in which CF network refinement is severely impaired, exhibit an abnormally high level of synchrony in PC population activity. These results suggest that CF network refinement is a structural basis for developmental desynchronization and maturation of PC population activity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. Modeling our understanding of the His-Purkinje system.

    PubMed

    Vigmond, Edward J; Stuyvers, Bruno D

    2016-01-01

    The His-Purkinje System (HPS) is responsible for the rapid electric conduction in the ventricles. It relays electrical impulses from the atrioventricular node to the muscle cells and, thus, coordinates the contraction of ventricles in order to ensure proper cardiac pump function. The HPS has been implicated in the genesis of ventricular tachycardia and fibrillation as a source of ectopic beats, as well as forming distinct portions of reentry circuitry. Despite its importance, it remains much less well characterized, structurally and functionally, than the myocardium. Notably, important differences exist with regard to cell structure and electrophysiology, including ion channels, intracellular calcium handling, and gap junctions. Very few computational models address the HPS, and the majority of organ level modeling studies omit it. This review will provide an overview of our current knowledge of structure and function (including electrophysiology) of the HPS. We will review the most recent advances in modeling of the system from the single cell to the organ level, with considerations for relevant interspecies distinctions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. The ionic bases of the action potential in isolated mouse cardiac Purkinje cell.

    PubMed

    Vaidyanathan, Ravi; O'Connell, Ryan P; Deo, Makarand; Milstein, Michelle L; Furspan, Philip; Herron, Todd J; Pandit, Sandeep V; Musa, Hassan; Berenfeld, Omer; Jalife, José; Anumonwo, Justus M B

    2013-01-01

    Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse. To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs. Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data. APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs. Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  16. Tilt and decentration of intraocular lenses in vivo from Purkinje and Scheimpflug imaging. Validation study.

    PubMed

    de Castro, Alberto; Rosales, Patricia; Marcos, Susana

    2007-03-01

    To measure tilt and decentration of intraocular lenses (IOLs) with Scheimpflug and Purkinje imaging systems in physical model eyes with known amounts of tilt and decentration and patients. Instituto de Optica Daza de Valdés, Consejo Superior de Investigaciones Científicas, Madrid, Spain. Measurements of IOL tilt and decentration were obtained using a commercial Scheimpflug system (Pentacam, Oculus), custom algorithms, and a custom-built Purkinje imaging apparatus. Twenty-five Scheimpflug images of the anterior segment of the eye were obtained at different meridians. Custom algorithms were used to process the images (correction of geometrical distortion, edge detection, and curve fittings). Intraocular lens tilt and decentration were estimated by fitting sinusoidal functions to the projections of the pupillary axis and IOL axis in each image. The Purkinje imaging system captures pupil images showing reflections of light from the anterior corneal surface and anterior and posterior lens surfaces. Custom algorithms were used to detect the Purkinje image locations and estimate IOL tilt and decentration based on a linear system equation and computer eye models with individual biometry. Both methods were validated with a physical model eye in which IOL tilt and decentration can be set nominally. Twenty-one eyes of 12 patients with IOLs were measured with both systems. Measurements of the physical model eye showed an absolute discrepancy between nominal and measured values of 0.279 degree (Purkinje) and 0.243 degree (Scheimpflug) for tilt and 0.094 mm (Purkinje) and 0.228 mm (Scheimpflug) for decentration. In patients, the mean tilt was less than 2.6 degrees and the mean decentration less than 0.4 mm. Both techniques showed mirror symmetry between right eyes and left eyes for tilt around the vertical axis and for decentration in the horizontal axis. Both systems showed high reproducibility. Validation experiments on physical model eyes showed slightly higher accuracy

  17. Purkinje Cell Protein 4 Expression Is Associated With DNA Methylation Status in Aldosterone-Producing Adenoma.

    PubMed

    Kobuke, Kazuhiro; Oki, Kenji; Gomez-Sanchez, Celso E; Ohno, Haruya; Itcho, Kiyotaka; Yoshii, Yoko; Yoneda, Masayasu; Hattori, Noboru

    2018-03-01

    Aldosterone production is stimulated by activation of calcium signaling in aldosterone-producing adenomas (APAs), and epigenetic factors such as DNA methylation may be associated with the expression of genes involved in aldosterone regulation. Our aim was to investigate the DNA methylation of genes related to calcium signaling cascades in APAs and the association of mutations in genes linked to APAs with DNA methylation levels. Nonfunctioning adrenocortical adenoma (n = 12) and APA (n = 35) samples were analyzed. The KCNJ5 T158A mutation was introduced into human adrenocortical cell lines (HAC15 cells) using lentiviral delivery. DNA methylation array analysis was conducted using adrenal tumor samples and HAC15 cells. The Purkinje cell protein 4 (PCP4) gene was one of the most hypomethylated in APAs. DNA methylation levels in two sites of PCP4 showed a significant inverse correlation with messenger RNA expression in adrenal tumors. Bioinformatics and multiple regression analysis revealed that CCAAT/enhancer binding protein alpha (CEBPA) may bind to the methylation site of the PCP4 promoter. According to chromatin immunoprecipitation assay, CEBPA was bound to the PCP4 hypomethylated region by chromatin immunoprecipitation assay. There were no significant differences in PCP4 methylation levels among APA genotypes. Moreover, KCNJ5 T158A did not influence PCP4 methylation levels in HAC15 cells. We showed that the PCP4 promoter was one of the most hypomethylated in APAs and that PCP4 transcription may be associated with demethylation as well as with CEBPA in APAs. KCNJ5 mutations known to result in aldosterone overproduction were not related to PCP4 methylation in either clinical or in vitro studies.

  18. Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

    PubMed Central

    Bielen, F V; Glitsch, H G; Verdonck, F

    1991-01-01

    1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that

  19. Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

    PubMed

    Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

    2014-11-01

    Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

  20. Posturography of ataxia induced by Coriolis- and Purkinje-effects.

    PubMed

    Fitger, C; Brandt, T

    1982-02-01

    Vestibular Coriolis- and Purkinje-effect, which are known to induce vertigo, were investigated with respect to body posture. One aim of this investigation was to provide information concerning clinical vertigo symptoms. Standing on a rotatable stabilometer, 25 healthy subjects had to execute lateral head tilts during (Coriolis), or after (Purkinje), rotation varied with different constant velocities. The conditions were varied with respect to eyes open vs. eyes closed, head upright vs. head tilt to the right and left, direction of rotation clockwise vs. counterclockwise, active vs. passive head tilt, and active vs. passive body rotation. The results supported the expectation that destabilization was less severe with open than with closed eyes and that sway amplitudes were increased after head tilt as well as with a higher velocity of rotation. The direction of the induced body shift was, as expected, opposite to the initial vestibular stimulus. A forward shift after stop without head tilt was frequently found, being independent of the previous direction of rotation. Reported perceptions coincided mostly not with the initial vestibular signal but rather with the actual movement of compensation. Active instead of passive movements did not produce clearly different effects. The Purkinje experiment appeared to be equivalent to the situation when a patient with an acute lesion of a horizontal vestibular canal bends his head. The stabilogram under this condition may allow a prediction of the side of the lesion.

  1. Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination.

    PubMed

    Zhou, Liang; Yang, Dong; Wang, De-Juan; Xie, Ya-Jun; Zhou, Jia-Huan; Zhou, Lin; Huang, Hao; Han, Shuo; Shao, Chong-Yu; Li, Hua-Shun; Zhu, J Julius; Qiu, Meng-Sheng; De Zeeuw, Chris I; Shen, Ying

    2015-12-15

    Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1.

  2. A Novel Marker for Purkinje Cells, Ribosomal Protein MPS1/S27: Expression of MPS1 in Human Cerebellum.

    PubMed

    Fernandez-Pol, J Alberto

    2016-01-01

    The ribosomal protein metallopanstimulin-1 (MPS1/S27) serves critical survival purposes in cell division, in normal and cancerous cells; for this reason, selective pressures of evolution have conserved the DNA sequences encoding MPS1/S27 in Archaea and Eukariotic cells. The expression of MPS1/S27 protein in human adult cerebellum has not been established. The presence of MPS1/S27, was screened in paraffin-embedded human adult brain specimens processed for tissue inmunohistochemistry. Affinity-purified specific antibodies were directed against the N-terminus of MPS1. The antibodies to MPS1 detected Purkinje cells (PC) and their dendrites. In PC, MPS1 antigen-positive staining was found in: the nucleolus, which was strongly stained; ribosomes attached to the external nuclear membrane; cytoplasm of PC, with strong staining in a punctuate fashion; the soma-attached large dendrite trunks of PC, which were MPS1 antigen-positive; and the granular cell layer, where cellular staining in a few cells that appeared to resemble smaller PC was observed. Since MPS1 is involved in cell division, DNA repair, and ribosomal biogenesis, it may be a useful antigen for studying processes such as protein synthesis, oncogenesis, regeneration, aging, and perhaps diseases of the human cerebellum. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  3. Robust transmission of rate coding in the inhibitory Purkinje cell to cerebellar nuclei pathway in awake mice

    PubMed Central

    Abbasi, Samira; Maran, Selva K.; Cao, Ying; Abbasi, Ataollah; Heck, Detlef H.

    2017-01-01

    Neural coding through inhibitory projection pathways remains poorly understood. We analyze the transmission properties of the Purkinje cell (PC) to cerebellar nucleus (CN) pathway in a modeling study using a data set recorded in awake mice containing respiratory rate modulation. We find that inhibitory transmission from tonically active PCs can transmit a behavioral rate code with high fidelity. We parameterized the required population code in PC activity and determined that 20% of PC inputs to a full compartmental CN neuron model need to be rate-comodulated for transmission of a rate code. Rate covariance in PC inputs also accounts for the high coefficient of variation in CN spike trains, while the balance between excitation and inhibition determines spike rate and local spike train variability. Overall, our modeling study can fully account for observed spike train properties of cerebellar output in awake mice, and strongly supports rate coding in the cerebellum. PMID:28617798

  4. Unravelling how βCaMKII controls the direction of plasticity at parallel fibre-Purkinje cell synapses

    NASA Astrophysics Data System (ADS)

    Pinto, Thiago M.; Schilstra, Maria J.; Steuber, Volker; Roque, Antonio C.

    2015-12-01

    Long-term plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses is thought to mediate cerebellar motor learning. It is known that calcium-calmodulin dependent protein kinase II (CaMKII) is essential for plasticity in the cerebellum. Recently, Van Woerden et al. demonstrated that the β isoform of CaMKII regulates the bidirectional inversion of PF-PC plasticity. Because the cellular events that underlie these experimental findings are still poorly understood, our work aims at unravelling how β CaMKII controls the direction of plasticity at PF-PC synapses. We developed a bidirectional plasticity model that replicates the experimental observations by Van Woerden et al. Simulation results obtained from this model indicate the mechanisms that underlie the bidirectional inversion of cerebellar plasticity. As suggested by Van Woerden et al., the filamentous actin binding enables β CaMKII to regulate the bidirectional plasticity at PF-PC synapses. Our model suggests that the reversal of long-term plasticity in PCs is based on a combination of mechanisms that occur at different calcium concentrations.

  5. Motor learning induces plastic changes in Purkinje cell dendritic spines in the rat cerebellum.

    PubMed

    González-Tapia, D; González-Ramírez, M M; Vázquez-Hernández, N; González-Burgos, I

    2017-12-14

    The paramedian lobule of the cerebellum is involved in learning to correctly perform motor skills through practice. Dendritic spines are dynamic structures that regulate excitatory synaptic stimulation. We studied plastic changes occurring in the dendritic spines of Purkinje cells from the paramedian lobule of rats during motor learning. Adult male rats were trained over a 6-day period using an acrobatic motor learning paradigm; the density and type of dendritic spines were determined every day during the study period using a modified version of the Golgi method. The learning curve reflected a considerable decrease in the number of errors made by rats as the training period progressed. We observed more dendritic spines on days 2 and 6, particularly more thin spines on days 1, 3, and 6, fewer mushroom spines on day 3, fewer stubby spines on day 1, and more thick spines on days 4 and 6. The initial stage of motor learning may be associated with fast processing of the underlying synaptic information combined with an apparent "silencing" of memory consolidation processes, based on the regulation of the neuronal excitability. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

  6. Action Potentials Initiate in the Axon Initial Segment and Propagate Through Axon Collaterals Reliably in Cerebellar Purkinje Neurons

    PubMed Central

    Foust, Amanda; Popovic, Marko; Zecevic, Dejan; McCormick, David A.

    2010-01-01

    Purkinje neurons are the output cells of the cerebellar cortex and generate spikes in two distinct modes, known as simple and complex spikes. Revealing the point of origin of these action potentials, and how they conduct into local axon collaterals, is important for understanding local and distal neuronal processing and communication. By utilizing a recent improvement in voltage sensitive dye imaging technique that provided exceptional spatial and temporal resolution, we were able to resolve the region of spike initiation as well as follow spike propagation into axon collaterals for each action potential initiated on single trials. All fast action potentials, for both simple and complex spikes, whether occurring spontaneously or in response to a somatic current pulse or synaptic input, initiated in the axon initial segment. At discharge frequencies of less than approximately 250 Hz, spikes propagated faithfully through the axon and axon collaterals, in a saltatory manner. Propagation failures were only observed for very high frequencies or for the spikelets associated with complex spikes. These results demonstrate that the axon initial segment is a critical decision point in Purkinje cell processing and that the properties of axon branch points are adjusted to maintain faithful transmission. PMID:20484631

  7. Calcium, Synaptic Plasticity and Intrinsic Homeostasis in Purkinje Neuron Models

    PubMed Central

    Achard, Pablo; De Schutter, Erik

    2008-01-01

    We recently reproduced the complex electrical activity of a Purkinje cell (PC) with very different combinations of ionic channel maximum conductances, suggesting that a large parameter space is available to homeostatic mechanisms. It has been hypothesized that cytoplasmic calcium concentrations control the homeostatic activity sensors. This raises many questions for PCs since in these neurons calcium plays an important role in the induction of synaptic plasticity. To address this question, we generated 148 new PC models. In these models the somatic membrane voltages are stable, but the somatic calcium dynamics are very variable, in agreement with experimental results. Conversely, the calcium signal in spiny dendrites shows only small variability. We demonstrate that this localized control of calcium conductances preserves the induction of long-term depression for all models. We conclude that calcium is unlikely to be the sole activity-sensor in this cell but that there is a strong relationship between activity homeostasis and synaptic plasticity. PMID:19129937

  8. A Critical Period in Purkinje Cell Development Is Mediated by Local Estradiol Synthesis, Disrupted by Inflammation, and Has Enduring Consequences Only for Males.

    PubMed

    Hoffman, Jessica F; Wright, Christopher L; McCarthy, Margaret M

    2016-09-28

    intrinsic program of gene expression determines the critical period. The enduring consequences of inflammation during the second postnatal week are stunted dendrites of the cerebellum's principal neurons, Purkinje cells, and impairments in later social behavior. These changes are not evident if inflammation occurs during the first or third week, highlighting the importance of fine-grained analyses of developmental processes and the factors that influence them. Copyright © 2016 the authors 0270-6474/16/3610039-11$15.00/0.

  9. Neural stem cells rescue nervous purkinje neurons by restoring molecular homeostasis of tissue plasminogen activator and downstream targets.

    PubMed

    Li, Jianxue; Imitola, Jaime; Snyder, Evan Y; Sidman, Richard L

    2006-07-26

    Neural stem cells (NSCs) offer special therapeutic prospects because they can be isolated from the CNS, expanded ex vivo, and re-implanted into diseased CNS where they not only migrate and differentiate according to cues from host tissue but also appear to be capable of affecting host cells. In nervous (nr) mutant mice Purkinje neuron (PN) mitochondria become abnormal by the second postnatal week, and a majority of PNs die in the fourth to fifth weeks. We previously identified in nr cerebellum a 10-fold increase in tissue plasminogen activator (tPA) as a key component of the mechanism causing nr PN death. Here we report that undifferentiated wild-type murine NSCs, when transplanted into the newborn nr cerebellar cortex, do not replace host PNs but contact imperiled PNs and support their mitochondrial function, dendritic growth, and synaptogenesis, subsequently leading to the rescue of host PNs and restoration of motor coordination. This protection of nr PNs also is verified by an in vitro organotypic slice model in which nr cerebellar slices are cocultured with NSCs. Most importantly, the integrated NSCs in young nr cerebellum rectify excessive tPA mRNA and protein to close to normal levels and protect the mitochondrial voltage-dependent anion channel and neurotrophins, downstream targets of the tPA/plasmin proteolytic system. This report demonstrates for the first time that NSCs can rescue imperiled host neurons by rectifying their gene expression, elevating somatic stem cell therapeutic potential beyond solely cell replacement strategy.

  10. Differential inhibition of Ca2+ channels in mature rat cerebellar Purkinje cells by sFTX-3.3 and FTX-3.3.

    PubMed

    Dupere, J R; Moya, E; Blagbrough, I S; Usowicz, M M

    1996-01-01

    Synthetic funnel web spider toxin (sFTX-3.3) is a polyamine amide analogue of FTX, a toxin fraction isolated from the venom of the funnel web spider, Agelenopsis aperta, that blocks P-type Ca2+ channels. The structures of these polyamine containing compounds are not identical: sFTX-3.3 contains an amide carbonyl oxygen that is absent from the predicted structure of native FTX. Recently, a compound called FTX-3.3 was synthesized with the structure predicted for native FTX. We have compared the effects of polyamine amide sFTX-3.3 and polyamine FTX-3.3, on Ca2+ channel currents in the soma of mature rat cerebellar Purkinje neurons, in which the predominant Ca2+ channels are defined as P-type. Differential inhibition by sFTX-3.3 and FTX-3.3 revealed three populations of Ca2+ channels. One group, mediating approximately 66% of the current, was blocked by sFTX-3.3 with an IC50 (concentration producing half maximal inhibition) of 33 nM or by FTX-3.3 with an IC50 of 55 pM. A second population (5-25% of the total current) was inhibited by sFTX-3.3 with an IC50 of 33 nM, but was insensitive to FTX-3.3, while a third (10-30%) was blocked by FTX-3.3 with an IC50 of 125 nM and was resistant to sFTX-3.3. These channels also showed distinctive current-voltage relationships. Our results suggest that P-type Ca2+ channels in mature rat cerebellar Purkinje cells may be subdivided according to pharmacological and biophysical properties.

  11. Electrophysiological effects of haloperidol on isolated rabbit Purkinje fibers and guinea pigs papillary muscles under normal and simulated ischemia.

    PubMed

    Yan, Dong; Cheng, Lu-feng; Song, Hong-Yan; Turdi, Subat; Kerram, Parhat

    2007-08-01

    Overdoses of haloperidol are associated with major ventricular arrhythmias, cardiac conduction block, and sudden death. The aim of this experiment was to study the effect of haloperidol on the action potentials in cardiac Purkinje fibers and papillary muscles under normal and simulated ischemia conditions in rabbits and guinea pigs. Using the standard intracellular microelectrode technique, we examined the effects of haloperidol on the action potential parameters [action potential amplitude (APA), phase 0 maximum upstroke velocity (V(max)), action potential amplitude at 90% of repolarization (APD(90)), and effective refractory period (ERP)] in rabbit cardiac Purkinje fibers and guinea pig cardiac papillary cells, in which both tissues were under simulated ischemic conditions. Under ischemic conditions, different concentrations of haloperidol depressed APA and prolonged APD(90) in a concentration-dependent manner in rabbit Purkinje fibers. Haloperidol (3 micromol/L) significantly depressed APA and prolonged APD(90), and from 1 micromol/L, haloperidol showed significant depression on V(max); ERP was not significantly affected. In guinea pig cardiac papillary muscles, the thresholds of significant reduction in APA, V(max), EPR, and APD(90) were 10, 0.3, 1, and 1 mumol/L, respectively, for haloperidol. Compared with cardiac conductive tissues, papillary muscles were more sensitive to ischemic conditions. Under ischemia, haloperidol prolonged ERP and APD(90) in a concentration-dependent manner and precipitated the decrease in V(max) induced by ischemia. The shortening of ERP and APD(90) in papillary muscle action potentials may be inhibited by haloperidol.

  12. Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

    PubMed

    Tsai, Peter T; Hull, Court; Chu, YunXiang; Greene-Colozzi, Emily; Sadowski, Abbey R; Leech, Jarrett M; Steinberg, Jason; Crawley, Jacqueline N; Regehr, Wade G; Sahin, Mustafa

    2012-08-30

    Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

  13. Collective gradient sensing: fundamental bounds, cluster mechanics, and cell-to-cell variability

    NASA Astrophysics Data System (ADS)

    Camley, Brian

    Many eukaryotic cells chemotax, sensing and following chemical gradients. However, experiments have shown that even under conditions when single cells do not chemotax, small clusters may still follow a gradient. Similar collective motion is also known to occur in response to gradients in substrate stiffness or electric potential (collective durotaxis or galvanotaxis). How can cell clusters sense a gradient that individual cells ignore? I discuss possible ``collective guidance'' mechanisms underlying this motion, where individual cells measure the mean value of the attractant, but need not measure its gradient to give rise to directional motility for a cell cluster. I show that the collective guidance hypothesis can be directly tested by looking for strong orientational effects in pairs of cells chemotaxing. Collective gradient sensing also has a new wrinkle in comparison to single-cell chemotaxis: to accurately determine a gradient direction, a cluster must integrate information from cells with highly variable properties. When is cell-to-cell variation a limiting factor in sensing accuracy? I provide some initial answers, and discuss how cell clusters can sense gradients in a way that is robust to cell-to-cell variation. Interestingly, these strategies may depend on the cluster's mechanics; I develop a bound that links the cluster's chemotactic accuracy and its rheology. This suggests that in some circumstances, mechanical transitions (e.g. unjamming) can control tactic accuracy. Work supported by NIH Grant No. P01 GM078586, NIH Grant No. F32GM110983.

  14. 3D gaze tracking method using Purkinje images on eye optical model and pupil

    NASA Astrophysics Data System (ADS)

    Lee, Ji Woo; Cho, Chul Woo; Shin, Kwang Yong; Lee, Eui Chul; Park, Kang Ryoung

    2012-05-01

    Gaze tracking is to detect the position a user is looking at. Most research on gaze estimation has focused on calculating the X, Y gaze position on a 2D plane. However, as the importance of stereoscopic displays and 3D applications has increased greatly, research into 3D gaze estimation of not only the X, Y gaze position, but also the Z gaze position has gained attention for the development of next-generation interfaces. In this paper, we propose a new method for estimating the 3D gaze position based on the illuminative reflections (Purkinje images) on the surface of the cornea and lens by considering the 3D optical structure of the human eye model. This research is novel in the following four ways compared with previous work. First, we theoretically analyze the generated models of Purkinje images based on the 3D human eye model for 3D gaze estimation. Second, the relative positions of the first and fourth Purkinje images to the pupil center, inter-distance between these two Purkinje images, and pupil size are used as the features for calculating the Z gaze position. The pupil size is used on the basis of the fact that pupil accommodation happens according to the gaze positions in the Z direction. Third, with these features as inputs, the final Z gaze position is calculated using a multi-layered perceptron (MLP). Fourth, the X, Y gaze position on the 2D plane is calculated by the position of the pupil center based on a geometric transform considering the calculated Z gaze position. Experimental results showed that the average errors of the 3D gaze estimation were about 0.96° (0.48 cm) on the X-axis, 1.60° (0.77 cm) on the Y-axis, and 4.59 cm along the Z-axis in 3D space.

  15. Activity-Dependent Gating of Calcium Spikes by A-type K+ Channels Controls Climbing Fiber Signaling in Purkinje Cell Dendrites

    PubMed Central

    Otsu, Yo; Marcaggi, Païkan; Feltz, Anne; Isope, Philippe; Kollo, Mihaly; Nusser, Zoltan; Mathieu, Benjamin; Kano, Masanobu; Tsujita, Mika; Sakimura, Kenji; Dieudonné, Stéphane

    2014-01-01

    Summary In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules. PMID:25220810

  16. Activity-dependent gating of calcium spikes by A-type K+ channels controls climbing fiber signaling in Purkinje cell dendrites.

    PubMed

    Otsu, Yo; Marcaggi, Païkan; Feltz, Anne; Isope, Philippe; Kollo, Mihaly; Nusser, Zoltan; Mathieu, Benjamin; Kano, Masanobu; Tsujita, Mika; Sakimura, Kenji; Dieudonné, Stéphane

    2014-10-01

    In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Sumoylation of FOXP2 regulates motor function and vocal communication through Purkinje cell development

    PubMed Central

    Usui, Noriyoshi; Co, Marissa; Harper, Matthew; Rieger, Michael A.; Dougherty, Joseph D.; Konopka, Genevieve

    2016-01-01

    Background Mutations in the gene encoding the transcription factor forkhead box P2, FOXP2, result in brain developmental abnormalities including reduced gray matter in both human patients and rodent models, and speech and language deficits. However, neither the region-specific function of FOXP2 in the brain, in particular the cerebellum, nor the effects of any post-translational modifications of FOXP2 in the brain and disorders have been explored. Methods We characterized sumoylation of FOXP2 biochemically, and analyzed the region-specific function and sumoylation of FOXP2 in the developing mouse cerebellum. Using in utero electroporation to manipulate the sumoylation-state of Foxp2 as well as Foxp2 expression levels in Purkinje cells (PCs) of the cerebellum in vivo, we reduced Foxp2 expression approximately 40% in the mouse cerebellum. Such a reduction approximates the haploinsufficiency observed in human patients who demonstrate speech and language impairments. Results We identified sumoylation of FOXP2 at K674 (K673 in mouse) in the cerebellum of neonates. In vitro co-immunoprecipitation and in vivo colocalization experiments suggest that PIAS3 acts as the SUMO E3 ligase for FOXP2 sumoylation. This sumoylation modifies transcriptional regulation by FOXP2. We demonstrate that Foxp2 sumoylation is required for regulation of cerebellar motor function and vocal communication, likely through dendritic outgrowth and arborization of PCs in the mouse cerebellum. Conclusions Sumoylation of Foxp2 in neonatal mouse cerebellum regulates PC development as well as motor functions and vocal communication, demonstrating evidence for sumoylation in regulating mammalian behaviors. PMID:27009683

  18. [Computer-assisted measurement of ocular misalignment in infants and young children using the digital Purkinje reflection pattern procedure].

    PubMed

    Barry, J C; Effert, R; Kaupp, A; Kleine, M; Reim, M

    1994-02-01

    A digital image recording and processing system is presented that allows a quick diagnosis of microstrabismus in non-cooperative children. It is thus particularly suited for screening purposes. The Purkinje Reflection Pattern Evaluation (RPE) method is used: three small flashes are used to produce the desired Purkinje images. Two horizontal rows of the three 1st Purkinje images (anterior corneal reflections) and of the three 4th Purkinje images (posterior crystalline lens reflections) stemming from the three light sources form the characteristic Purkinje image reflection pattern. Each eye's position is calculated from the shift between the upper and lower rows of reflections by means of two simple formulae. From the angles obtained in binocular fixation and monocular fixation the manifest angle of strabismus corresponding to the angle measured in the simultaneous prism-and-cover test is computed. The measurement is performed at a fixation distance of 50 cm under natural viewing conditions. To obtain a picture one only has to get the child's attention for a short moment. The primary position is triggered with the fixation light, which is operated by a switch. The digital image recording is done with a hand-held device comprising two miniaturized video cameras, three photo flashes and a fixation light that is operated manually. An IBM-compatible PC equipped with a hard disk and two frame grabbers was adapted for the storage and processing of the pictures. The pictures are evaluated interactively in a few minutes on the workstation's monitor immediately after the measurement. To this end specially designed menu-driven software was implemented. Examples of the measuring procedure and clinical results in infants with microtropic highlight the potential of the system as a screening apparatus and for the exact measurement of small and large squint angles. Usually even 1-year-old children can cooperate well enough to get good-quality pictures in binocular fixation. The new

  19. Nutrient shielding in clusters of cells

    NASA Astrophysics Data System (ADS)

    Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

    2013-06-01

    Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells' spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude among different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ.

  20. Nutrient Shielding in Clusters of Cells

    PubMed Central

    Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

    2014-01-01

    Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells’ spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude between different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ. PMID:23848711

  1. Antioxidant supplementation upregulates calbindin expression in cerebellar Purkinje cells of rat pups subjected to post natal exposure to sodium arsenite.

    PubMed

    Dhar, Pushpa; Kaushal, Parul; Kumar, Pavan

    2018-07-01

    Optimal cytoplasmic calcium (Ca 2+ ) levels have been associated with adequate cell functioning and neuronal survival. Altered intracellular Ca 2+ levels following impaired Ca 2+ homeostasis could induce neuronal degeneration or even cell death. There are reports of arsenite induced oxidative stress and the associated disturbances in intracellular calcium homeostasis. The present study focused on determining the strategies that would modulate tissue redox status and calcium binding protein (CaBP) (Calbindin D28k-CB) expression affected adversely by sodium arsenite (NaAsO 2 ) exposure (postnatal) of rat pups. NaAsO 2 alone or along with antioxidants (AOXs) (alpha lipoic acid or curcumin) was administered by intraperitoneal (i.p.) route from postnatal day (PND) 1-21 (covering rapid brain growth period - RBGP) to experimental groups and animals receiving sterile water by the same route served as the controls. At the end of the experimental period, the animals were subjected to euthanasia and the cerebellar tissue obtained therefrom was processed for immunohistochemical localization and western blot analysis of CB protein. CB was diffusely expressed in cell body as well as dendritic processes of Purkinje cells (PCs) along the PC Layer (PCL) in all cerebellar folia of the control and the experimental animals. The multilayered pattern of CB +ve cells along with their downregulated expression and low packing density was significantly evident in the arsenic (iAs) alone exposed group as against the controls and AOX supplemented groups. The observations are suggestive of AOX induced restoration of CaBP expression in rat cerebellum following early postnatal exposure to NaAsO 2 . Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses

    PubMed Central

    Silver, R Angus; Momiyama, Akiko; Cull-Candy, Stuart G

    1998-01-01

    EPSCs were recorded under whole-cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12- to 14-day-old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired-pulse depression and all-or-none appearance in response to a graded stimulus. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non-uniform release probability. This ‘multiple-probability fluctuation’ (MPF) analysis gave an estimate of 510 ± 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 ± 0.03 nS (n = 6). Control experiments, and simulations examining the effects of non-uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mm Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 ± 0.01 nS and a CV of 0.37 ± 0.01 (n = 4). Similar estimates of q were obtained in 2 mm Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 μm) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. We used MPF analysis to identify those quantal parameters that change during frequency-dependent depression at climbing fibre-Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (P¯r) was unusually high (0.90 ± 0.03 at 0.033 Hz

  3. Optimization of cerebellar purkinje neuron cultures and development of a plasmid-based method for purkinje neuron-specific, miRNA-mediated protein knockdown.

    PubMed

    Alexander, C J; Hammer, J A

    2016-01-01

    We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    NASA Astrophysics Data System (ADS)

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

  5. Generation-V dual-Purkinje-image eyetracker

    NASA Technical Reports Server (NTRS)

    Crane, H. D.; Steele, C. M.

    1985-01-01

    Major advances characterize the Generation-V dual-Purkinje-image eyetracker compared with the Generation-III version previously described. These advances include a large reduction in size, major improvements in frequency response and noise level, automatic alignment to a subject, and automatic adjustment for different separation between the visual and optic axes, which can vary considerably from subject to subject. In a number of applications described in the paper, the eyetracker is coupled with other highly specialized optical devices. These applications include accurately stabilizing an image on a subject's retina; accurately simulating a visually dead retinal region (i.e., a scotoma) of arbitrary shape, size, and position; and, for clinical purposes, stabilizing the position of a laser coagulator beam on a patient's retina so that the point of contact is unaffected by the patient's own eye movements.

  6. Gap junctions contribute to anchorage-independent clustering of breast cancer cells.

    PubMed

    Gava, Fabien; Rigal, Lise; Mondesert, Odile; Pesce, Elise; Ducommun, Bernard; Lobjois, Valérie

    2018-02-27

    Cancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. We previously reported that cell-cell adhesion proteins, such as E-cadherin, and desmosomal proteins are involved in cell aggregation to form clusters independently of cell migration or matrix adhesion. Here, we investigated the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells. We used live cell image acquisition and analysis to monitor the kinetics of MCF7 cell clustering in the presence/absence of GJIC pharmacological inhibitors and to screen a LOPAC® bioactive compound library. We also used a calcein transfer assay and flow cytometry to evaluate GJIC involvement in cancer cell clustering. We first demonstrated that functional GJIC are established in the early phase of cancer cell aggregation. We then showed that pharmacological inhibition of GJIC using tonabersat and meclofenamate delayed MCF7 cell clustering and reduced calcein transfer. We also found that brefeldin A, an inhibitor of vesicular trafficking, which we identified by screening a small compound library, and latrunculin A, an actin cytoskeleton-disrupting agent, both impaired MCF7 cell clustering and calcein transfer. Our results demonstrate that GJIC are involved from the earliest stages of anchorage-independent cancer cell aggregation. They also give insights into the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.

  7. Patterning C. elegans: homeotic cluster genes, cell fates and cell migrations.

    PubMed

    Salser, S J; Kenyon, C

    1994-05-01

    Despite its simple body form, the nematode C. elegans expresses homeotic cluster genes similar to those of insects and vertebrates in the patterning of many cell types and tissues along the anteroposterior axis. In the ventral nerve cord, these genes program spatial patterns of cell death, fusion, division and neurotransmitter production; in migrating cells they regulate the direction and extent of movement. Nematode development permits an analysis at the cellular level of how homeotic cluster genes interact to specify cell fates, and how cell behavior can be regulated to assemble an organism.

  8. Clusters of circulating tumor cells traverse capillary-sized vessels

    PubMed Central

    Au, Sam H.; Storey, Brian D.; Moore, John C.; Tang, Qin; Chen, Yeng-Long; Javaid, Sarah; Sarioglu, A. Fatih; Sullivan, Ryan; Madden, Marissa W.; O’Keefe, Ryan; Haber, Daniel A.; Maheswaran, Shyamala; Langenau, David M.; Stott, Shannon L.; Toner, Mehmet

    2016-01-01

    Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis. PMID:27091969

  9. Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells

    PubMed Central

    Miyazaki, Taisuke; Yamasaki, Miwako; Hashimoto, Kouichi; Kohda, Kazuhisa; Yuzaki, Michisuke; Shimamoto, Keiko; Tanaka, Kohichi; Kano, Masanobu; Watanabe, Masahiko

    2017-01-01

    Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the l-glutamate/l-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and nonsynaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF–PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10–90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs. PMID:28655840

  10. IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

    PubMed Central

    Hisatsune, Chihiro; Miyamoto, Hiroyuki; Hirono, Moritoshi; Yamaguchi, Naohide; Sugawara, Takeyuki; Ogawa, Naoko; Ebisui, Etsuko; Ohshima, Toshio; Yamada, Masahisa; Hensch, Takao K.; Hattori, Mitsuharu; Mikoshiba, Katsuhiko

    2013-01-01

    The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway. PMID:24109434

  11. Changes in Simple Spike Activity of some Purkinje cells in the Oculomotor Vermis during Saccade Adaptation are Appropriate to Participate in Motor Learning

    PubMed Central

    Kojima, Yoshiko; Soetedjo, Robijanto; Fuchs, Albert F.

    2010-01-01

    Adaptation of saccadic eye movements provides an excellent motor learning model to study theories of neuronal plasticity. When primates make saccades to a jumping target, a backward step of the target during the saccade can make it appear to overshoot. If this deception continues for many trials, saccades gradually decrease in amplitude to go directly to the back-stepped target location. We used this adaptation paradigm to evaluate the Marr-Albus hypothesis that such motor learning occurs at the Purkinje (P-) cell of the cerebellum. We recorded the activity of identified P-cells in the oculomotor vermis, lobules VIc and VII. After determining the on and off error directions of a P-cell’s complex spike activity, we determined whether its saccade-related simple spike (SS) activity changed during saccade adaptation in those two directions. Before adaptation, 57 of 61 P-cells exhibited a clear burst, pause or a combination of both for saccades in one or both directions. Sixty-two percent of all cells, including 2 of the 4 initially unresponsive ones, behaved differently for saccades whose size changed because of adaptation than for saccades of similar sizes gathered before adaptation. In at least 42% of these, the changes were appropriate to decrease saccade amplitude based on our current knowledge of cerebellum and brain stem saccade circuitry. Changes in activity during adaptation were not compensating for the potential fatigue associated with performing many saccades. Therefore, many P-cells in the oculomotor vermis exhibit changes in SS activity specific to adapted saccades and therefore appropriate to induce adaptation. PMID:20220005

  12. Cadherins in cerebellar development: translation of embryonic patterning into mature functional compartmentalization.

    PubMed

    Redies, Christoph; Neudert, Franziska; Lin, Juntang

    2011-09-01

    Cadherins are cell adhesion molecules with multiple morphogenic functions in brain development, for example, in neuroblast migration and aggregation, axon navigation, neural circuit formation, and synaptogenesis. More than 100 members of the cadherin superfamily are expressed in the developing and mature brain. Most of the cadherins investigated, in particular classic cadherins and δ-protocadherins, are expressed in the cerebellum. For several cadherin subtypes, expression begins at early embryonic stages and persists until mature stages of cerebellar development. At intermediate stages, distinct Purkinje cell clusters exhibit unique rostrocaudal and mediolateral expression profiles for each cadherin. In the chicken, mouse, and other species, the Purkinje cell clusters are separated by intervening raphes of migrating granule cells. This pattern of Purkinje cell clusters/raphes is, at least in part, continuous with the parasagittal striping pattern that is apparent in the mature cerebellar cortex, for example, for zebrin II/aldolase C. Moreover, subregions of the deep cerebellar nuclei, vestibular nuclei and the olivary complex also express cadherins differentially. Neuroanatomical evidence suggests that the nuclear subregions and cortical domains that express the same cadherin subtype are connected to each other, to form neural subcircuits of the cerebellar system. Cadherins thus provide a molecular code that specifies not only embryonic structures but also functional cerebellar compartmentalization. By following the implementation of this code, it can be revealed how mature functional architecture emerges from embryonic patterning during cerebellar development. Dysfunction of some cadherins is associated with psychiatric diseases and developmental impairments and may also affect cerebellar function.

  13. Estimation of Local Orientations in Fibrous Structures With Applications to the Purkinje System

    PubMed Central

    Plank, Gernot; Trayanova, Natalia A.; Vidal, René

    2011-01-01

    The extraction of the cardiac Purkinje system (PS) from intensity images is a critical step toward the development of realistic structural models of the heart. Such models are important for uncovering the mechanisms of cardiac disease and improving its treatment and prevention. Unfortunately, the manual extraction of the PS is a challenging and error-prone task due to the presence of image noise and numerous fiber junctions. To deal with these challenges, we propose a framework that estimates local fiber orientations with high accuracy and reconstructs the fibers via tracking. Our key contribution is the development of a descriptor for estimating the orientation distribution function (ODF), a spherical function encoding the local geometry of the fibers at a point of interest. The fiber/branch orientations are identified as the modes of the ODFs via spherical clustering and guide the extraction of the fiber centerlines. Experiments on synthetic data evaluate the sensitivity of our approach to image noise, width of the fiber, and choice of the mode detection strategy, and show its superior performance compared to those of the existing descriptors. Experiments on the free-running PS in an MR image also demonstrate the accuracy of our method in reconstructing such sparse fibrous structures. PMID:21335301

  14. SC3 - consensus clustering of single-cell RNA-Seq data

    PubMed Central

    Kiselev, Vladimir Yu.; Kirschner, Kristina; Schaub, Michael T.; Andrews, Tallulah; Yiu, Andrew; Chandra, Tamir; Natarajan, Kedar N; Reik, Wolf; Barahona, Mauricio; Green, Anthony R; Hemberg, Martin

    2017-01-01

    Single-cell RNA-seq (scRNA-seq) enables a quantitative cell-type characterisation based on global transcriptome profiles. We present Single-Cell Consensus Clustering (SC3), a user-friendly tool for unsupervised clustering which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach. We demonstrate that SC3 is capable of identifying subclones based on the transcriptomes from neoplastic cells collected from patients. PMID:28346451

  15. Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters.

    PubMed

    Bithi, Swastika S; Vanapalli, Siva A

    2017-02-02

    Drug assays with patient-derived cells such as circulating tumor cells requires manipulating small sample volumes without loss of rare disease-causing cells. Here, we report an effective technology for isolating and analyzing individual tumor cells and their clusters from minute sample volumes using an optimized microfluidic device integrated with pipettes. The method involves using hand pipetting to create an array of cell-laden nanoliter-sized droplets immobilized in a microfluidic device without loss of tumor cells during the pipetting process. Using this technology, we demonstrate single-cell analysis of tumor cell response to the chemotherapy drug doxorubicin. We find that even though individual tumor cells display diverse uptake profiles of the drug, the onset of apoptosis is determined by accumulation of a critical intracellular concentration of doxorubicin. Experiments with clusters of tumor cells compartmentalized in microfluidic drops reveal that cells within a cluster have higher viability than their single-cell counterparts when exposed to doxorubicin. This result suggests that circulating tumor cell clusters might be able to better survive chemotherapy drug treatment. Our technology is a promising tool for understanding tumor cell-drug interactions in patient-derived samples including rare cells.

  16. Differentiation of human-induced pluripotent stem cells into insulin-producing clusters.

    PubMed

    Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

    2015-02-01

    In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by "stimulating" certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells. A human-induced pluripotent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic β-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test. Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test. This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.

  17. Absence of Rapid Propagation through the Purkinje Network as a Potential Cause of Line Block in the Human Heart with Left Bundle Branch Block.

    PubMed

    Okada, Jun-Ichi; Washio, Takumi; Nakagawa, Machiko; Watanabe, Masahiro; Kadooka, Yoshimasa; Kariya, Taro; Yamashita, Hiroshi; Yamada, Yoko; Momomura, Shin-Ichi; Nagai, Ryozo; Hisada, Toshiaki; Sugiura, Seiryo

    2018-01-01

    Background: Cardiac resynchronization therapy is an effective device therapy for heart failure patients with conduction block. However, a problem with this invasive technique is the nearly 30% of non-responders. A number of studies have reported a functional line of block of cardiac excitation propagation in responders. However, this can only be detected using non-contact endocardial mapping. Further, although the line of block is considered a sign of responders to therapy, the mechanism remains unclear. Methods: Herein, we created two patient-specific heart models with conduction block and simulated the propagation of excitation based on a cellmodel of electrophysiology. In one model with a relatively narrow QRS width (176 ms), we modeled the Purkinje network using a thin endocardial layer with rapid conduction. To reproduce a wider QRS complex (200 ms) in the second model, we eliminated the Purkinje network, and we simulated the endocardial mapping by solving the inverse problem according to the actual mapping system. Results: We successfully observed the line of block using non-contact mapping in the model without the rapid propagation of excitation through the Purkinje network, although the excitation in the wall propagated smoothly. This model of slow conduction also reproduced the characteristic properties of the line of block, including dense isochronal lines and fractionated local electrocardiograms. Further, simulation of ventricular pacing from the lateral wall shifted the location of the line of block. By contrast, in the model with the Purkinje network, propagation of excitation in the endocardial map faithfully followed the actual propagation in the wall, without showing the line of block. Finally, switching the mode of propagation between the two models completely reversed these findings. Conclusions: Our simulation data suggest that the absence of rapid propagation of excitation through the Purkinje network is the major cause of the functional line

  18. Calbindin-D28k is a more reliable marker of human Purkinje cells than standard Nissl stains: a stereological experiment.

    PubMed

    Whitney, Elizabeth R; Kemper, Thomas L; Rosene, Douglas L; Bauman, Margaret L; Blatt, Gene J

    2008-02-15

    In a study of human Purkinje cell (PC) number, a striking mismatch between the number of PCs observed with the Nissl stain and the number of PCs immunopositive for calbindin-D28k (CB) was identified in 2 of the 10 brains examined. In the remaining eight brains this mismatch was not observed. Further, in these eight brains, analysis of CB immunostained sections counterstained with the Nissl stain revealed that more than 99% Nissl stained PCs were also immunopositive for CB. In contrast, in the two discordant brains, only 10-20% of CB immunopositive PCs were also identified with the Nissl stain. Although this finding was unexpected, a historical survey of the literature revealed that Spielmeyer [Spielmeyer W. Histopathologie des nervensystems. Julius Springer: Berlin; 1922. p. 56-79] described human cases with PCs that lacked the expected Nissl staining intensity, an important historical finding and critical issue when studying postmortem human brains. The reason for this failure in Nissl staining is not entirely clear, but it may result from premortem circumstances since it is not accounted for by postmortem delay or processing variables. Regardless of the exact cause, these observations suggest that Nissl staining may not be a reliable marker for PCs and that CB is an excellent alternative marker.

  19. Chemical ablation of the Purkinje system causes early termination and activation rate slowing of long-duration ventricular fibrillation in dogs.

    PubMed

    Dosdall, Derek J; Tabereaux, Paul B; Kim, Jong J; Walcott, Gregory P; Rogers, Jack M; Killingsworth, Cheryl R; Huang, Jian; Robertson, Peter G; Smith, William M; Ideker, Raymond E

    2008-08-01

    Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long-duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the ventricular endocardium was exposed and treated with Lugol solution (n = 6) or normal Tyrode solution as a control (n = 6). The left anterior papillary muscle endocardium was mapped with a 504-electrode (21 x 24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a six-electrode plunge needle on each side of the plaque. Ventricular fibrillation (VF) was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol-ablated hearts than in control hearts (4.9 +/- 1.5 vs. 9.2 +/- 3.2 min, P = 0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol-ablated and control hearts but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wave fronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wave front direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.

  20. Action potential processing in a detailed Purkinje cell model reveals a critical role for axonal compartmentalization

    PubMed Central

    Masoli, Stefano; Solinas, Sergio; D'Angelo, Egidio

    2015-01-01

    The Purkinje cell (PC) is among the most complex neurons in the brain and plays a critical role for cerebellar functioning. PCs operate as fast pacemakers modulated by synaptic inputs but can switch from simple spikes to complex bursts and, in some conditions, show bistability. In contrast to original works emphasizing dendritic Ca-dependent mechanisms, recent experiments have supported a primary role for axonal Na-dependent processing, which could effectively regulate spike generation and transmission to deep cerebellar nuclei (DCN). In order to account for the numerous ionic mechanisms involved (at present including Nav1.6, Cav2.1, Cav3.1, Cav3.2, Cav3.3, Kv1.1, Kv1.5, Kv3.3, Kv3.4, Kv4.3, KCa1.1, KCa2.2, KCa3.1, Kir2.x, HCN1), we have elaborated a multicompartmental model incorporating available knowledge on localization and gating of PC ionic channels. The axon, including initial segment (AIS) and Ranvier nodes (RNs), proved critical to obtain appropriate pacemaking and firing frequency modulation. Simple spikes initiated in the AIS and protracted discharges were stabilized in the soma through Na-dependent mechanisms, while somato-dendritic Ca channels contributed to sustain pacemaking and to generate complex bursting at high discharge regimes. Bistability occurred only following Na and Ca channel down-regulation. In addition, specific properties in RNs K currents were required to limit spike transmission frequency along the axon. The model showed how organized electroresponsive functions could emerge from the molecular complexity of PCs and showed that the axon is fundamental to complement ionic channel compartmentalization enabling action potential processing and transmission of specific spike patterns to DCN. PMID:25759640

  1. Inflammation-induced reversible switch of the neuron-specific enolase promoter from Purkinje neurons to Bergmann glia.

    PubMed

    Sawada, Yusuke; Konno, Ayumu; Nagaoka, Jun; Hirai, Hirokazu

    2016-06-13

    Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs.

  2. Segmentation of clustered cells in negative phase contrast images with integrated light intensity and cell shape information.

    PubMed

    Wang, Y; Wang, C; Zhang, Z

    2018-05-01

    Automated cell segmentation plays a key role in characterisations of cell behaviours for both biology research and clinical practices. Currently, the segmentation of clustered cells still remains as a challenge and is the main reason for false segmentation. In this study, the emphasis was put on the segmentation of clustered cells in negative phase contrast images. A new method was proposed to combine both light intensity and cell shape information through the construction of grey-weighted distance transform (GWDT) within preliminarily segmented areas. With the constructed GWDT, the clustered cells can be detected and then separated with a modified region skeleton-based method. Moreover, a contour expansion operation was applied to get optimised detection of cell boundaries. In this paper, the working principle and detailed procedure of the proposed method are described, followed by the evaluation of the method on clustered cell segmentation. Results show that the proposed method achieves an improved performance in clustered cell segmentation compared with other methods, with 85.8% and 97.16% accuracy rate for clustered cells and all cells, respectively. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  3. Clustering in Cell Cycle Dynamics with General Response/Signaling Feedback

    PubMed Central

    Young, Todd R.; Fernandez, Bastien; Buckalew, Richard; Moses, Gregory; Boczko, Erik M.

    2011-01-01

    Motivated by experimental and theoretical work on autonomous oscillations in yeast, we analyze ordinary differential equations models of large populations of cells with cell-cycle dependent feedback. We assume a particular type of feedback that we call Responsive/Signaling (RS), but do not specify a functional form of the feedback. We study the dynamics and emergent behaviour of solutions, particularly temporal clustering and stability of clustered solutions. We establish the existence of certain periodic clustered solutions as well as “uniform” solutions and add to the evidence that cell-cycle dependent feedback robustly leads to cell-cycle clustering. We highlight the fundamental differences in dynamics between systems with negative and positive feedback. For positive feedback systems the most important mechanism seems to be the stability of individual isolated clusters. On the other hand we find that in negative feedback systems, clusters must interact with each other to reinforce coherence. We conclude from various details of the mathematical analysis that negative feedback is most consistent with observations in yeast experiments. PMID:22001733

  4. Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

    PubMed

    Klisch, Karl; Schraner, Elisabeth M; Boos, Alois

    2017-01-01

    Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering. © 2016 S. Karger AG, Basel.

  5. Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell

    PubMed Central

    1985-01-01

    A microprocessor-controlled system of microinjections and microaspirations has been developed to change, within approximately 1 ms, the [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils (0.3-0.4 micron radius) of a skinned canine cardiac Purkinje cell (2.5-4.5 micron overall radius) at different phases of a Ca2+ transient. Simultaneously monitoring tension and aequorin bioluminescence provided two methods for estimating the peak myoplasmic [free Ca2+] reached during the spontaneous cyclic Ca2+ release from the SR obtained in the continuous presence of a bulk solution [free Ca2+] sufficiently high to overload the SR. These methods gave results in excellent agreement for the spontaneous Ca2+ release under a variety of conditions of pH and [free Mg2+], and of enhancement of Ca2+ release by calmodulin. Disagreement was observed, however, when the Ca2+ transient was modified during its ascending phase. The experiments also permitted quantification of the aequorin binding within the myofibrils and determination of its operational apparent affinity constant for Ca2+ at various [free Mg2+] levels. An increase of [free Ca2+] at the outer surface of the SR during the ascending phase of the Ca2+ transient induced further release of Ca2+. In contrast, an increase of [free Ca2+] during the descending phase of the Ca2+ transient did not cause further Ca2+ release. Varying [free H+], [free Mg2+], or the [Na+]/[K+] ratio had no significant effect on the Ca2+ transient during which the modification was applied, but it altered the subsequent Ca2+ transient. Therefore, Ca2+ appears to be the major, if not the only, ion controlling Ca2+ release from the SR rapidly enough to alter a Ca2+ transient during its course. PMID:3981128

  6. Purkinje cell-specific males absent on the first (mMof) gene deletion results in an ataxia-telangiectasia-like neurological phenotype and backward walking in mice

    PubMed Central

    Kumar, Rakesh; Hunt, Clayton R.; Gupta, Arun; Nannepaga, Suraj; Pandita, Raj K.; Shay, Jerry W.; Bachoo, Robert; Ludwig, Thomas; Burns, Dennis K.; Pandita, Tej K.

    2011-01-01

    The brains of ataxia telangiectasia (AT) patients display an aberrant loss of Purkinje cells (PCs) that is postulated to contribute to the observed deficits in motor coordination as well as in learning and cognitive function. AT patients have mutations in the ataxia telangiectasia mutated (ATM) gene [Savitsky et al. (1995) Science 268:1749–1753]. However, in Atm-deficient mice, the neurological defects are limited, and the PCs are not deformed or lost as observed in AT patients [Barlow et al. (1996) Cell 86:159–171]. Here we report that PC-specific deletion of the mouse males absent on the first (mMof) gene (Cre−), which encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac) and influences ATM function, is critical for PC longevity. Mice deficient for PC-specific Mof display impaired motor coordination, ataxia, a backward-walking phenotype, and a reduced life span. Treatment of MofF/F/Pcp2-Cre+ mice with histone deacetylase inhibitors modestly prolongs PC survival and delays death. Therefore, Mof expression and H4K16 acetylation are essential for PC survival and function, and their absence leads to PC loss and cerebellar dysfunction similar to that observed in AT patients. PMID:21321203

  7. Catheter ablation of fatal ventricular tachyarrhythmias storm in acute coronary syndrome--role of Purkinje fiber network.

    PubMed

    Enjoji, Yoshihisa; Mizobuchi, Masahiro; Muranishi, Hiromi; Miyamoto, Chinae; Utsunomiya, Makoto; Funatsu, Atsushi; Kobayashi, Tomoko; Nakamura, Shigeru

    2009-12-01

    Ventricular fibrillation (VF) or ventricular tachycardia (VT) storm is a life-threatening arrhythmia. Antiarrhythmic drugs (AADs) are not necessarily effective to rescue life from such conditions. Catheter ablation (CA) targeting triggering premature ventricular contractions (PVCs) of VF or VT that originates from Purkinje fiber network (PFN) is reported to be effective, especially in idiopathic patients. However, in condition of acute coronary syndrome (ACS), the efficacy of CA is not well understood. To clarify the usefulness of CA as an alternative way to AADs, we performed CA in four patients with VF or VT storm. The Purkinje potential was seen just before the myocardial ventricular wave during sinus rhythm that became more prominent and double components during the initiating PVC at the targeted area. Following CA, spontaneous episodes of VF or VT were no longer observed. CA is an efficacious way to bail out PFN-related VF or VT storm even in ACS.

  8. Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.

    PubMed

    Schweizer, Patrick A; Darche, Fabrice F; Ullrich, Nina D; Geschwill, Pascal; Greber, Boris; Rivinius, Rasmus; Seyler, Claudia; Müller-Decker, Karin; Draguhn, Andreas; Utikal, Jochen; Koenen, Michael; Katus, Hugo A; Thomas, Dierk

    2017-10-16

    Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca 2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized

  9. Acute hypoxia-induced alterations of calbindin-D28k immunoreactivity in cerebellar Purkinje cells of the guinea pig fetus at term.

    PubMed

    Katsetos, C D; Spandou, E; Legido, A; Taylor, M L; Zanelli, S A; de Chadarevian, J P; Christakos, S; Mishra, O P; Delivoria-Papadopoulos, M

    2001-05-01

    Purkinje cells (PCs) are vulnerable to hypoxic/ischemic insults and rich in calcium and calcium-buffering/sequestering systems, including calcium-binding proteins (CaBPs). Calbindin-D28k is an EF-hand CaBP, which is highly expressed in PCs where it acts primarily as a cellular Ca++ buffer. Elevation of [Ca++] in the cytosol and nuclei of PCs is pivotal in hypoxic/ischemic cell death. We hypothesize that hypoxia results in decreased concentration, or availability of calbindin-D28k in PCs, thereby decreasing their buffering capacity and resulting in increase of intracellular and intranuclear [Ca++]. Cerebellar tissues from normoxic fetuses were compared to fetuses obtained from term pregnant guinea pigs exposed to hypoxia [7% FiO2] for 60 min. The pregnant guinea pigs were either killed upon delivery immediately following hypoxia (Hx0h) or were subsequently allowed to recover for 24 h (Hx24h) or 72 h (Hx72h). Fetal brain hypoxia was documented biochemically by a decrease in brain tissue levels of ATP and phosphocreatine. Compared to normoxic fetuses, there is a predominantly somatodendritic loss or decrease of calbindin-D28k immunohistochemical staining in PCs of Hx0h (p < 0.005), Hx24h (p < 0.05), and Hx72h (p < 0.005) fetuses. Hypoxia-induced alterations of calbindin-D28k immunoreactivity are qualitatively similar at all time points and include a distinctive intranuclear localization in subpopulations of PCs. A similar trend is demonstrated by immunoblotting. Subpopulations of TUNEL+/calbindin-D28k- PCs lacking morphologic features of apoptosis or necrosis are demonstrated in Hx24h and Hx72h fetuses. The present study demonstrates an abrogating effect of perinatal hypoxia on calbindin-D28k immunoreactivity in cerebellar PCs. The perturbation of this Ca++ buffer protein in hypoxia-induced neuronal injury may herald delayed cell death or degeneration.

  10. Intractable Electrical Storm After Coronary Artery Bypass Grafting Originating in Abnormal Purkinje Fibers.

    PubMed

    Tokunaga, Chiho; Tsukada, Toru; Sakamoto, Hiroaki; Naruse, Yoshihisa; Yoshida, Kentaro; Sekiguchi, Yukio; Imai, Akito; Aonuma, Kazutaka; Hiramatsu, Yuji

    2016-01-01

    Electrical storm is a rare but critical complication following revascularization in patients with ischemic heart disease. We report the case of a 67-year-old man who developed drug refractory intractable electrical storm after emergent coronary artery bypass grafting for ischemic cardiomyopathy. The electrical storm was successfully eliminated by percutaneous endocardial radiofrequency catheter ablation targeting the abnormal Purkinje-related triggering ventricular premature contractions in a low-voltage zone. © 2015 Wiley Periodicals, Inc.

  11. Running and rotating: modelling the dynamics of migrating cell clusters

    NASA Astrophysics Data System (ADS)

    Copenhagen, Katherine; Gov, Nir; Gopinathan, Ajay

    Collective motion of cells is a common occurrence in many biological systems, including tissue development and repair, and tumor formation. Recent experiments have shown cells form clusters in a chemical gradient, which display three different phases of motion: translational, rotational, and random. We present a model for cell clusters based loosely on other models seen in the literature that involves a Vicsek-like alignment as well as physical collisions and adhesions between cells. With this model we show that a mechanism for driving rotational motion in this kind of system is an increased motility of rim cells. Further, we examine the details of the relationship between rim and core cells, and find that the phases of the cluster as a whole are correlated with the creation and annihilation of topological defects in the tangential component of the velocity field.

  12. [Measuring the effect of eyeglasses on determination of squint angle with Purkinje reflexes and the prism cover test].

    PubMed

    Barry, J C; Backes, A

    1998-04-01

    The alternating prism and cover test is the conventional test for the measurement of the angle of strabismus. The error induced by the prismatic effect of glasses is typically about 27-30%/10 D. Alternatively, the angle of strabismus can be measured with methods based on Purkinje reflex positions. This study examines the differences between three such options, taking into account the influence of glasses. The studied system comprised the eyes with or without glasses, a fixation object and a device for recording the eye position: in the case of the alternate prism and cover test, a prism bar was required; in the case of a Purkinje reflex based device, light sources for generation of reflexes and a camera for the documentation of the reflex positions were used. Measurements performed on model eyes and computer ray traces were used to analyze and compare the options. When a single corneal reflex is used, the misalignment of the corneal axis can be measured; the error in this measurement due to the prismatic effect of glasses was 7.6%/10 D, the smallest found in this study. The individual Hirschberg ratio can be determined by monocular measurements in three gaze directions. The angle of strabismus can be measured with Purkinje reflex based methods if the fundamental differences between these methods and the alternate prism and cover test, and if the influence of glasses and other sources of error are accounted for.

  13. A phase cell cluster expansion for Euclidean field theories

    NASA Astrophysics Data System (ADS)

    Battle, Guy A., III; Federbush, Paul

    1982-08-01

    We adapt the cluster expansion first used to treat infrared problems for lattice models (a mass zero cluster expansion) to the usual field theory situation. The field is expanded in terms of special block spin functions and the cluster expansion given in terms of the expansion coefficients (phase cell variables); the cluster expansion expresses correlation functions in terms of contributions from finite coupled subsets of these variables. Most of the present work is carried through in d space time dimensions (for φ24 the details of the cluster expansion are pursued and convergence is proven). Thus most of the results in the present work will apply to a treatment of φ34 to which we hope to return in a succeeding paper. Of particular interest in this paper is a substitute for the stability of the vacuum bound appropriate to this cluster expansion (for d = 2 and d = 3), and a new method for performing estimates with tree graphs. The phase cell cluster expansions have the renormalization group incorporated intimately into their structure. We hope they will be useful ultimately in treating four dimensional field theories.

  14. Mean-cluster approach indicates cell sorting time scales are determined by collective dynamics

    NASA Astrophysics Data System (ADS)

    Beatrici, Carine P.; de Almeida, Rita M. C.; Brunnet, Leonardo G.

    2017-03-01

    Cell migration is essential to cell segregation, playing a central role in tissue formation, wound healing, and tumor evolution. Considering random mixtures of two cell types, it is still not clear which cell characteristics define clustering time scales. The mass of diffusing clusters merging with one another is expected to grow as td /d +2 when the diffusion constant scales with the inverse of the cluster mass. Cell segregation experiments deviate from that behavior. Explanations for that could arise from specific microscopic mechanisms or from collective effects, typical of active matter. Here we consider a power law connecting diffusion constant and cluster mass to propose an analytic approach to model cell segregation where we explicitly take into account finite-size corrections. The results are compared with active matter model simulations and experiments available in the literature. To investigate the role played by different mechanisms we considered different hypotheses describing cell-cell interaction: differential adhesion hypothesis and different velocities hypothesis. We find that the simulations yield normal diffusion for long time intervals. Analytic and simulation results show that (i) cluster evolution clearly tends to a scaling regime, disrupted only at finite-size limits; (ii) cluster diffusion is greatly enhanced by cell collective behavior, such that for high enough tendency to follow the neighbors, cluster diffusion may become independent of cluster size; (iii) the scaling exponent for cluster growth depends only on the mass-diffusion relation, not on the detailed local segregation mechanism. These results apply for active matter systems in general and, in particular, the mechanisms found underlying the increase in cell sorting speed certainly have deep implications in biological evolution as a selection mechanism.

  15. Graph analysis of cell clusters forming vascular networks

    NASA Astrophysics Data System (ADS)

    Alves, A. P.; Mesquita, O. N.; Gómez-Gardeñes, J.; Agero, U.

    2018-03-01

    This manuscript describes the experimental observation of vasculogenesis in chick embryos by means of network analysis. The formation of the vascular network was observed in the area opaca of embryos from 40 to 55 h of development. In the area opaca endothelial cell clusters self-organize as a primitive and approximately regular network of capillaries. The process was observed by bright-field microscopy in control embryos and in embryos treated with Bevacizumab (Avastin), an antibody that inhibits the signalling of the vascular endothelial growth factor (VEGF). The sequence of images of the vascular growth were thresholded, and used to quantify the forming network in control and Avastin-treated embryos. This characterization is made by measuring vessels density, number of cell clusters and the largest cluster density. From the original images, the topology of the vascular network was extracted and characterized by means of the usual network metrics such as: the degree distribution, average clustering coefficient, average short path length and assortativity, among others. This analysis allows to monitor how the largest connected cluster of the vascular network evolves in time and provides with quantitative evidence of the disruptive effects that Avastin has on the tree structure of vascular networks.

  16. Unrepaired clustered DNA lesions induce chromosome breakage in human cells

    PubMed Central

    Asaithamby, Aroumougame; Hu, Burong; Chen, David J.

    2011-01-01

    Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis. PMID:21527720

  17. The histone deacetylase HDAC3 is essential for Purkinje cell function, potentially complicating the use of HDAC inhibitors in SCA1

    PubMed Central

    Venkatraman, Anand; Hu, Yuan-Shih; Didonna, Alessandro; Cvetanovic, Marija; Krbanjevic, Aleksandar; Bilesimo, Patrice; Opal, Puneet

    2014-01-01

    Spinocerebellar ataxia type 1 (SCA1) is an incurable neurodegenerative disease caused by a pathogenic glutamine repeat expansion in the protein ataxin-1 (ATXN1). One likely mechanism mediating pathogenesis is excessive transcriptional repression induced by the expanded ATXN-1. Because ATXN1 binds HDAC3, a Class I histone deacetylase (HDAC) that we have found to be required for ATXN1-induced transcriptional repression, we tested whether genetically depleting HDAC3 improves the phenotype of the SCA1 knock-in mouse (SCA1154Q/2Q), the most physiologically relevant model of SCA1. Given that HDAC3 null mice are embryonic lethal, we used for our analyses a combination of HDAC3 haploinsufficient and Purkinje cell (PC)-specific HDAC3 null mice. Although deleting a single allele of HDAC3 in the context of SCA1 was insufficient to improve cerebellar and cognitive deficits of the disease, a complete loss of PC HDAC3 was highly deleterious both behaviorally, with mice showing early onset ataxia, and pathologically, with progressive histologic evidence of degeneration. Inhibition of HDAC3 may yet have a role in SCA1 therapy, but our study provides cautionary evidence that this approach could produce untoward effects. Indeed, the neurotoxic consequences of HDAC3 depletion could prove relevant, wherever pharmacologic inhibition of HDAC3 is being contemplated, in disorders ranging from cancer to neurodegeneration. PMID:24594842

  18. Clustering of brain tumor cells: a first step for understanding tumor recurrence

    NASA Astrophysics Data System (ADS)

    Khain, Evgeniy; Nowicki, M. O.; Chiocca, E. A.; Lawler, S. E.; Schneider-Mizell, C. M.; Sander, L. M.

    2012-02-01

    Glioblastoma tumors are highly invasive; therefore the overall prognosis of patients remains poor, despite major improvements in treatment techniques. Cancer cells detach from the inner tumor core and actively migrate away [1]; eventually these invasive cells might form clusters, which can develop to recurrent tumors. In vitro experiments in collagen gel [1] followed the clustering dynamics of different glioma cell lines. Based on the experimental data, we formulated a stochastic model for cell dynamics, which identified two mechanisms of clustering. First, there is a critical value of the strength of adhesion; above the threshold, large clusters grow from a homogeneous suspension of cells; below it, the system remains homogeneous, similarly to the ordinary phase separation. Second, when cells form a cluster, there is evidence that their proliferation rate increases. We confirmed the theoretical predictions in a separate cell migration experiment on a substrate and found that both mechanisms are crucial for cluster formation and growth [2]. In addition to their medical importance, these phenomena present exciting examples of pattern formation and collective cell behavior in intrinsically non-equilibrium systems [3]. [4pt] [1] A. M. Stein et al, Biophys. J., 92, 356 (2007). [0pt] [2] E. Khain et al, EPL 88, 28006 (2009). [0pt] [3] E. Khain et al, Phys. Rev. E. 83, 031920 (2011).

  19. A microfluidic device for label-free, physical capture of circulating tumor cell-clusters

    PubMed Central

    Sarioglu, A. Fatih; Aceto, Nicola; Kojic, Nikola; Donaldson, Maria C.; Zeinali, Mahnaz; Hamza, Bashar; Engstrom, Amanda; Zhu, Huili; Sundaresan, Tilak K.; Miyamoto, David T.; Luo, Xi; Bardia, Aditya; Wittner, Ben S.; Ramaswamy, Sridhar; Shioda, Toshi; Ting, David T.; Stott, Shannon L.; Kapur, Ravi; Maheswaran, Shyamala; Haber, Daniel A.; Toner, Mehmet

    2015-01-01

    Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identify CTC-clusters in 30–40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. PMID:25984697

  20. Topological cell clustering in the ATLAS calorimeters and its performance in LHC Run 1

    NASA Astrophysics Data System (ADS)

    Aad, G.; Abbott, B.; Abdallah, J.; Abdinov, O.; Aben, R.; Abolins, M.; AbouZeid, O. S.; Abramowicz, H.; Abreu, H.; Abreu, R.; Abulaiti, Y.; Acharya, B. S.; Adamczyk, L.; Adams, D. L.; Adelman, J.; Adomeit, S.; Adye, T.; Affolder, A. A.; Agatonovic-Jovin, T.; Agricola, J.; Aguilar-Saavedra, J. A.; Ahlen, S. P.; Ahmadov, F.; Aielli, G.; Akerstedt, H.; Åkesson, T. P. A.; Akimov, A. V.; Alberghi, G. L.; Albert, J.; Albrand, S.; Verzini, M. J. Alconada; Aleksa, M.; Aleksandrov, I. N.; Alexa, C.; Alexander, G.; Alexopoulos, T.; Alhroob, M.; Alimonti, G.; Alio, L.; Alison, J.; Alkire, S. P.; Allbrooke, B. M. M.; Allport, P. P.; Aloisio, A.; Alonso, A.; Alonso, F.; Alpigiani, C.; Altheimer, A.; Gonzalez, B. Alvarez; Piqueras, D. Álvarez; Alviggi, M. G.; Amadio, B. T.; Amako, K.; Coutinho, Y. Amaral; Amelung, C.; Amidei, D.; Santos, S. P. Amor Dos; Amorim, A.; Amoroso, S.; Amram, N.; Amundsen, G.; Anastopoulos, C.; Ancu, L. S.; Andari, N.; Andeen, T.; Anders, C. F.; Anders, G.; Anders, J. K.; Anderson, K. J.; Andreazza, A.; Andrei, V.; Angelidakis, S.; Angelozzi, I.; Anger, P.; Angerami, A.; Anghinolfi, F.; Anisenkov, A. V.; Anjos, N.; Annovi, A.; Antonelli, M.; Antonov, A.; Antos, J.; Anulli, F.; Aoki, M.; Bella, L. Aperio; Arabidze, G.; Arai, Y.; Araque, J. P.; Arce, A. T. H.; Arduh, F. A.; Arguin, J.-F.; Argyropoulos, S.; Arik, M.; Armbruster, A. J.; Arnaez, O.; Arnold, H.; Arratia, M.; Arslan, O.; Artamonov, A.; Artoni, G.; Artz, S.; Asai, S.; Asbah, N.; Ashkenazi, A.; Åsman, B.; Asquith, L.; Assamagan, K.; Astalos, R.; Atkinson, M.; Atlay, N. B.; Augsten, K.; Aurousseau, M.; Avolio, G.; Axen, B.; Ayoub, M. K.; Azuelos, G.; Baak, M. A.; Baas, A. E.; Baca, M. J.; Bacci, C.; Bachacou, H.; Bachas, K.; Backes, M.; Backhaus, M.; Bagiacchi, P.; Bagnaia, P.; Bai, Y.; Bain, T.; Baines, J. T.; Baker, O. K.; Baldin, E. M.; Balek, P.; Balestri, T.; Balli, F.; Balunas, W. K.; Banas, E.; Banerjee, Sw.; Bannoura, A. A. E.; Barak, L.; Barberio, E. L.; Barberis, D.; Barbero, M.; Barillari, T.; Barisonzi, M.; Barklow, T.; Barlow, N.; Barnes, S. L.; Barnett, B. M.; Barnett, R. M.; Barnovska, Z.; Baroncelli, A.; Barone, G.; Barr, A. J.; Barreiro, F.; da Costa, J. Barreiro Guimarães; Bartoldus, R.; Barton, A. E.; Bartos, P.; Basalaev, A.; Bassalat, A.; Basye, A.; Bates, R. L.; Batista, S. J.; Batley, J. R.; Battaglia, M.; Bauce, M.; Bauer, F.; Bawa, H. S.; Beacham, J. B.; Beattie, M. D.; Beau, T.; Beauchemin, P. H.; Beccherle, R.; Bechtle, P.; Beck, H. P.; Becker, K.; Becker, M.; Beckingham, M.; Becot, C.; Beddall, A. J.; Beddall, A.; Bednyakov, V. A.; Bee, C. P.; Beemster, L. J.; Beermann, T. A.; Begel, M.; Behr, J. K.; Belanger-Champagne, C.; Bell, W. H.; Bella, G.; Bellagamba, L.; Bellerive, A.; Bellomo, M.; Belotskiy, K.; Beltramello, O.; Benary, O.; Benchekroun, D.; Bender, M.; Bendtz, K.; Benekos, N.; Benhammou, Y.; Noccioli, E. Benhar; Garcia, J. A. Benitez; Benjamin, D. P.; Bensinger, J. R.; Bentvelsen, S.; Beresford, L.; Beretta, M.; Berge, D.; Kuutmann, E. Bergeaas; Berger, N.; Berghaus, F.; Beringer, J.; Bernard, C.; Bernard, N. R.; Bernius, C.; Bernlochner, F. U.; Berry, T.; Berta, P.; Bertella, C.; Bertoli, G.; Bertolucci, F.; Bertsche, C.; Bertsche, D.; Besana, M. I.; Besjes, G. J.; Bylund, O. Bessidskaia; Bessner, M.; Besson, N.; Betancourt, C.; Bethke, S.; Bevan, A. J.; Bhimji, W.; Bianchi, R. M.; Bianchini, L.; Bianco, M.; Biebel, O.; Biedermann, D.; Biesuz, N. V.; Biglietti, M.; De Mendizabal, J. Bilbao; Bilokon, H.; Bindi, M.; Binet, S.; Bingul, A.; Bini, C.; Biondi, S.; Bjergaard, D. M.; Black, C. W.; Black, J. E.; Black, K. M.; Blackburn, D.; Blair, R. E.; Blanchard, J.-B.; Blanco, J. E.; Blazek, T.; Bloch, I.; Blocker, C.; Blum, W.; Blumenschein, U.; Blunier, S.; Bobbink, G. J.; Bobrovnikov, V. S.; Bocchetta, S. S.; Bocci, A.; Bock, C.; Boehler, M.; Bogaerts, J. A.; Bogavac, D.; Bogdanchikov, A. G.; Bohm, C.; Boisvert, V.; Bold, T.; Boldea, V.; Boldyrev, A. S.; Bomben, M.; Bona, M.; Boonekamp, M.; Borisov, A.; Borissov, G.; Borroni, S.; Bortfeldt, J.; Bortolotto, V.; Bos, K.; Boscherini, D.; Bosman, M.; Boudreau, J.; Bouffard, J.; Bouhova-Thacker, E. V.; Boumediene, D.; Bourdarios, C.; Bousson, N.; Boutle, S. K.; Boveia, A.; Boyd, J.; Boyko, I. R.; Bozic, I.; Bracinik, J.; Brandt, A.; Brandt, G.; Brandt, O.; Bratzler, U.; Brau, B.; Brau, J. E.; Braun, H. M.; Madden, W. D. Breaden; Brendlinger, K.; Brennan, A. J.; Brenner, L.; Brenner, R.; Bressler, S.; Bristow, T. M.; Britton, D.; Britzger, D.; Brochu, F. M.; Brock, I.; Brock, R.; Bronner, J.; Brooijmans, G.; Brooks, T.; Brooks, W. K.; Brosamer, J.; Brost, E.; de Renstrom, P. A. Bruckman; Bruncko, D.; Bruneliere, R.; Bruni, A.; Bruni, G.; Bruschi, M.; Bruscino, N.; Bryngemark, L.; Buanes, T.; Buat, Q.; Buchholz, P.; Buckley, A. G.; Budagov, I. A.; Buehrer, F.; Bugge, L.; Bugge, M. K.; Bulekov, O.; Bullock, D.; Burckhart, H.; Burdin, S.; Burgard, C. D.; Burghgrave, B.; Burke, S.; Burmeister, I.; Busato, E.; Büscher, D.; Büscher, V.; Bussey, P.; Butler, J. M.; Butt, A. I.; Buttar, C. M.; Butterworth, J. M.; Butti, P.; Buttinger, W.; Buzatu, A.; Buzykaev, A. R.; Urbán, S. Cabrera; Caforio, D.; Cairo, V. M.; Cakir, O.; Calace, N.; Calafiura, P.; Calandri, A.; Calderini, G.; Calfayan, P.; Caloba, L. P.; Calvet, D.; Calvet, S.; Toro, R. Camacho; Camarda, S.; Camarri, P.; Cameron, D.; Armadans, R. Caminal; Campana, S.; Campanelli, M.; Campoverde, A.; Canale, V.; Canepa, A.; Bret, M. Cano; Cantero, J.; Cantrill, R.; Cao, T.; Garrido, M. D. M. Capeans; Caprini, I.; Caprini, M.; Capua, M.; Caputo, R.; Carbone, R. M.; Cardarelli, R.; Cardillo, F.; Carli, T.; Carlino, G.; Carminati, L.; Caron, S.; Carquin, E.; Carrillo-Montoya, G. D.; Carter, J. R.; Carvalho, J.; Casadei, D.; Casado, M. P.; Casolino, M.; Casper, D. W.; Castaneda-Miranda, E.; Castelli, A.; Gimenez, V. Castillo; Castro, N. F.; Catastini, P.; Catinaccio, A.; Catmore, J. R.; Cattai, A.; Caudron, J.; Cavaliere, V.; Cavalli, D.; Cavalli-Sforza, M.; Cavasinni, V.; Ceradini, F.; Alberich, L. Cerda; Cerio, B. C.; Cerny, K.; Cerqueira, A. S.; Cerri, A.; Cerrito, L.; Cerutti, F.; Cerv, M.; Cervelli, A.; Cetin, S. A.; Chafaq, A.; Chakraborty, D.; Chalupkova, I.; Chan, Y. L.; Chang, P.; Chapman, J. D.; Charlton, D. G.; Chau, C. C.; Barajas, C. A. Chavez; Che, S.; Cheatham, S.; Chegwidden, A.; Chekanov, S.; Chekulaev, S. V.; Chelkov, G. A.; Chelstowska, M. A.; Chen, C.; Chen, H.; Chen, K.; Chen, L.; Chen, S.; Chen, S.; Chen, X.; Chen, Y.; Cheng, H. C.; Cheng, Y.; Cheplakov, A.; Cheremushkina, E.; Moursli, R. Cherkaoui El; Chernyatin, V.; Cheu, E.; Chevalier, L.; Chiarella, V.; Chiarelli, G.; Chiodini, G.; Chisholm, A. S.; Chislett, R. T.; Chitan, A.; Chizhov, M. V.; Choi, K.; Chouridou, S.; Chow, B. K. B.; Christodoulou, V.; Chromek-Burckhart, D.; Chudoba, J.; Chuinard, A. J.; Chwastowski, J. J.; Chytka, L.; Ciapetti, G.; Ciftci, A. K.; Cinca, D.; Cindro, V.; Cioara, I. A.; Ciocio, A.; Cirotto, F.; Citron, Z. H.; Ciubancan, M.; Clark, A.; Clark, B. L.; Clark, P. J.; Clarke, R. N.; Clement, C.; Coadou, Y.; Cobal, M.; Coccaro, A.; Cochran, J.; Coffey, L.; Cogan, J. G.; Colasurdo, L.; Cole, B.; Cole, S.; Colijn, A. P.; Collot, J.; Colombo, T.; Compostella, G.; Muiño, P. Conde; Coniavitis, E.; Connell, S. H.; Connelly, I. A.; Consorti, V.; Constantinescu, S.; Conta, C.; Conti, G.; Conventi, F.; Cooke, M.; Cooper, B. D.; Cooper-Sarkar, A. M.; Cornelissen, T.; Corradi, M.; Corriveau, F.; Corso-Radu, A.; Cortes-Gonzalez, A.; Cortiana, G.; Costa, G.; Costa, M. J.; Costanzo, D.; Côté, D.; Cottin, G.; Cowan, G.; Cox, B. E.; Cranmer, K.; Cree, G.; Crépé-Renaudin, S.; Crescioli, F.; Cribbs, W. A.; Ortuzar, M. Crispin; Cristinziani, M.; Croft, V.; Crosetti, G.; Donszelmann, T. Cuhadar; Cummings, J.; Curatolo, M.; Cúth, J.; Cuthbert, C.; Czirr, H.; Czodrowski, P.; D'Auria, S.; D'Onofrio, M.; De Sousa, M. J. Da Cunha Sargedas; Via, C. Da; Dabrowski, W.; Dafinca, A.; Dai, T.; Dale, O.; Dallaire, F.; Dallapiccola, C.; Dam, M.; Dandoy, J. R.; Dang, N. P.; Daniells, A. C.; Danninger, M.; Hoffmann, M. Dano; Dao, V.; Darbo, G.; Darmora, S.; Dassoulas, J.; Dattagupta, A.; Davey, W.; David, C.; Davidek, T.; Davies, E.; Davies, M.; Davison, P.; Davygora, Y.; Dawe, E.; Dawson, I.; Daya-Ishmukhametova, R. K.; De, K.; de Asmundis, R.; De Benedetti, A.; De Castro, S.; De Cecco, S.; De Groot, N.; de Jong, P.; De la Torre, H.; De Lorenzi, F.; De Pedis, D.; De Salvo, A.; De Sanctis, U.; De Santo, A.; De Regie, J. B. De Vivie; Dearnaley, W. J.; Debbe, R.; Debenedetti, C.; Dedovich, D. V.; Deigaard, I.; Del Peso, J.; Del Prete, T.; Delgove, D.; Deliot, F.; Delitzsch, C. M.; Deliyergiyev, M.; Dell'Acqua, A.; Dell'Asta, L.; Dell'Orso, M.; Della Pietra, M.; della Volpe, D.; Delmastro, M.; Delsart, P. A.; Deluca, C.; DeMarco, D. A.; Demers, S.; Demichev, M.; Demilly, A.; Denisov, S. P.; Derendarz, D.; Derkaoui, J. E.; Derue, F.; Dervan, P.; Desch, K.; Deterre, C.; Dette, K.; Deviveiros, P. O.; Dewhurst, A.; Dhaliwal, S.; Di Ciaccio, A.; Di Ciaccio, L.; Di Domenico, A.; Di Donato, C.; Di Girolamo, A.; Di Girolamo, B.; Di Mattia, A.; Di Micco, B.; Di Nardo, R.; Di Simone, A.; Di Sipio, R.; Di Valentino, D.; Diaconu, C.; Diamond, M.; Dias, F. A.; Diaz, M. A.; Diehl, E. B.; Dietrich, J.; Diglio, S.; Dimitrievska, A.; Dingfelder, J.; Dita, P.; Dita, S.; Dittus, F.; Djama, F.; Djobava, T.; Djuvsland, J. I.; do Vale, M. A. B.; Dobos, D.; Dobre, M.; Doglioni, C.; Dohmae, T.; Dolejsi, J.; Dolezal, Z.; Dolgoshein, B. A.; Donadelli, M.; Donati, S.; Dondero, P.; Donini, J.; Dopke, J.; Doria, A.; Dova, M. T.; Doyle, A. T.; Drechsler, E.; Dris, M.; Du, Y.; Dubreuil, E.; Duchovni, E.; Duckeck, G.; Ducu, O. A.; Duda, D.; Dudarev, A.; Duflot, L.; Duguid, L.; Dührssen, M.; Dunford, M.; Yildiz, H. Duran; Düren, M.; Durglishvili, A.; Duschinger, D.; Dutta, B.; Dyndal, M.; Eckardt, C.; Ecker, K. M.; Edgar, R. C.; Edson, W.; Edwards, N. C.; Ehrenfeld, W.; Eifert, T.; Eigen, G.; Einsweiler, K.; Ekelof, T.; Kacimi, M. El; Ellert, M.; Elles, S.; Ellinghaus, F.; Elliot, A. A.; Ellis, N.; Elmsheuser, J.; Elsing, M.; Emeliyanov, D.; Enari, Y.; Endner, O. C.; Endo, M.; Erdmann, J.; Ereditato, A.; Ernis, G.; Ernst, J.; Ernst, M.; Errede, S.; Ertel, E.; Escalier, M.; Esch, H.; Escobar, C.; Esposito, B.; Etienvre, A. I.; Etzion, E.; Evans, H.; Ezhilov, A.; Fabbri, L.; Facini, G.; Fakhrutdinov, R. M.; Falciano, S.; Falla, R. J.; Faltova, J.; Fang, Y.; Fanti, M.; Farbin, A.; Farilla, A.; Farooque, T.; Farrell, S.; Farrington, S. M.; Farthouat, P.; Fassi, F.; Fassnacht, P.; Fassouliotis, D.; Giannelli, M. Faucci; Favareto, A.; Fayard, L.; Fedin, O. L.; Fedorko, W.; Feigl, S.; Feligioni, L.; Feng, C.; Feng, E. J.; Feng, H.; Fenyuk, A. B.; Feremenga, L.; Martinez, P. Fernandez; Perez, S. Fernandez; Ferrando, J.; Ferrari, A.; Ferrari, P.; Ferrari, R.; de Lima, D. E. Ferreira; Ferrer, A.; Ferrere, D.; Ferretti, C.; Parodi, A. Ferretto; Fiascaris, M.; Fiedler, F.; Filipčič, A.; Filipuzzi, M.; Filthaut, F.; Fincke-Keeler, M.; Finelli, K. D.; Fiolhais, M. C. N.; Fiorini, L.; Firan, A.; Fischer, A.; Fischer, C.; Fischer, J.; Fisher, W. C.; Flaschel, N.; Fleck, I.; Fleischmann, P.; Fletcher, G. T.; Fletcher, G.; Fletcher, R. R. M.; Flick, T.; Floderus, A.; Castillo, L. R. Flores; Flowerdew, M. J.; Formica, A.; Forti, A.; Fournier, D.; Fox, H.; Fracchia, S.; Francavilla, P.; Franchini, M.; Francis, D.; Franconi, L.; Franklin, M.; Frate, M.; Fraternali, M.; Freeborn, D.; French, S. T.; Fressard-Batraneanu, S. M.; Friedrich, F.; Froidevaux, D.; Frost, J. A.; Fukunaga, C.; Torregrosa, E. Fullana; Fulsom, B. G.; Fusayasu, T.; Fuster, J.; Gabaldon, C.; Gabizon, O.; Gabrielli, A.; Gabrielli, A.; Gach, G. P.; Gadatsch, S.; Gadomski, S.; Gagliardi, G.; Gagnon, P.; Galea, C.; Galhardo, B.; Gallas, E. J.; Gallop, B. J.; Gallus, P.; Galster, G.; Gan, K. K.; Gao, J.; Gao, Y.; Gao, Y. S.; Walls, F. M. Garay; Garberson, F.; García, C.; Navarro, J. E. García; Garcia-Sciveres, M.; Gardner, R. W.; Garelli, N.; Garonne, V.; Gatti, C.; Gaudiello, A.; Gaudio, G.; Gaur, B.; Gauthier, L.; Gauzzi, P.; Gavrilenko, I. L.; Gay, C.; Gaycken, G.; Gazis, E. N.; Ge, P.; Gecse, Z.; Gee, C. N. P.; Geich-Gimbel, Ch.; Geisler, M. P.; Gemme, C.; Genest, M. H.; Geng, C.; Gentile, S.; George, M.; George, S.; Gerbaudo, D.; Gershon, A.; Ghasemi, S.; Ghazlane, H.; Giacobbe, B.; Giagu, S.; Giangiobbe, V.; Giannetti, P.; Gibbard, B.; Gibson, S. M.; Gignac, M.; Gilchriese, M.; Gillam, T. P. S.; Gillberg, D.; Gilles, G.; Gingrich, D. M.; Giokaris, N.; Giordani, M. P.; Giorgi, F. M.; Giorgi, F. M.; Giraud, P. F.; Giromini, P.; Giugni, D.; Giuliani, C.; Giulini, M.; Gjelsten, B. K.; Gkaitatzis, S.; Gkialas, I.; Gkougkousis, E. L.; Gladilin, L. K.; Glasman, C.; Glatzer, J.; Glaysher, P. C. F.; Glazov, A.; Goblirsch-Kolb, M.; Goddard, J. R.; Godlewski, J.; Goldfarb, S.; Golling, T.; Golubkov, D.; Gomes, A.; Gonçalo, R.; Costa, J. Goncalves Pinto Firmino Da; Gonella, L.; de la Hoz, S. González; Parra, G. Gonzalez; Gonzalez-Sevilla, S.; Goossens, L.; Gorbounov, P. A.; Gordon, H. A.; Gorelov, I.; Gorini, B.; Gorini, E.; Gorišek, A.; Gornicki, E.; Goshaw, A. T.; Gössling, C.; Gostkin, M. I.; Goujdami, D.; Goussiou, A. G.; Govender, N.; Gozani, E.; Grabas, H. M. X.; Graber, L.; Grabowska-Bold, I.; Gradin, P. O. J.; Grafström, P.; Gramling, J.; Gramstad, E.; Grancagnolo, S.; Gratchev, V.; Gray, H. M.; Graziani, E.; Greenwood, Z. D.; Grefe, C.; Gregersen, K.; Gregor, I. M.; Grenier, P.; Griffiths, J.; Grillo, A. A.; Grimm, K.; Grinstein, S.; Gris, Ph.; Grivaz, J.-F.; Groh, S.; Grohs, J. P.; Grohsjean, A.; Gross, E.; Grosse-Knetter, J.; Grossi, G. C.; Grout, Z. J.; Guan, L.; Guenther, J.; Guescini, F.; Guest, D.; Gueta, O.; Guido, E.; Guillemin, T.; Guindon, S.; Gul, U.; Gumpert, C.; Guo, J.; Guo, Y.; Gupta, S.; Gustavino, G.; Gutierrez, P.; Ortiz, N. G. Gutierrez; Gutschow, C.; Guyot, C.; Gwenlan, C.; Gwilliam, C. B.; Haas, A.; Haber, C.; Hadavand, H. K.; Haddad, N.; Haefner, P.; Hageböck, S.; Hajduk, Z.; Hakobyan, H.; Haleem, M.; Haley, J.; Hall, D.; Halladjian, G.; Hallewell, G. D.; Hamacher, K.; Hamal, P.; Hamano, K.; Hamilton, A.; Hamity, G. N.; Hamnett, P. G.; Han, L.; Hanagaki, K.; Hanawa, K.; Hance, M.; Haney, B.; Hanke, P.; Hanna, R.; Hansen, J. B.; Hansen, J. D.; Hansen, M. C.; Hansen, P. H.; Hara, K.; Hard, A. S.; Harenberg, T.; Hariri, F.; Harkusha, S.; Harrington, R. D.; Harrison, P. F.; Hartjes, F.; Hasegawa, M.; Hasegawa, Y.; Hasib, A.; Hassani, S.; Haug, S.; Hauser, R.; Hauswald, L.; Havranek, M.; Hawkes, C. M.; Hawkings, R. J.; Hawkins, A. D.; Hayashi, T.; Hayden, D.; Hays, C. P.; Hays, J. M.; Hayward, H. S.; Haywood, S. J.; Head, S. J.; Heck, T.; Hedberg, V.; Heelan, L.; Heim, S.; Heim, T.; Heinemann, B.; Heinrich, L.; Hejbal, J.; Helary, L.; Hellman, S.; Helsens, C.; Henderson, J.; Henderson, R. C. W.; Heng, Y.; Hengler, C.; Henkelmann, S.; Henrichs, A.; Correia, A. M. Henriques; Henrot-Versille, S.; Herbert, G. H.; Jiménez, Y. Hernández; Herten, G.; Hertenberger, R.; Hervas, L.; Hesketh, G. G.; Hessey, N. P.; Hetherly, J. W.; Hickling, R.; Higón-Rodriguez, E.; Hill, E.; Hill, J. C.; Hiller, K. H.; Hillier, S. J.; Hinchliffe, I.; Hines, E.; Hinman, R. R.; Hirose, M.; Hirschbuehl, D.; Hobbs, J.; Hod, N.; Hodgkinson, M. C.; Hodgson, P.; Hoecker, A.; Hoeferkamp, M. R.; Hoenig, F.; Hohlfeld, M.; Hohn, D.; Holmes, T. R.; Homann, M.; Hong, T. M.; Hopkins, W. H.; Horii, Y.; Horton, A. J.; Hostachy, J.-Y.; Hou, S.; Hoummada, A.; Howard, J.; Howarth, J.; Hrabovsky, M.; Hristova, I.; Hrivnac, J.; Hryn'ova, T.; Hrynevich, A.; Hsu, C.; Hsu, P. J.; Hsu, S.-C.; Hu, D.; Hu, Q.; Hu, X.; Huang, Y.; Hubacek, Z.; Hubaut, F.; Huegging, F.; Huffman, T. B.; Hughes, E. W.; Hughes, G.; Huhtinen, M.; Hülsing, T. A.; Huseynov, N.; Huston, J.; Huth, J.; Iacobucci, G.; Iakovidis, G.; Ibragimov, I.; Iconomidou-Fayard, L.; Ideal, E.; Idrissi, Z.; Iengo, P.; Igonkina, O.; Iizawa, T.; Ikegami, Y.; Ikeno, M.; Ilchenko, Y.; Iliadis, D.; Ilic, N.; Ince, T.; Introzzi, G.; Ioannou, P.; Iodice, M.; Iordanidou, K.; Ippolito, V.; Quiles, A. Irles; Isaksson, C.; Ishino, M.; Ishitsuka, M.; Ishmukhametov, R.; Issever, C.; Istin, S.; Ponce, J. M. Iturbe; Iuppa, R.; Ivarsson, J.; Iwanski, W.; Iwasaki, H.; Izen, J. M.; Izzo, V.; Jabbar, S.; Jackson, B.; Jackson, M.; Jackson, P.; Jaekel, M. R.; Jain, V.; Jakobi, K. B.; Jakobs, K.; Jakobsen, S.; Jakoubek, T.; Jakubek, J.; Jamin, D. O.; Jana, D. K.; Jansen, E.; Jansky, R.; Janssen, J.; Janus, M.; Jarlskog, G.; Javadov, N.; Javůrek, T.; Jeanty, L.; Jejelava, J.; Jeng, G.-Y.; Jennens, D.; Jenni, P.; Jentzsch, J.; Jeske, C.; Jézéquel, S.; Ji, H.; Jia, J.; Jiang, H.; Jiang, Y.; Jiggins, S.; Pena, J. Jimenez; Jin, S.; Jinaru, A.; Jinnouchi, O.; Joergensen, M. D.; Johansson, P.; Johns, K. A.; Johnson, W. J.; Jon-And, K.; Jones, G.; Jones, R. W. L.; Jones, T. J.; Jongmanns, J.; Jorge, P. M.; Joshi, K. D.; Jovicevic, J.; Ju, X.; Rozas, A. Juste; Kaci, M.; Kaczmarska, A.; Kado, M.; Kagan, H.; Kagan, M.; Kahn, S. J.; Kajomovitz, E.; Kalderon, C. W.; Kaluza, A.; Kama, S.; Kamenshchikov, A.; Kanaya, N.; Kaneti, S.; Kantserov, V. A.; Kanzaki, J.; Kaplan, B.; Kaplan, L. S.; Kapliy, A.; Kar, D.; Karakostas, K.; Karamaoun, A.; Karastathis, N.; Kareem, M. J.; Karentzos, E.; Karnevskiy, M.; Karpov, S. N.; Karpova, Z. M.; Karthik, K.; Kartvelishvili, V.; Karyukhin, A. N.; Kasahara, K.; Kashif, L.; Kass, R. D.; Kastanas, A.; Kataoka, Y.; Kato, C.; Katre, A.; Katzy, J.; Kawade, K.; Kawagoe, K.; Kawamoto, T.; Kawamura, G.; Kazama, S.; Kazanin, V. F.; Keeler, R.; Kehoe, R.; Keller, J. S.; Kempster, J. J.; Keoshkerian, H.; Kepka, O.; Kerševan, B. P.; Kersten, S.; Keyes, R. A.; Khalil-zada, F.; Khandanyan, H.; Khanov, A.; Kharlamov, A. G.; Khoo, T. J.; Khovanskiy, V.; Khramov, E.; Khubua, J.; Kido, S.; Kim, H. Y.; Kim, S. H.; Kim, Y. K.; Kimura, N.; Kind, O. M.; King, B. T.; King, M.; King, S. B.; Kirk, J.; Kiryunin, A. E.; Kishimoto, T.; Kisielewska, D.; Kiss, F.; Kiuchi, K.; Kivernyk, O.; Kladiva, E.; Klein, M. H.; Klein, M.; Klein, U.; Kleinknecht, K.; Klimek, P.; Klimentov, A.; Klingenberg, R.; Klinger, J. A.; Klioutchnikova, T.; Kluge, E.-E.; Kluit, P.; Kluth, S.; Knapik, J.; Kneringer, E.; Knoops, E. B. F. G.; Knue, A.; Kobayashi, A.; Kobayashi, D.; Kobayashi, T.; Kobel, M.; Kocian, M.; Kodys, P.; Koffas, T.; Koffeman, E.; Kogan, L. A.; Kohlmann, S.; Kohout, Z.; Kohriki, T.; Koi, T.; Kolanoski, H.; Kolb, M.; Koletsou, I.; Komar, A. A.; Komori, Y.; Kondo, T.; Kondrashova, N.; Köneke, K.; König, A. C.; Kono, T.; Konoplich, R.; Konstantinidis, N.; Kopeliansky, R.; Koperny, S.; Köpke, L.; Kopp, A. K.; Korcyl, K.; Kordas, K.; Korn, A.; Korol, A. A.; Korolkov, I.; Korolkova, E. V.; Kortner, O.; Kortner, S.; Kosek, T.; Kostyukhin, V. V.; Kotov, V. M.; Kotwal, A.; Kourkoumeli-Charalampidi, A.; Kourkoumelis, C.; Kouskoura, V.; Koutsman, A.; Kowalewski, R.; Kowalski, T. Z.; Kozanecki, W.; Kozhin, A. S.; Kramarenko, V. A.; Kramberger, G.; Krasnopevtsev, D.; Krasny, M. W.; Krasznahorkay, A.; Kraus, J. K.; Kravchenko, A.; Kreiss, S.; Kretz, M.; Kretzschmar, J.; Kreutzfeldt, K.; Krieger, P.; Krizka, K.; Kroeninger, K.; Kroha, H.; Kroll, J.; Kroseberg, J.; Krstic, J.; Kruchonak, U.; Krüger, H.; Krumnack, N.; Kruse, A.; Kruse, M. C.; Kruskal, M.; Kubota, T.; Kucuk, H.; Kuday, S.; Kuehn, S.; Kugel, A.; Kuger, F.; Kuhl, A.; Kuhl, T.; Kukhtin, V.; Kukla, R.; Kulchitsky, Y.; Kuleshov, S.; Kuna, M.; Kunigo, T.; Kupco, A.; Kurashige, H.; Kurochkin, Y. A.; Kus, V.; Kuwertz, E. S.; Kuze, M.; Kvita, J.; Kwan, T.; Kyriazopoulos, D.; Rosa, A. La; Navarro, J. L. La Rosa; Rotonda, L. La; Lacasta, C.; Lacava, F.; Lacey, J.; Lacker, H.; Lacour, D.; Lacuesta, V. R.; Ladygin, E.; Lafaye, R.; Laforge, B.; Lagouri, T.; Lai, S.; Lambourne, L.; Lammers, S.; Lampen, C. L.; Lampl, W.; Lançon, E.; Landgraf, U.; Landon, M. P. J.; Lang, V. S.; Lange, J. C.; Lankford, A. J.; Lanni, F.; Lantzsch, K.; Lanza, A.; Laplace, S.; Lapoire, C.; Laporte, J. F.; Lari, T.; Manghi, F. Lasagni; Lassnig, M.; Laurelli, P.; Lavrijsen, W.; Law, A. T.; Laycock, P.; Lazovich, T.; Dortz, O. Le; Guirriec, E. Le; Menedeu, E. Le; LeBlanc, M.; LeCompte, T.; Ledroit-Guillon, F.; Lee, C. A.; Lee, S. C.; Lee, L.; Lefebvre, G.; Lefebvre, M.; Legger, F.; Leggett, C.; Lehan, A.; Miotto, G. Lehmann; Lei, X.; Leight, W. A.; Leisos, A.; Leister, A. G.; Leite, M. A. L.; Leitner, R.; Lellouch, D.; Lemmer, B.; Leney, K. J. C.; Lenz, T.; Lenzi, B.; Leone, R.; Leone, S.; Leonidopoulos, C.; Leontsinis, S.; Leroy, C.; Lester, C. G.; Levchenko, M.; Levêque, J.; Levin, D.; Levinson, L. J.; Levy, M.; Lewis, A.; Leyko, A. M.; Leyton, M.; Li, B.; Li, H.; Li, H. L.; Li, L.; Li, L.; Li, S.; Li, X.; Li, Y.; Liang, Z.; Liao, H.; Liberti, B.; Liblong, A.; Lichard, P.; Lie, K.; Liebal, J.; Liebig, W.; Limbach, C.; Limosani, A.; Lin, S. C.; Lin, T. H.; Linde, F.; Lindquist, B. E.; Linnemann, J. T.; Lipeles, E.; Lipniacka, A.; Lisovyi, M.; Liss, T. M.; Lissauer, D.; Lister, A.; Litke, A. M.; Liu, B.; Liu, D.; Liu, H.; Liu, J.; Liu, J. B.; Liu, K.; Liu, L.; Liu, M.; Liu, M.; Liu, Y.; Livan, M.; Lleres, A.; Merino, J. Llorente; Lloyd, S. L.; Sterzo, F. Lo; Lobodzinska, E.; Loch, P.; Lockman, W. S.; Loebinger, F. K.; Loevschall-Jensen, A. E.; Loew, K. M.; Loginov, A.; Lohse, T.; Lohwasser, K.; Lokajicek, M.; Long, B. A.; Long, J. D.; Long, R. E.; Looper, K. A.; Lopes, L.; Mateos, D. Lopez; Paredes, B. Lopez; Paz, I. Lopez; Lorenz, J.; Martinez, N. Lorenzo; Losada, M.; Lösel, P. J.; Lou, X.; Lounis, A.; Love, J.; Love, P. A.; Lu, H.; Lu, N.; Lubatti, H. J.; Luci, C.; Lucotte, A.; Luedtke, C.; Luehring, F.; Lukas, W.; Luminari, L.; Lundberg, O.; Lund-Jensen, B.; Lynn, D.; Lysak, R.; Lytken, E.; Ma, H.; Ma, L. L.; Maccarrone, G.; Macchiolo, A.; Macdonald, C. M.; Maček, B.; Miguens, J. Machado; Macina, D.; Madaffari, D.; Madar, R.; Maddocks, H. J.; Mader, W. F.; Madsen, A.; Maeda, J.; Maeland, S.; Maeno, T.; Maevskiy, A.; Magradze, E.; Mahboubi, K.; Mahlstedt, J.; Maiani, C.; Maidantchik, C.; Maier, A. A.; Maier, T.; Maio, A.; Majewski, S.; Makida, Y.; Makovec, N.; Malaescu, B.; Malecki, Pa.; Maleev, V. P.; Malek, F.; Mallik, U.; Malon, D.; Malone, C.; Maltezos, S.; Malyshev, V. M.; Malyukov, S.; Mamuzic, J.; Mancini, G.; Mandelli, B.; Mandelli, L.; Mandić, I.; Mandrysch, R.; Maneira, J.; Filho, L. Manhaes de Andrade; Ramos, J. Manjarres; Mann, A.; Manousakis-Katsikakis, A.; Mansoulie, B.; Mantifel, R.; Mantoani, M.; Mapelli, L.; March, L.; Marchiori, G.; Marcisovsky, M.; Marino, C. P.; Marjanovic, M.; Marley, D. E.; Marroquim, F.; Marsden, S. P.; Marshall, Z.; Marti, L. F.; Marti-Garcia, S.; Martin, B.; Martin, T. A.; Martin, V. J.; Latour, B. Martin dit; Martinez, M.; Martin-Haugh, S.; Martoiu, V. S.; Martyniuk, A. C.; Marx, M.; Marzano, F.; Marzin, A.; Masetti, L.; Mashimo, T.; Mashinistov, R.; Masik, J.; Maslennikov, A. L.; Massa, I.; Massa, L.; Mastrandrea, P.; Mastroberardino, A.; Masubuchi, T.; Mättig, P.; Mattmann, J.; Maurer, J.; Maxfield, S. J.; Maximov, D. A.; Mazini, R.; Mazza, S. M.; Goldrick, G. Mc; Kee, S. P. Mc; McCarn, A.; McCarthy, R. L.; McCarthy, T. G.; McCubbin, N. A.; McFarlane, K. W.; Mcfayden, J. A.; Mchedlidze, G.; McMahon, S. J.; McPherson, R. A.; Medinnis, M.; Meehan, S.; Mehlhase, S.; Mehta, A.; Meier, K.; Meineck, C.; Meirose, B.; Garcia, B. R. Mellado; Meloni, F.; Mengarelli, A.; Menke, S.; Meoni, E.; Mercurio, K. M.; Mergelmeyer, S.; Mermod, P.; Merola, L.; Meroni, C.; Merritt, F. S.; Messina, A.; Metcalfe, J.; Mete, A. S.; Meyer, C.; Meyer, C.; Meyer, J.-P.; Meyer, J.; Theenhausen, H. Meyer Zu; Middleton, R. P.; Miglioranzi, S.; Mijović, L.; Mikenberg, G.; Mikestikova, M.; Mikuž, M.; Milesi, M.; Milic, A.; Miller, D. W.; Mills, C.; Milov, A.; Milstead, D. A.; Minaenko, A. A.; Minami, Y.; Minashvili, I. A.; Mincer, A. I.; Mindur, B.; Mineev, M.; Ming, Y.; Mir, L. M.; Mistry, K. P.; Mitani, T.; Mitrevski, J.; Mitsou, V. A.; Miucci, A.; Miyagawa, P. S.; Mjörnmark, J. U.; Moa, T.; Mochizuki, K.; Mohapatra, S.; Mohr, W.; Molander, S.; Moles-Valls, R.; Monden, R.; Mondragon, M. C.; Mönig, K.; Monini, C.; Monk, J.; Monnier, E.; Montalbano, A.; Berlingen, J. Montejo; Monticelli, F.; Monzani, S.; Moore, R. W.; Morange, N.; Moreno, D.; Llácer, M. Moreno; Morettini, P.; Mori, D.; Mori, T.; Morii, M.; Morinaga, M.; Morisbak, V.; Moritz, S.; Morley, A. K.; Mornacchi, G.; Morris, J. D.; Mortensen, S. S.; Morton, A.; Morvaj, L.; Mosidze, M.; Moss, J.; Motohashi, K.; Mount, R.; Mountricha, E.; Mouraviev, S. V.; Moyse, E. J. W.; Muanza, S.; Mudd, R. D.; Mueller, F.; Mueller, J.; Mueller, R. S. P.; Mueller, T.; Muenstermann, D.; Mullen, P.; Mullier, G. A.; Sanchez, F. J. Munoz; Quijada, J. A. Murillo; Murray, W. J.; Musheghyan, H.; Musto, E.; Myagkov, A. G.; Myska, M.; Nachman, B. P.; Nackenhorst, O.; Nadal, J.; Nagai, K.; Nagai, R.; Nagai, Y.; Nagano, K.; Nagarkar, A.; Nagasaka, Y.; Nagata, K.; Nagel, M.; Nagy, E.; Nairz, A. M.; Nakahama, Y.; Nakamura, K.; Nakamura, T.; Nakano, I.; Namasivayam, H.; Garcia, R. F. Naranjo; Narayan, R.; Villar, D. I. Narrias; Naumann, T.; Navarro, G.; Nayyar, R.; Neal, H. A.; Nechaeva, P. Yu.; Neep, T. J.; Nef, P. D.; Negri, A.; Negrini, M.; Nektarijevic, S.; Nellist, C.; Nelson, A.; Nemecek, S.; Nemethy, P.; Nepomuceno, A. A.; Nessi, M.; Neubauer, M. S.; Neumann, M.; Neves, R. M.; Nevski, P.; Newman, P. R.; Nguyen, D. H.; Nickerson, R. B.; Nicolaidou, R.; Nicquevert, B.; Nielsen, J.; Nikiforou, N.; Nikiforov, A.; Nikolaenko, V.; Nikolic-Audit, I.; Nikolopoulos, K.; Nilsen, J. K.; Nilsson, P.; Ninomiya, Y.; Nisati, A.; Nisius, R.; Nobe, T.; Nodulman, L.; Nomachi, M.; Nomidis, I.; Nooney, T.; Norberg, S.; Nordberg, M.; Novgorodova, O.; Nowak, S.; Nozaki, M.; Nozka, L.; Ntekas, K.; Hanninger, G. Nunes; Nunnemann, T.; Nurse, E.; Nuti, F.; O'grady, F.; O'Neil, D. C.; O'Shea, V.; Oakham, F. G.; Oberlack, H.; Obermann, T.; Ocariz, J.; Ochi, A.; Ochoa, I.; Ochoa-Ricoux, J. P.; Oda, S.; Odaka, S.; Ogren, H.; Oh, A.; Oh, S. H.; Ohm, C. C.; Ohman, H.; Oide, H.; Okamura, W.; Okawa, H.; Okumura, Y.; Okuyama, T.; Olariu, A.; Pino, S. A. Olivares; Damazio, D. Oliveira; Olszewski, A.; Olszowska, J.; Onofre, A.; Onogi, K.; Onyisi, P. U. E.; Oram, C. J.; Oreglia, M. J.; Oren, Y.; Orestano, D.; Orlando, N.; Barrera, C. Oropeza; Orr, R. S.; Osculati, B.; Ospanov, R.; Garzon, G. Otero y.; Otono, H.; Ouchrif, M.; Ould-Saada, F.; Ouraou, A.; Oussoren, K. P.; Ouyang, Q.; Ovcharova, A.; Owen, M.; Owen, R. E.; Ozcan, V. E.; Ozturk, N.; Pachal, K.; Pages, A. Pacheco; Aranda, C. Padilla; Pagáčová, M.; Griso, S. Pagan; Paganis, E.; Paige, F.; Pais, P.; Pajchel, K.; Palacino, G.; Palestini, S.; Palka, M.; Pallin, D.; Palma, A.; Pan, Y. B.; Panagiotopoulou, E. St.; Pandini, C. E.; Vazquez, J. G. Panduro; Pani, P.; Panitkin, S.; Pantea, D.; Paolozzi, L.; Papadopoulou, Th. D.; Papageorgiou, K.; Paramonov, A.; Hernandez, D. Paredes; Parker, M. A.; Parker, K. A.; Parodi, F.; Parsons, J. A.; Parzefall, U.; Pasqualucci, E.; Passaggio, S.; Pastore, F.; Pastore, Fr.; Pásztor, G.; Pataraia, S.; Patel, N. D.; Pater, J. R.; Pauly, T.; Pearce, J.; Pearson, B.; Pedersen, L. E.; Pedersen, M.; Lopez, S. Pedraza; Pedro, R.; Peleganchuk, S. V.; Pelikan, D.; Penc, O.; Peng, C.; Peng, H.; Penning, B.; Penwell, J.; Perepelitsa, D. V.; Codina, E. Perez; García-Estañ, M. T. Pérez; Perini, L.; Pernegger, H.; Perrella, S.; Peschke, R.; Peshekhonov, V. D.; Peters, K.; Peters, R. F. Y.; Petersen, B. A.; Petersen, T. C.; Petit, E.; Petridis, A.; Petridou, C.; Petroff, P.; Petrolo, E.; Petrucci, F.; Pettersson, N. E.; Pezoa, R.; Phillips, P. W.; Piacquadio, G.; Pianori, E.; Picazio, A.; Piccaro, E.; Piccinini, M.; Pickering, M. A.; Piegaia, R.; Pignotti, D. T.; Pilcher, J. E.; Pilkington, A. D.; Pin, A. W. J.; Pina, J.; Pinamonti, M.; Pinfold, J. L.; Pingel, A.; Pires, S.; Pirumov, H.; Pitt, M.; Pizio, C.; Plazak, L.; Pleier, M.-A.; Pleskot, V.; Plotnikova, E.; Plucinski, P.; Pluth, D.; Poettgen, R.; Poggioli, L.; Pohl, D.; Polesello, G.; Poley, A.; Policicchio, A.; Polifka, R.; Polini, A.; Pollard, C. S.; Polychronakos, V.; Pommès, K.; Pontecorvo, L.; Pope, B. G.; Popeneciu, G. A.; Popovic, D. S.; Poppleton, A.; Pospisil, S.; Potamianos, K.; Potrap, I. N.; Potter, C. J.; Potter, C. T.; Poulard, G.; Poveda, J.; Pozdnyakov, V.; Astigarraga, M. E. Pozo; Pralavorio, P.; Pranko, A.; Prasad, S.; Prell, S.; Price, D.; Price, L. E.; Primavera, M.; Prince, S.; Proissl, M.; Prokofiev, K.; Prokoshin, F.; Protopapadaki, E.; Protopopescu, S.; Proudfoot, J.; Przybycien, M.; Ptacek, E.; Puddu, D.; Pueschel, E.; Puldon, D.; Purohit, M.; Puzo, P.; Qian, J.; Qin, G.; Qin, Y.; Quadt, A.; Quarrie, D. R.; Quayle, W. B.; Queitsch-Maitland, M.; Quilty, D.; Raddum, S.; Radeka, V.; Radescu, V.; Radhakrishnan, S. K.; Radloff, P.; Rados, P.; Ragusa, F.; Rahal, G.; Rajagopalan, S.; Rammensee, M.; Rangel-Smith, C.; Rauscher, F.; Rave, S.; Ravenscroft, T.; Raymond, M.; Read, A. L.; Readioff, N. P.; Rebuzzi, D. M.; Redelbach, A.; Redlinger, G.; Reece, R.; Reeves, K.; Rehnisch, L.; Reichert, J.; Reisin, H.; Rembser, C.; Ren, H.; Renaud, A.; Rescigno, M.; Resconi, S.; Rezanova, O. L.; Reznicek, P.; Rezvani, R.; Richter, R.; Richter, S.; Richter-Was, E.; Ricken, O.; Ridel, M.; Rieck, P.; Riegel, C. J.; Rieger, J.; Rifki, O.; Rijssenbeek, M.; Rimoldi, A.; Rinaldi, L.; Ristić, B.; Ritsch, E.; Riu, I.; Rizatdinova, F.; Rizvi, E.; Robertson, S. H.; Robichaud-Veronneau, A.; Robinson, D.; Robinson, J. E. M.; Robson, A.; Roda, C.; Roe, S.; Røhne, O.; Romaniouk, A.; Romano, M.; Saez, S. M. Romano; Adam, E. Romero; Rompotis, N.; Ronzani, M.; Roos, L.; Ros, E.; Rosati, S.; Rosbach, K.; Rose, P.; Rosenthal, O.; Rossetti, V.; Rossi, E.; Rossi, L. P.; Rosten, J. H. N.; Rosten, R.; Rotaru, M.; Roth, I.; Rothberg, J.; Rousseau, D.; Royon, C. R.; Rozanov, A.; Rozen, Y.; Ruan, X.; Rubbo, F.; Rubinskiy, I.; Rud, V. I.; Rudolph, C.; Rudolph, M. S.; Rühr, F.; Ruiz-Martinez, A.; Rurikova, Z.; Rusakovich, N. A.; Ruschke, A.; Russell, H. L.; Rutherfoord, J. P.; Ruthmann, N.; Ryabov, Y. F.; Rybar, M.; Rybkin, G.; Ryder, N. C.; Ryzhov, A.; Saavedra, A. F.; Sabato, G.; Sacerdoti, S.; Saddique, A.; Sadrozinski, H. F.-W.; Sadykov, R.; Tehrani, F. Safai; Saha, P.; Sahinsoy, M.; Saimpert, M.; Saito, T.; Sakamoto, H.; Sakurai, Y.; Salamanna, G.; Salamon, A.; Loyola, J. E. Salazar; Saleem, M.; Salek, D.; De Bruin, P. H. Sales; Salihagic, D.; Salnikov, A.; Salt, J.; Salvatore, D.; Salvatore, F.; Salvucci, A.; Salzburger, A.; Sammel, D.; Sampsonidis, D.; Sanchez, A.; Sánchez, J.; Martinez, V. Sanchez; Sandaker, H.; Sandbach, R. L.; Sander, H. G.; Sanders, M. P.; Sandhoff, M.; Sandoval, C.; Sandstroem, R.; Sankey, D. P. C.; Sannino, M.; Sansoni, A.; Santoni, C.; Santonico, R.; Santos, H.; Castillo, I. Santoyo; Sapp, K.; Sapronov, A.; Saraiva, J. G.; Sarrazin, B.; Sasaki, O.; Sasaki, Y.; Sato, K.; Sauvage, G.; Sauvan, E.; Savage, G.; Savard, P.; Sawyer, C.; Sawyer, L.; Saxon, J.; Sbarra, C.; Sbrizzi, A.; Scanlon, T.; Scannicchio, D. A.; Scarcella, M.; Scarfone, V.; Schaarschmidt, J.; Schacht, P.; Schaefer, D.; Schaefer, R.; Schaeffer, J.; Schaepe, S.; Schaetzel, S.; Schäfer, U.; Schaffer, A. C.; Schaile, D.; Schamberger, R. D.; Scharf, V.; Schegelsky, V. A.; Scheirich, D.; Schernau, M.; Schiavi, C.; Schillo, C.; Schioppa, M.; Schlenker, S.; Schmieden, K.; Schmitt, C.; Schmitt, S.; Schmitt, S.; Schmitz, S.; Schneider, B.; Schnellbach, Y. J.; Schnoor, U.; Schoeffel, L.; Schoening, A.; Schoenrock, B. D.; Schopf, E.; Schorlemmer, A. L. S.; Schott, M.; Schouten, D.; Schovancova, J.; Schramm, S.; Schreyer, M.; Schuh, N.; Schultens, M. J.; Schultz-Coulon, H.-C.; Schulz, H.; Schumacher, M.; Schumm, B. A.; Schune, Ph.; Schwanenberger, C.; Schwartzman, A.; Schwarz, T. A.; Schwegler, Ph.; Schweiger, H.; Schwemling, Ph.; Schwienhorst, R.; Schwindling, J.; Schwindt, T.; Scifo, E.; Sciolla, G.; Scuri, F.; Scutti, F.; Searcy, J.; Sedov, G.; Sedykh, E.; Seema, P.; Seidel, S. C.; Seiden, A.; Seifert, F.; Seixas, J. M.; Sekhniaidze, G.; Sekhon, K.; Sekula, S. J.; Seliverstov, D. M.; Semprini-Cesari, N.; Serfon, C.; Serin, L.; Serkin, L.; Serre, T.; Sessa, M.; Seuster, R.; Severini, H.; Sfiligoj, T.; Sforza, F.; Sfyrla, A.; Shabalina, E.; Shamim, M.; Shan, L. Y.; Shang, R.; Shank, J. T.; Shapiro, M.; Shatalov, P. B.; Shaw, K.; Shaw, S. M.; Shcherbakova, A.; Shehu, C. Y.; Sherwood, P.; Shi, L.; Shimizu, S.; Shimmin, C. O.; Shimojima, M.; Shiyakova, M.; Shmeleva, A.; Saadi, D. Shoaleh; Shochet, M. J.; Shojaii, S.; Shrestha, S.; Shulga, E.; Shupe, M. A.; Sicho, P.; Sidebo, P. E.; Sidiropoulou, O.; Sidorov, D.; Sidoti, A.; Siegert, F.; Sijacki, Dj.; Silva, J.; Silver, Y.; Silverstein, S. B.; Simak, V.; Simard, O.; Simic, Lj.; Simion, S.; Simioni, E.; Simmons, B.; Simon, D.; Simon, M.; Sinervo, P.; Sinev, N. B.; Sioli, M.; Siragusa, G.; Sisakyan, A. N.; Sivoklokov, S. Yu.; Sjölin, J.; Sjursen, T. B.; Skinner, M. B.; Skottowe, H. P.; Skubic, P.; Slater, M.; Slavicek, T.; Slawinska, M.; Sliwa, K.; Smakhtin, V.; Smart, B. H.; Smestad, L.; Smirnov, S. Yu.; Smirnov, Y.; Smirnova, L. N.; Smirnova, O.; Smith, M. N. K.; Smith, R. W.; Smizanska, M.; Smolek, K.; Snesarev, A. A.; Snidero, G.; Snyder, S.; Sobie, R.; Socher, F.; Soffer, A.; Soh, D. A.; Sokhrannyi, G.; Sanchez, C. A. Solans; Solar, M.; Solc, J.; Soldatov, E. Yu.; Soldevila, U.; Solodkov, A. A.; Soloshenko, A.; Solovyanov, O. V.; Solovyev, V.; Sommer, P.; Song, H. Y.; Soni, N.; Sood, A.; Sopczak, A.; Sopko, B.; Sopko, V.; Sorin, V.; Sosa, D.; Sosebee, M.; Sotiropoulou, C. L.; Soualah, R.; Soukharev, A. M.; South, D.; Sowden, B. C.; Spagnolo, S.; Spalla, M.; Spangenberg, M.; Spanò, F.; Spearman, W. R.; Sperlich, D.; Spettel, F.; Spighi, R.; Spigo, G.; Spiller, L. A.; Spousta, M.; Denis, R. D. St.; Stabile, A.; Staerz, S.; Stahlman, J.; Stamen, R.; Stamm, S.; Stanecka, E.; Stanek, R. W.; Stanescu, C.; Stanescu-Bellu, M.; Stanitzki, M. M.; Stapnes, S.; Starchenko, E. A.; Stark, J.; Staroba, P.; Starovoitov, P.; Staszewski, R.; Steinberg, P.; Stelzer, B.; Stelzer, H. J.; Stelzer-Chilton, O.; Stenzel, H.; Stewart, G. A.; Stillings, J. A.; Stockton, M. C.; Stoebe, M.; Stoicea, G.; Stolte, P.; Stonjek, S.; Stradling, A. R.; Straessner, A.; Stramaglia, M. E.; Strandberg, J.; Strandberg, S.; Strandlie, A.; Strauss, E.; Strauss, M.; Strizenec, P.; Ströhmer, R.; Strom, D. M.; Stroynowski, R.; Strubig, A.; Stucci, S. A.; Stugu, B.; Styles, N. A.; Su, D.; Su, J.; Subramaniam, R.; Succurro, A.; Suchek, S.; Sugaya, Y.; Suk, M.; Sulin, V. V.; Sultansoy, S.; Sumida, T.; Sun, S.; Sun, X.; Sundermann, J. E.; Suruliz, K.; Susinno, G.; Sutton, M. R.; Suzuki, S.; Svatos, M.; Swiatlowski, M.; Sykora, I.; Sykora, T.; Ta, D.; Taccini, C.; Tackmann, K.; Taenzer, J.; Taffard, A.; Tafirout, R.; Taiblum, N.; Takai, H.; Takashima, R.; Takeda, H.; Takeshita, T.; Takubo, Y.; Talby, M.; Talyshev, A. A.; Tam, J. Y. C.; Tan, K. G.; Tanaka, J.; Tanaka, R.; Tanaka, S.; Tannenwald, B. B.; Araya, S. Tapia; Tapprogge, S.; Tarem, S.; Tarrade, F.; Tartarelli, G. F.; Tas, P.; Tasevsky, M.; Tashiro, T.; Tassi, E.; Delgado, A. Tavares; Tayalati, Y.; Taylor, A. C.; Taylor, F. E.; Taylor, G. N.; Taylor, P. T. E.; Taylor, W.; Teischinger, F. A.; Teixeira-Dias, P.; Temming, K. K.; Temple, D.; Kate, H. Ten; Teng, P. K.; Teoh, J. J.; Tepel, F.; Terada, S.; Terashi, K.; Terron, J.; Terzo, S.; Testa, M.; Teuscher, R. J.; Theveneaux-Pelzer, T.; Thomas, J. P.; Thomas-Wilsker, J.; Thompson, E. N.; Thompson, P. D.; Thompson, R. J.; Thompson, A. S.; Thomsen, L. A.; Thomson, E.; Thomson, M.; Thun, R. P.; Tibbetts, M. J.; Torres, R. E. Ticse; Tikhomirov, V. O.; Tikhonov, Yu. A.; Timoshenko, S.; Tiouchichine, E.; Tipton, P.; Tisserant, S.; Todome, K.; Todorov, T.; Todorova-Nova, S.; Tojo, J.; Tokár, S.; Tokushuku, K.; Tollefson, K.; Tolley, E.; Tomlinson, L.; Tomoto, M.; Tompkins, L.; Toms, K.; Torrence, E.; Torres, H.; Pastor, E. Torró; Toth, J.; Touchard, F.; Tovey, D. R.; Trefzger, T.; Tremblet, L.; Tricoli, A.; Trigger, I. M.; Trincaz-Duvoid, S.; Tripiana, M. F.; Trischuk, W.; Trocmé, B.; Troncon, C.; Trottier-McDonald, M.; Trovatelli, M.; Truong, L.; Trzebinski, M.; Trzupek, A.; Tsarouchas, C.; Tseng, J. C.-L.; Tsiareshka, P. V.; Tsionou, D.; Tsipolitis, G.; Tsirintanis, N.; Tsiskaridze, S.; Tsiskaridze, V.; Tskhadadze, E. G.; Tsui, K. M.; Tsukerman, I. I.; Tsulaia, V.; Tsuno, S.; Tsybychev, D.; Tudorache, A.; Tudorache, V.; Tuna, A. N.; Tupputi, S. A.; Turchikhin, S.; Turecek, D.; Turra, R.; Turvey, A. J.; Tuts, P. M.; Tykhonov, A.; Tylmad, M.; Tyndel, M.; Ueda, I.; Ueno, R.; Ughetto, M.; Ukegawa, F.; Unal, G.; Undrus, A.; Unel, G.; Ungaro, F. C.; Unno, Y.; Unverdorben, C.; Urban, J.; Urquijo, P.; Urrejola, P.; Usai, G.; Usanova, A.; Vacavant, L.; Vacek, V.; Vachon, B.; Valderanis, C.; Valencic, N.; Valentinetti, S.; Valero, A.; Valery, L.; Valkar, S.; Vallecorsa, S.; Ferrer, J. A. Valls; Van Den Wollenberg, W.; Van Der Deijl, P. C.; van der Geer, R.; van der Graaf, H.; van Eldik, N.; van Gemmeren, P.; Van Nieuwkoop, J.; van Vulpen, I.; van Woerden, M. C.; Vanadia, M.; Vandelli, W.; Vanguri, R.; Vaniachine, A.; Vannucci, F.; Vardanyan, G.; Vari, R.; Varnes, E. W.; Varol, T.; Varouchas, D.; Vartapetian, A.; Varvell, K. E.; Vazeille, F.; Schroeder, T. Vazquez; Veatch, J.; Veloce, L. M.; Veloso, F.; Velz, T.; Veneziano, S.; Ventura, A.; Ventura, D.; Venturi, M.; Venturi, N.; Venturini, A.; Vercesi, V.; Verducci, M.; Verkerke, W.; Vermeulen, J. C.; Vest, A.; Vetterli, M. C.; Viazlo, O.; Vichou, I.; Vickey, T.; Boeriu, O. E. Vickey; Viehhauser, G. H. A.; Viel, S.; Vigne, R.; Villa, M.; Perez, M. Villaplana; Vilucchi, E.; Vincter, M. G.; Vinogradov, V. B.; Vivarelli, I.; Vlachos, S.; Vladoiu, D.; Vlasak, M.; Vogel, M.; Vokac, P.; Volpi, G.; Volpi, M.; von der Schmitt, H.; von Radziewski, H.; von Toerne, E.; Vorobel, V.; Vorobev, K.; Vos, M.; Voss, R.; Vossebeld, J. H.; Vranjes, N.; Milosavljevic, M. Vranjes; Vrba, V.; Vreeswijk, M.; Vuillermet, R.; Vukotic, I.; Vykydal, Z.; Wagner, P.; Wagner, W.; Wahlberg, H.; Wahrmund, S.; Wakabayashi, J.; Walder, J.; Walker, R.; Walkowiak, W.; Wang, C.; Wang, F.; Wang, H.; Wang, H.; Wang, J.; Wang, J.; Wang, K.; Wang, R.; Wang, S. M.; Wang, T.; Wang, T.; Wang, X.; Wanotayaroj, C.; Warburton, A.; Ward, C. P.; Wardrope, D. R.; Washbrook, A.; Wasicki, C.; Watkins, P. M.; Watson, A. T.; Watson, I. J.; Watson, M. F.; Watts, G.; Watts, S.; Waugh, B. M.; Webb, S.; Weber, M. S.; Weber, S. W.; Webster, J. S.; Weidberg, A. R.; Weinert, B.; Weingarten, J.; Weiser, C.; Weits, H.; Wells, P. S.; Wenaus, T.; Wengler, T.; Wenig, S.; Wermes, N.; Werner, M.; Werner, P.; Wessels, M.; Wetter, J.; Whalen, K.; Wharton, A. M.; White, A.; White, M. J.; White, R.; White, S.; Whiteson, D.; Wickens, F. J.; Wiedenmann, W.; Wielers, M.; Wienemann, P.; Wiglesworth, C.; Wiik-Fuchs, L. A. M.; Wildauer, A.; Wilkens, H. G.; Williams, H. H.; Williams, S.; Willis, C.; Willocq, S.; Wilson, A.; Wilson, J. A.; Wingerter-Seez, I.; Winklmeier, F.; Winter, B. T.; Wittgen, M.; Wittkowski, J.; Wollstadt, S. J.; Wolter, M. W.; Wolters, H.; Wosiek, B. K.; Wotschack, J.; Woudstra, M. J.; Wozniak, K. W.; Wu, M.; Wu, M.; Wu, S. L.; Wu, X.; Wu, Y.; Wyatt, T. R.; Wynne, B. M.; Xella, S.; Xu, D.; Xu, L.; Yabsley, B.; Yacoob, S.; Yakabe, R.; Yamada, M.; Yamaguchi, D.; Yamaguchi, Y.; Yamamoto, A.; Yamamoto, S.; Yamanaka, T.; Yamauchi, K.; Yamazaki, Y.; Yan, Z.; Yang, H.; Yang, H.; Yang, Y.; Yao, W.-M.; Yap, Y. C.; Yasu, Y.; Yatsenko, E.; Wong, K. H. Yau; Ye, J.; Ye, S.; Yeletskikh, I.; Yen, A. L.; Yildirim, E.; Yorita, K.; Yoshida, R.; Yoshihara, K.; Young, C.; Young, C. J. S.; Youssef, S.; Yu, D. R.; Yu, J.; Yu, J. M.; Yu, J.; Yuan, L.; Yuen, S. P. Y.; Yurkewicz, A.; Yusuff, I.; Zabinski, B.; Zaidan, R.; Zaitsev, A. M.; Zalieckas, J.; Zaman, A.; Zambito, S.; Zanello, L.; Zanzi, D.; Zeitnitz, C.; Zeman, M.; Zemla, A.; Zeng, J. C.; Zeng, Q.; Zengel, K.; Zenin, O.; Ženiš, T.; Zerwas, D.; Zhang, D.; Zhang, F.; Zhang, G.; Zhang, H.; Zhang, J.; Zhang, L.; Zhang, R.; Zhang, X.; Zhang, Z.; Zhao, X.; Zhao, Y.; Zhao, Z.; Zhemchugov, A.; Zhong, J.; Zhou, B.; Zhou, C.; Zhou, L.; Zhou, L.; Zhou, M.; Zhou, N.; Zhu, C. G.; Zhu, H.; Zhu, J.; Zhu, Y.; Zhuang, X.; Zhukov, K.; Zibell, A.; Zieminska, D.; Zimine, N. I.; Zimmermann, C.; Zimmermann, S.; Zinonos, Z.; Zinser, M.; Ziolkowski, M.; Živković, L.; Zobernig, G.; Zoccoli, A.; Nedden, M. zur; Zurzolo, G.; Zwalinski, L.

    2017-07-01

    The reconstruction of the signal from hadrons and jets emerging from the proton-proton collisions at the Large Hadron Collider (LHC) and entering the ATLAS calorimeters is based on a three-dimensional topological clustering of individual calorimeter cell signals. The cluster formation follows cell signal-significance patterns generated by electromagnetic and hadronic showers. In this, the clustering algorithm implicitly performs a topological noise suppression by removing cells with insignificant signals which are not in close proximity to cells with significant signals. The resulting topological cell clusters have shape and location information, which is exploited to apply a local energy calibration and corrections depending on the nature of the cluster. Topological cell clustering is established as a well-performing calorimeter signal definition for jet and missing transverse momentum reconstruction in ATLAS.

  1. Topological cell clustering in the ATLAS calorimeters and its performance in LHC Run 1.

    PubMed

    Aad, G; Abbott, B; Abdallah, J; Abdinov, O; Aben, R; Abolins, M; AbouZeid, O S; Abramowicz, H; Abreu, H; Abreu, R; Abulaiti, Y; Acharya, B S; Adamczyk, L; Adams, D L; Adelman, J; Adomeit, S; Adye, T; Affolder, A A; Agatonovic-Jovin, T; Agricola, J; Aguilar-Saavedra, J A; Ahlen, S P; Ahmadov, F; Aielli, G; Akerstedt, H; Åkesson, T P A; Akimov, A V; Alberghi, G L; Albert, J; Albrand, S; Verzini, M J Alconada; Aleksa, M; Aleksandrov, I N; Alexa, C; Alexander, G; Alexopoulos, T; Alhroob, M; Alimonti, G; Alio, L; Alison, J; Alkire, S P; Allbrooke, B M M; Allport, P P; Aloisio, A; Alonso, A; Alonso, F; Alpigiani, C; Altheimer, A; Gonzalez, B Alvarez; Piqueras, D Álvarez; Alviggi, M G; Amadio, B T; Amako, K; Coutinho, Y Amaral; Amelung, C; Amidei, D; Santos, S P Amor Dos; Amorim, A; Amoroso, S; Amram, N; Amundsen, G; Anastopoulos, C; Ancu, L S; Andari, N; Andeen, T; Anders, C F; Anders, G; Anders, J K; Anderson, K J; Andreazza, A; Andrei, V; Angelidakis, S; Angelozzi, I; Anger, P; Angerami, A; Anghinolfi, F; Anisenkov, A V; Anjos, N; Annovi, A; Antonelli, M; Antonov, A; Antos, J; Anulli, F; Aoki, M; Bella, L Aperio; Arabidze, G; Arai, Y; Araque, J P; Arce, A T H; Arduh, F A; Arguin, J-F; Argyropoulos, S; Arik, M; Armbruster, A J; Arnaez, O; Arnold, H; Arratia, M; Arslan, O; Artamonov, A; Artoni, G; Artz, S; Asai, S; Asbah, N; Ashkenazi, A; Åsman, B; Asquith, L; Assamagan, K; Astalos, R; Atkinson, M; Atlay, N B; Augsten, K; Aurousseau, M; Avolio, G; Axen, B; Ayoub, M K; Azuelos, G; Baak, M A; Baas, A E; Baca, M J; Bacci, C; Bachacou, H; Bachas, K; Backes, M; Backhaus, M; Bagiacchi, P; Bagnaia, P; Bai, Y; Bain, T; Baines, J T; Baker, O K; Baldin, E M; Balek, P; Balestri, T; Balli, F; Balunas, W K; Banas, E; Banerjee, Sw; Bannoura, A A E; Barak, L; Barberio, E L; Barberis, D; Barbero, M; Barillari, T; Barisonzi, M; Barklow, T; Barlow, N; Barnes, S L; Barnett, B M; Barnett, R M; Barnovska, Z; Baroncelli, A; Barone, G; Barr, A J; Barreiro, F; da Costa, J Barreiro Guimarães; Bartoldus, R; Barton, A E; Bartos, P; Basalaev, A; Bassalat, A; Basye, A; Bates, R L; Batista, S J; Batley, J R; Battaglia, M; Bauce, M; Bauer, F; Bawa, H S; Beacham, J B; Beattie, M D; Beau, T; Beauchemin, P H; Beccherle, R; Bechtle, P; Beck, H P; Becker, K; Becker, M; Beckingham, M; Becot, C; Beddall, A J; Beddall, A; Bednyakov, V A; Bee, C P; Beemster, L J; Beermann, T A; Begel, M; Behr, J K; Belanger-Champagne, C; Bell, W H; Bella, G; Bellagamba, L; Bellerive, A; Bellomo, M; Belotskiy, K; Beltramello, O; Benary, O; Benchekroun, D; Bender, M; Bendtz, K; Benekos, N; Benhammou, Y; Noccioli, E Benhar; Garcia, J A Benitez; Benjamin, D P; Bensinger, J R; Bentvelsen, S; Beresford, L; Beretta, M; Berge, D; Kuutmann, E Bergeaas; Berger, N; Berghaus, F; Beringer, J; Bernard, C; Bernard, N R; Bernius, C; Bernlochner, F U; Berry, T; Berta, P; Bertella, C; Bertoli, G; Bertolucci, F; Bertsche, C; Bertsche, D; Besana, M I; Besjes, G J; Bylund, O Bessidskaia; Bessner, M; Besson, N; Betancourt, C; Bethke, S; Bevan, A J; Bhimji, W; Bianchi, R M; Bianchini, L; Bianco, M; Biebel, O; Biedermann, D; Biesuz, N V; Biglietti, M; De Mendizabal, J Bilbao; Bilokon, H; Bindi, M; Binet, S; Bingul, A; Bini, C; Biondi, S; Bjergaard, D M; Black, C W; Black, J E; Black, K M; Blackburn, D; Blair, R E; Blanchard, J-B; Blanco, J E; Blazek, T; Bloch, I; Blocker, C; Blum, W; Blumenschein, U; Blunier, S; Bobbink, G J; Bobrovnikov, V S; Bocchetta, S S; Bocci, A; Bock, C; Boehler, M; Bogaerts, J A; Bogavac, D; Bogdanchikov, A G; Bohm, C; Boisvert, V; Bold, T; Boldea, V; Boldyrev, A S; Bomben, M; Bona, M; Boonekamp, M; Borisov, A; Borissov, G; Borroni, S; Bortfeldt, J; Bortolotto, V; Bos, K; Boscherini, D; Bosman, M; Boudreau, J; Bouffard, J; Bouhova-Thacker, E V; Boumediene, D; Bourdarios, C; Bousson, N; Boutle, S K; Boveia, A; Boyd, J; Boyko, I R; Bozic, I; Bracinik, J; Brandt, A; Brandt, G; Brandt, O; Bratzler, U; Brau, B; Brau, J E; Braun, H M; Madden, W D Breaden; Brendlinger, K; Brennan, A J; Brenner, L; Brenner, R; Bressler, S; Bristow, T M; Britton, D; Britzger, D; Brochu, F M; Brock, I; Brock, R; Bronner, J; Brooijmans, G; Brooks, T; Brooks, W K; Brosamer, J; Brost, E; de Renstrom, P A Bruckman; Bruncko, D; Bruneliere, R; Bruni, A; Bruni, G; Bruschi, M; Bruscino, N; Bryngemark, L; Buanes, T; Buat, Q; Buchholz, P; Buckley, A G; Budagov, I A; Buehrer, F; Bugge, L; Bugge, M K; Bulekov, O; Bullock, D; Burckhart, H; Burdin, S; Burgard, C D; Burghgrave, B; Burke, S; Burmeister, I; Busato, E; Büscher, D; Büscher, V; Bussey, P; Butler, J M; Butt, A I; Buttar, C M; Butterworth, J M; Butti, P; Buttinger, W; Buzatu, A; Buzykaev, A R; Urbán, S Cabrera; Caforio, D; Cairo, V M; Cakir, O; Calace, N; Calafiura, P; Calandri, A; Calderini, G; Calfayan, P; Caloba, L P; Calvet, D; Calvet, S; Toro, R Camacho; Camarda, S; Camarri, P; Cameron, D; Armadans, R Caminal; Campana, S; Campanelli, M; Campoverde, A; Canale, V; Canepa, A; Bret, M Cano; Cantero, J; Cantrill, R; Cao, T; Garrido, M D M Capeans; Caprini, I; Caprini, M; Capua, M; Caputo, R; Carbone, R M; Cardarelli, R; Cardillo, F; Carli, T; Carlino, G; Carminati, L; Caron, S; Carquin, E; Carrillo-Montoya, G D; Carter, J R; Carvalho, J; Casadei, D; Casado, M P; Casolino, M; Casper, D W; Castaneda-Miranda, E; Castelli, A; Gimenez, V Castillo; Castro, N F; Catastini, P; Catinaccio, A; Catmore, J R; Cattai, A; Caudron, J; Cavaliere, V; Cavalli, D; Cavalli-Sforza, M; Cavasinni, V; Ceradini, F; Alberich, L Cerda; Cerio, B C; Cerny, K; Cerqueira, A S; Cerri, A; Cerrito, L; Cerutti, F; Cerv, M; Cervelli, A; Cetin, S A; Chafaq, A; Chakraborty, D; Chalupkova, I; Chan, Y L; Chang, P; Chapman, J D; Charlton, D G; Chau, C C; Barajas, C A Chavez; Che, S; Cheatham, S; Chegwidden, A; Chekanov, S; Chekulaev, S V; Chelkov, G A; Chelstowska, M A; Chen, C; Chen, H; Chen, K; Chen, L; Chen, S; Chen, S; Chen, X; Chen, Y; Cheng, H C; Cheng, Y; Cheplakov, A; Cheremushkina, E; Moursli, R Cherkaoui El; Chernyatin, V; Cheu, E; Chevalier, L; Chiarella, V; Chiarelli, G; Chiodini, G; Chisholm, A S; Chislett, R T; Chitan, A; Chizhov, M V; Choi, K; Chouridou, S; Chow, B K B; Christodoulou, V; Chromek-Burckhart, D; Chudoba, J; Chuinard, A J; Chwastowski, J J; Chytka, L; Ciapetti, G; Ciftci, A K; Cinca, D; Cindro, V; Cioara, I A; Ciocio, A; Cirotto, F; Citron, Z H; Ciubancan, M; Clark, A; Clark, B L; Clark, P J; Clarke, R N; Clement, C; Coadou, Y; Cobal, M; Coccaro, A; Cochran, J; Coffey, L; Cogan, J G; Colasurdo, L; Cole, B; Cole, S; Colijn, A P; Collot, J; Colombo, T; Compostella, G; Muiño, P Conde; Coniavitis, E; Connell, S H; Connelly, I A; Consorti, V; Constantinescu, S; Conta, C; Conti, G; Conventi, F; Cooke, M; Cooper, B D; Cooper-Sarkar, A M; Cornelissen, T; Corradi, M; Corriveau, F; Corso-Radu, A; Cortes-Gonzalez, A; Cortiana, G; Costa, G; Costa, M J; Costanzo, D; Côté, D; Cottin, G; Cowan, G; Cox, B E; Cranmer, K; Cree, G; Crépé-Renaudin, S; Crescioli, F; Cribbs, W A; Ortuzar, M Crispin; Cristinziani, M; Croft, V; Crosetti, G; Donszelmann, T Cuhadar; Cummings, J; Curatolo, M; Cúth, J; Cuthbert, C; Czirr, H; Czodrowski, P; D'Auria, S; D'Onofrio, M; De Sousa, M J Da Cunha Sargedas; Via, C Da; Dabrowski, W; Dafinca, A; Dai, T; Dale, O; Dallaire, F; Dallapiccola, C; Dam, M; Dandoy, J R; Dang, N P; Daniells, A C; Danninger, M; Hoffmann, M Dano; Dao, V; Darbo, G; Darmora, S; Dassoulas, J; Dattagupta, A; Davey, W; David, C; Davidek, T; Davies, E; Davies, M; Davison, P; Davygora, Y; Dawe, E; Dawson, I; Daya-Ishmukhametova, R K; De, K; de Asmundis, R; De Benedetti, A; De Castro, S; De Cecco, S; De Groot, N; de Jong, P; De la Torre, H; De Lorenzi, F; De Pedis, D; De Salvo, A; De Sanctis, U; De Santo, A; De Regie, J B De Vivie; Dearnaley, W J; Debbe, R; Debenedetti, C; Dedovich, D V; Deigaard, I; Del Peso, J; Del Prete, T; Delgove, D; Deliot, F; Delitzsch, C M; Deliyergiyev, M; Dell'Acqua, A; Dell'Asta, L; Dell'Orso, M; Della Pietra, M; Della Volpe, D; Delmastro, M; Delsart, P A; Deluca, C; DeMarco, D A; Demers, S; Demichev, M; Demilly, A; Denisov, S P; Derendarz, D; Derkaoui, J E; Derue, F; Dervan, P; Desch, K; Deterre, C; Dette, K; Deviveiros, P O; Dewhurst, A; Dhaliwal, S; Di Ciaccio, A; Di Ciaccio, L; Di Domenico, A; Di Donato, C; Di Girolamo, A; Di Girolamo, B; Di Mattia, A; Di Micco, B; Di Nardo, R; Di Simone, A; Di Sipio, R; Di Valentino, D; Diaconu, C; Diamond, M; Dias, F A; Diaz, M A; Diehl, E B; Dietrich, J; Diglio, S; Dimitrievska, A; Dingfelder, J; Dita, P; Dita, S; Dittus, F; Djama, F; Djobava, T; Djuvsland, J I; do Vale, M A B; Dobos, D; Dobre, M; Doglioni, C; Dohmae, T; Dolejsi, J; Dolezal, Z; Dolgoshein, B A; Donadelli, M; Donati, S; Dondero, P; Donini, J; Dopke, J; Doria, A; Dova, M T; Doyle, A T; Drechsler, E; Dris, M; Du, Y; Dubreuil, E; Duchovni, E; Duckeck, G; Ducu, O A; Duda, D; Dudarev, A; Duflot, L; Duguid, L; Dührssen, M; Dunford, M; Yildiz, H Duran; Düren, M; Durglishvili, A; Duschinger, D; Dutta, B; Dyndal, M; Eckardt, C; Ecker, K M; Edgar, R C; Edson, W; Edwards, N C; Ehrenfeld, W; Eifert, T; Eigen, G; Einsweiler, K; Ekelof, T; Kacimi, M El; Ellert, M; Elles, S; Ellinghaus, F; Elliot, A A; Ellis, N; Elmsheuser, J; Elsing, M; Emeliyanov, D; Enari, Y; Endner, O C; Endo, M; Erdmann, J; Ereditato, A; Ernis, G; Ernst, J; Ernst, M; Errede, S; Ertel, E; Escalier, M; Esch, H; Escobar, C; Esposito, B; Etienvre, A I; Etzion, E; Evans, H; Ezhilov, A; Fabbri, L; Facini, G; Fakhrutdinov, R M; Falciano, S; Falla, R J; Faltova, J; Fang, Y; Fanti, M; Farbin, A; Farilla, A; Farooque, T; Farrell, S; Farrington, S M; Farthouat, P; Fassi, F; Fassnacht, P; Fassouliotis, D; Giannelli, M Faucci; Favareto, A; Fayard, L; Fedin, O L; Fedorko, W; Feigl, S; Feligioni, L; Feng, C; Feng, E J; Feng, H; Fenyuk, A B; Feremenga, L; Martinez, P Fernandez; Perez, S Fernandez; Ferrando, J; Ferrari, A; Ferrari, P; Ferrari, R; de Lima, D E Ferreira; Ferrer, A; Ferrere, D; Ferretti, C; Parodi, A Ferretto; Fiascaris, M; Fiedler, F; Filipčič, A; Filipuzzi, M; Filthaut, F; Fincke-Keeler, M; Finelli, K D; Fiolhais, M C N; Fiorini, L; Firan, A; Fischer, A; Fischer, C; Fischer, J; Fisher, W C; Flaschel, N; Fleck, I; Fleischmann, P; Fletcher, G T; Fletcher, G; Fletcher, R R M; Flick, T; Floderus, A; Castillo, L R Flores; Flowerdew, M J; Formica, A; Forti, A; Fournier, D; Fox, H; Fracchia, S; Francavilla, P; Franchini, M; Francis, D; Franconi, L; Franklin, M; Frate, M; Fraternali, M; Freeborn, D; French, S T; Fressard-Batraneanu, S M; Friedrich, F; Froidevaux, D; Frost, J A; Fukunaga, C; Torregrosa, E Fullana; Fulsom, B G; Fusayasu, T; Fuster, J; Gabaldon, C; Gabizon, O; Gabrielli, A; Gabrielli, A; Gach, G P; Gadatsch, S; Gadomski, S; Gagliardi, G; Gagnon, P; Galea, C; Galhardo, B; Gallas, E J; Gallop, B J; Gallus, P; Galster, G; Gan, K K; Gao, J; Gao, Y; Gao, Y S; Walls, F M Garay; Garberson, F; García, C; Navarro, J E García; Garcia-Sciveres, M; Gardner, R W; Garelli, N; Garonne, V; Gatti, C; Gaudiello, A; Gaudio, G; Gaur, B; Gauthier, L; Gauzzi, P; Gavrilenko, I L; Gay, C; Gaycken, G; Gazis, E N; Ge, P; Gecse, Z; Gee, C N P; Geich-Gimbel, Ch; Geisler, M P; Gemme, C; Genest, M H; Geng, C; Gentile, S; George, M; George, S; Gerbaudo, D; Gershon, A; Ghasemi, S; Ghazlane, H; Giacobbe, B; Giagu, S; Giangiobbe, V; Giannetti, P; Gibbard, B; Gibson, S M; Gignac, M; Gilchriese, M; Gillam, T P S; Gillberg, D; Gilles, G; Gingrich, D M; Giokaris, N; Giordani, M P; Giorgi, F M; Giorgi, F M; Giraud, P F; Giromini, P; Giugni, D; Giuliani, C; Giulini, M; Gjelsten, B K; Gkaitatzis, S; Gkialas, I; Gkougkousis, E L; Gladilin, L K; Glasman, C; Glatzer, J; Glaysher, P C F; Glazov, A; Goblirsch-Kolb, M; Goddard, J R; Godlewski, J; Goldfarb, S; Golling, T; Golubkov, D; Gomes, A; Gonçalo, R; Costa, J Goncalves Pinto Firmino Da; Gonella, L; de la Hoz, S González; Parra, G Gonzalez; Gonzalez-Sevilla, S; Goossens, L; Gorbounov, P A; Gordon, H A; Gorelov, I; Gorini, B; Gorini, E; Gorišek, A; Gornicki, E; Goshaw, A T; Gössling, C; Gostkin, M I; Goujdami, D; Goussiou, A G; Govender, N; Gozani, E; Grabas, H M X; Graber, L; Grabowska-Bold, I; Gradin, P O J; Grafström, P; Gramling, J; Gramstad, E; Grancagnolo, S; Gratchev, V; Gray, H M; Graziani, E; Greenwood, Z D; Grefe, C; Gregersen, K; Gregor, I M; Grenier, P; Griffiths, J; Grillo, A A; Grimm, K; Grinstein, S; Gris, Ph; Grivaz, J-F; Groh, S; Grohs, J P; Grohsjean, A; Gross, E; Grosse-Knetter, J; Grossi, G C; Grout, Z J; Guan, L; Guenther, J; Guescini, F; Guest, D; Gueta, O; Guido, E; Guillemin, T; Guindon, S; Gul, U; Gumpert, C; Guo, J; Guo, Y; Gupta, S; Gustavino, G; Gutierrez, P; Ortiz, N G Gutierrez; Gutschow, C; Guyot, C; Gwenlan, C; Gwilliam, C B; Haas, A; Haber, C; Hadavand, H K; Haddad, N; Haefner, P; Hageböck, S; Hajduk, Z; Hakobyan, H; Haleem, M; Haley, J; Hall, D; Halladjian, G; Hallewell, G D; Hamacher, K; Hamal, P; Hamano, K; Hamilton, A; Hamity, G N; Hamnett, P G; Han, L; Hanagaki, K; Hanawa, K; Hance, M; Haney, B; Hanke, P; Hanna, R; Hansen, J B; Hansen, J D; Hansen, M C; Hansen, P H; Hara, K; Hard, A S; Harenberg, T; Hariri, F; Harkusha, S; Harrington, R D; Harrison, P F; Hartjes, F; Hasegawa, M; Hasegawa, Y; Hasib, A; Hassani, S; Haug, S; Hauser, R; Hauswald, L; Havranek, M; Hawkes, C M; Hawkings, R J; Hawkins, A D; Hayashi, T; Hayden, D; Hays, C P; Hays, J M; Hayward, H S; Haywood, S J; Head, S J; Heck, T; Hedberg, V; Heelan, L; Heim, S; Heim, T; Heinemann, B; Heinrich, L; Hejbal, J; Helary, L; Hellman, S; Helsens, C; Henderson, J; Henderson, R C W; Heng, Y; Hengler, C; Henkelmann, S; Henrichs, A; Correia, A M Henriques; Henrot-Versille, S; Herbert, G H; Jiménez, Y Hernández; Herten, G; Hertenberger, R; Hervas, L; Hesketh, G G; Hessey, N P; Hetherly, J W; Hickling, R; Higón-Rodriguez, E; Hill, E; Hill, J C; Hiller, K H; Hillier, S J; Hinchliffe, I; Hines, E; Hinman, R R; Hirose, M; Hirschbuehl, D; Hobbs, J; Hod, N; Hodgkinson, M C; Hodgson, P; Hoecker, A; Hoeferkamp, M R; Hoenig, F; Hohlfeld, M; Hohn, D; Holmes, T R; Homann, M; Hong, T M; Hopkins, W H; Horii, Y; Horton, A J; Hostachy, J-Y; Hou, S; Hoummada, A; Howard, J; Howarth, J; Hrabovsky, M; Hristova, I; Hrivnac, J; Hryn'ova, T; Hrynevich, A; Hsu, C; Hsu, P J; Hsu, S-C; Hu, D; Hu, Q; Hu, X; Huang, Y; Hubacek, Z; Hubaut, F; Huegging, F; Huffman, T B; Hughes, E W; Hughes, G; Huhtinen, M; Hülsing, T A; Huseynov, N; Huston, J; Huth, J; Iacobucci, G; Iakovidis, G; Ibragimov, I; Iconomidou-Fayard, L; Ideal, E; Idrissi, Z; Iengo, P; Igonkina, O; Iizawa, T; Ikegami, Y; Ikeno, M; Ilchenko, Y; Iliadis, D; Ilic, N; Ince, T; Introzzi, G; Ioannou, P; Iodice, M; Iordanidou, K; Ippolito, V; Quiles, A Irles; Isaksson, C; Ishino, M; Ishitsuka, M; Ishmukhametov, R; Issever, C; Istin, S; Ponce, J M Iturbe; Iuppa, R; Ivarsson, J; Iwanski, W; Iwasaki, H; Izen, J M; Izzo, V; Jabbar, S; Jackson, B; Jackson, M; Jackson, P; Jaekel, M R; Jain, V; Jakobi, K B; Jakobs, K; Jakobsen, S; Jakoubek, T; Jakubek, J; Jamin, D O; Jana, D K; Jansen, E; Jansky, R; Janssen, J; Janus, M; Jarlskog, G; Javadov, N; Javůrek, T; Jeanty, L; Jejelava, J; Jeng, G-Y; Jennens, D; Jenni, P; Jentzsch, J; Jeske, C; Jézéquel, S; Ji, H; Jia, J; Jiang, H; Jiang, Y; Jiggins, S; Pena, J Jimenez; Jin, S; Jinaru, A; Jinnouchi, O; Joergensen, M D; Johansson, P; Johns, K A; Johnson, W J; Jon-And, K; Jones, G; Jones, R W L; Jones, T J; Jongmanns, J; Jorge, P M; Joshi, K D; Jovicevic, J; Ju, X; Rozas, A Juste; Kaci, M; Kaczmarska, A; Kado, M; Kagan, H; Kagan, M; Kahn, S J; Kajomovitz, E; Kalderon, C W; Kaluza, A; Kama, S; Kamenshchikov, A; Kanaya, N; Kaneti, S; Kantserov, V A; Kanzaki, J; Kaplan, B; Kaplan, L S; Kapliy, A; Kar, D; Karakostas, K; Karamaoun, A; Karastathis, N; Kareem, M J; Karentzos, E; Karnevskiy, M; Karpov, S N; Karpova, Z M; Karthik, K; Kartvelishvili, V; Karyukhin, A N; Kasahara, K; Kashif, L; Kass, R D; Kastanas, A; Kataoka, Y; Kato, C; Katre, A; Katzy, J; Kawade, K; Kawagoe, K; Kawamoto, T; Kawamura, G; Kazama, S; Kazanin, V F; Keeler, R; Kehoe, R; Keller, J S; Kempster, J J; Keoshkerian, H; Kepka, O; Kerševan, B P; Kersten, S; Keyes, R A; Khalil-Zada, F; Khandanyan, H; Khanov, A; Kharlamov, A G; Khoo, T J; Khovanskiy, V; Khramov, E; Khubua, J; Kido, S; Kim, H Y; Kim, S H; Kim, Y K; Kimura, N; Kind, O M; King, B T; King, M; King, S B; Kirk, J; Kiryunin, A E; Kishimoto, T; Kisielewska, D; Kiss, F; Kiuchi, K; Kivernyk, O; Kladiva, E; Klein, M H; Klein, M; Klein, U; Kleinknecht, K; Klimek, P; Klimentov, A; Klingenberg, R; Klinger, J A; Klioutchnikova, T; Kluge, E-E; Kluit, P; Kluth, S; Knapik, J; Kneringer, E; Knoops, E B F G; Knue, A; Kobayashi, A; Kobayashi, D; Kobayashi, T; Kobel, M; Kocian, M; Kodys, P; Koffas, T; Koffeman, E; Kogan, L A; Kohlmann, S; Kohout, Z; Kohriki, T; Koi, T; Kolanoski, H; Kolb, M; Koletsou, I; Komar, A A; Komori, Y; Kondo, T; Kondrashova, N; Köneke, K; König, A C; Kono, T; Konoplich, R; Konstantinidis, N; Kopeliansky, R; Koperny, S; Köpke, L; Kopp, A K; Korcyl, K; Kordas, K; Korn, A; Korol, A A; Korolkov, I; Korolkova, E V; Kortner, O; Kortner, S; Kosek, T; Kostyukhin, V V; Kotov, V M; Kotwal, A; Kourkoumeli-Charalampidi, A; Kourkoumelis, C; Kouskoura, V; Koutsman, A; Kowalewski, R; Kowalski, T Z; Kozanecki, W; Kozhin, A S; Kramarenko, V A; Kramberger, G; Krasnopevtsev, D; Krasny, M W; Krasznahorkay, A; Kraus, J K; Kravchenko, A; Kreiss, S; Kretz, M; Kretzschmar, J; Kreutzfeldt, K; Krieger, P; Krizka, K; Kroeninger, K; Kroha, H; Kroll, J; Kroseberg, J; Krstic, J; Kruchonak, U; Krüger, H; Krumnack, N; Kruse, A; Kruse, M C; Kruskal, M; Kubota, T; Kucuk, H; Kuday, S; Kuehn, S; Kugel, A; Kuger, F; Kuhl, A; Kuhl, T; Kukhtin, V; Kukla, R; Kulchitsky, Y; Kuleshov, S; Kuna, M; Kunigo, T; Kupco, A; Kurashige, H; Kurochkin, Y A; Kus, V; Kuwertz, E S; Kuze, M; Kvita, J; Kwan, T; Kyriazopoulos, D; Rosa, A La; Navarro, J L La Rosa; Rotonda, L La; Lacasta, C; Lacava, F; Lacey, J; Lacker, H; Lacour, D; Lacuesta, V R; Ladygin, E; Lafaye, R; Laforge, B; Lagouri, T; Lai, S; Lambourne, L; Lammers, S; Lampen, C L; Lampl, W; Lançon, E; Landgraf, U; Landon, M P J; Lang, V S; Lange, J C; Lankford, A J; Lanni, F; Lantzsch, K; Lanza, A; Laplace, S; Lapoire, C; Laporte, J F; Lari, T; Manghi, F Lasagni; Lassnig, M; Laurelli, P; Lavrijsen, W; Law, A T; Laycock, P; Lazovich, T; Dortz, O Le; Guirriec, E Le; Menedeu, E Le; LeBlanc, M; LeCompte, T; Ledroit-Guillon, F; Lee, C A; Lee, S C; Lee, L; Lefebvre, G; Lefebvre, M; Legger, F; Leggett, C; Lehan, A; Miotto, G Lehmann; Lei, X; Leight, W A; Leisos, A; Leister, A G; Leite, M A L; Leitner, R; Lellouch, D; Lemmer, B; Leney, K J C; Lenz, T; Lenzi, B; Leone, R; Leone, S; Leonidopoulos, C; Leontsinis, S; Leroy, C; Lester, C G; Levchenko, M; Levêque, J; Levin, D; Levinson, L J; Levy, M; Lewis, A; Leyko, A M; Leyton, M; Li, B; Li, H; Li, H L; Li, L; Li, L; Li, S; Li, X; Li, Y; Liang, Z; Liao, H; Liberti, B; Liblong, A; Lichard, P; Lie, K; Liebal, J; Liebig, W; Limbach, C; Limosani, A; Lin, S C; Lin, T H; Linde, F; Lindquist, B E; Linnemann, J T; Lipeles, E; Lipniacka, A; Lisovyi, M; Liss, T M; Lissauer, D; Lister, A; Litke, A M; Liu, B; Liu, D; Liu, H; Liu, J; Liu, J B; Liu, K; Liu, L; Liu, M; Liu, M; Liu, Y; Livan, M; Lleres, A; Merino, J Llorente; Lloyd, S L; Sterzo, F Lo; Lobodzinska, E; Loch, P; Lockman, W S; Loebinger, F K; Loevschall-Jensen, A E; Loew, K M; Loginov, A; Lohse, T; Lohwasser, K; Lokajicek, M; Long, B A; Long, J D; Long, R E; Looper, K A; Lopes, L; Mateos, D Lopez; Paredes, B Lopez; Paz, I Lopez; Lorenz, J; Martinez, N Lorenzo; Losada, M; Lösel, P J; Lou, X; Lounis, A; Love, J; Love, P A; Lu, H; Lu, N; Lubatti, H J; Luci, C; Lucotte, A; Luedtke, C; Luehring, F; Lukas, W; Luminari, L; Lundberg, O; Lund-Jensen, B; Lynn, D; Lysak, R; Lytken, E; Ma, H; Ma, L L; Maccarrone, G; Macchiolo, A; Macdonald, C M; Maček, B; Miguens, J Machado; Macina, D; Madaffari, D; Madar, R; Maddocks, H J; Mader, W F; Madsen, A; Maeda, J; Maeland, S; Maeno, T; Maevskiy, A; Magradze, E; Mahboubi, K; Mahlstedt, J; Maiani, C; Maidantchik, C; Maier, A A; Maier, T; Maio, A; Majewski, S; Makida, Y; Makovec, N; Malaescu, B; Malecki, Pa; Maleev, V P; Malek, F; Mallik, U; Malon, D; Malone, C; Maltezos, S; Malyshev, V M; Malyukov, S; Mamuzic, J; Mancini, G; Mandelli, B; Mandelli, L; Mandić, I; Mandrysch, R; Maneira, J; Filho, L Manhaes de Andrade; Ramos, J Manjarres; Mann, A; Manousakis-Katsikakis, A; Mansoulie, B; Mantifel, R; Mantoani, M; Mapelli, L; March, L; Marchiori, G; Marcisovsky, M; Marino, C P; Marjanovic, M; Marley, D E; Marroquim, F; Marsden, S P; Marshall, Z; Marti, L F; Marti-Garcia, S; Martin, B; Martin, T A; Martin, V J; Latour, B Martin Dit; Martinez, M; Martin-Haugh, S; Martoiu, V S; Martyniuk, A C; Marx, M; Marzano, F; Marzin, A; Masetti, L; Mashimo, T; Mashinistov, R; Masik, J; Maslennikov, A L; Massa, I; Massa, L; Mastrandrea, P; Mastroberardino, A; Masubuchi, T; Mättig, P; Mattmann, J; Maurer, J; Maxfield, S J; Maximov, D A; Mazini, R; Mazza, S M; Goldrick, G Mc; Kee, S P Mc; McCarn, A; McCarthy, R L; McCarthy, T G; McCubbin, N A; McFarlane, K W; Mcfayden, J A; Mchedlidze, G; McMahon, S J; McPherson, R A; Medinnis, M; Meehan, S; Mehlhase, S; Mehta, A; Meier, K; Meineck, C; Meirose, B; Garcia, B R Mellado; Meloni, F; Mengarelli, A; Menke, S; Meoni, E; Mercurio, K M; Mergelmeyer, S; Mermod, P; Merola, L; Meroni, C; Merritt, F S; Messina, A; Metcalfe, J; Mete, A S; Meyer, C; Meyer, C; Meyer, J-P; Meyer, J; Theenhausen, H Meyer Zu; Middleton, R P; Miglioranzi, S; Mijović, L; Mikenberg, G; Mikestikova, M; Mikuž, M; Milesi, M; Milic, A; Miller, D W; Mills, C; Milov, A; Milstead, D A; Minaenko, A A; Minami, Y; Minashvili, I A; Mincer, A I; Mindur, B; Mineev, M; Ming, Y; Mir, L M; Mistry, K P; Mitani, T; Mitrevski, J; Mitsou, V A; Miucci, A; Miyagawa, P S; Mjörnmark, J U; Moa, T; Mochizuki, K; Mohapatra, S; Mohr, W; Molander, S; Moles-Valls, R; Monden, R; Mondragon, M C; Mönig, K; Monini, C; Monk, J; Monnier, E; Montalbano, A; Berlingen, J Montejo; Monticelli, F; Monzani, S; Moore, R W; Morange, N; Moreno, D; Llácer, M Moreno; Morettini, P; Mori, D; Mori, T; Morii, M; Morinaga, M; Morisbak, V; Moritz, S; Morley, A K; Mornacchi, G; Morris, J D; Mortensen, S S; Morton, A; Morvaj, L; Mosidze, M; Moss, J; Motohashi, K; Mount, R; Mountricha, E; Mouraviev, S V; Moyse, E J W; Muanza, S; Mudd, R D; Mueller, F; Mueller, J; Mueller, R S P; Mueller, T; Muenstermann, D; Mullen, P; Mullier, G A; Sanchez, F J Munoz; Quijada, J A Murillo; Murray, W J; Musheghyan, H; Musto, E; Myagkov, A G; Myska, M; Nachman, B P; Nackenhorst, O; Nadal, J; Nagai, K; Nagai, R; Nagai, Y; Nagano, K; Nagarkar, A; Nagasaka, Y; Nagata, K; Nagel, M; Nagy, E; Nairz, A M; Nakahama, Y; Nakamura, K; Nakamura, T; Nakano, I; Namasivayam, H; Garcia, R F Naranjo; Narayan, R; Villar, D I Narrias; Naumann, T; Navarro, G; Nayyar, R; Neal, H A; Nechaeva, P Yu; Neep, T J; Nef, P D; Negri, A; Negrini, M; Nektarijevic, S; Nellist, C; Nelson, A; Nemecek, S; Nemethy, P; Nepomuceno, A A; Nessi, M; Neubauer, M S; Neumann, M; Neves, R M; Nevski, P; Newman, P R; Nguyen, D H; Nickerson, R B; Nicolaidou, R; Nicquevert, B; Nielsen, J; Nikiforou, N; Nikiforov, A; Nikolaenko, V; Nikolic-Audit, I; Nikolopoulos, K; Nilsen, J K; Nilsson, P; Ninomiya, Y; Nisati, A; Nisius, R; Nobe, T; Nodulman, L; Nomachi, M; Nomidis, I; Nooney, T; Norberg, S; Nordberg, M; Novgorodova, O; Nowak, S; Nozaki, M; Nozka, L; Ntekas, K; Hanninger, G Nunes; Nunnemann, T; Nurse, E; Nuti, F; O'grady, F; O'Neil, D C; O'Shea, V; Oakham, F G; Oberlack, H; Obermann, T; Ocariz, J; Ochi, A; Ochoa, I; Ochoa-Ricoux, J P; Oda, S; Odaka, S; Ogren, H; Oh, A; Oh, S H; Ohm, C C; Ohman, H; Oide, H; Okamura, W; Okawa, H; Okumura, Y; Okuyama, T; Olariu, A; Pino, S A Olivares; Damazio, D Oliveira; Olszewski, A; Olszowska, J; Onofre, A; Onogi, K; Onyisi, P U E; Oram, C J; Oreglia, M J; Oren, Y; Orestano, D; Orlando, N; Barrera, C Oropeza; Orr, R S; Osculati, B; Ospanov, R; Garzon, G Otero Y; Otono, H; Ouchrif, M; Ould-Saada, F; Ouraou, A; Oussoren, K P; Ouyang, Q; Ovcharova, A; Owen, M; Owen, R E; Ozcan, V E; Ozturk, N; Pachal, K; Pages, A Pacheco; Aranda, C Padilla; Pagáčová, M; Griso, S Pagan; Paganis, E; Paige, F; Pais, P; Pajchel, K; Palacino, G; Palestini, S; Palka, M; Pallin, D; Palma, A; Pan, Y B; Panagiotopoulou, E St; Pandini, C E; Vazquez, J G Panduro; Pani, P; Panitkin, S; Pantea, D; Paolozzi, L; Papadopoulou, Th D; Papageorgiou, K; Paramonov, A; Hernandez, D Paredes; Parker, M A; Parker, K A; Parodi, F; Parsons, J A; Parzefall, U; Pasqualucci, E; Passaggio, S; Pastore, F; Pastore, Fr; Pásztor, G; Pataraia, S; Patel, N D; Pater, J R; Pauly, T; Pearce, J; Pearson, B; Pedersen, L E; Pedersen, M; Lopez, S Pedraza; Pedro, R; Peleganchuk, S V; Pelikan, D; Penc, O; Peng, C; Peng, H; Penning, B; Penwell, J; Perepelitsa, D V; Codina, E Perez; García-Estañ, M T Pérez; Perini, L; Pernegger, H; Perrella, S; Peschke, R; Peshekhonov, V D; Peters, K; Peters, R F Y; Petersen, B A; Petersen, T C; Petit, E; Petridis, A; Petridou, C; Petroff, P; Petrolo, E; Petrucci, F; Pettersson, N E; Pezoa, R; Phillips, P W; Piacquadio, G; Pianori, E; Picazio, A; Piccaro, E; Piccinini, M; Pickering, M A; Piegaia, R; Pignotti, D T; Pilcher, J E; Pilkington, A D; Pin, A W J; Pina, J; Pinamonti, M; Pinfold, J L; Pingel, A; Pires, S; Pirumov, H; Pitt, M; Pizio, C; Plazak, L; Pleier, M-A; Pleskot, V; Plotnikova, E; Plucinski, P; Pluth, D; Poettgen, R; Poggioli, L; Pohl, D; Polesello, G; Poley, A; Policicchio, A; Polifka, R; Polini, A; Pollard, C S; Polychronakos, V; Pommès, K; Pontecorvo, L; Pope, B G; Popeneciu, G A; Popovic, D S; Poppleton, A; Pospisil, S; Potamianos, K; Potrap, I N; Potter, C J; Potter, C T; Poulard, G; Poveda, J; Pozdnyakov, V; Astigarraga, M E Pozo; Pralavorio, P; Pranko, A; Prasad, S; Prell, S; Price, D; Price, L E; Primavera, M; Prince, S; Proissl, M; Prokofiev, K; Prokoshin, F; Protopapadaki, E; Protopopescu, S; Proudfoot, J; Przybycien, M; Ptacek, E; Puddu, D; Pueschel, E; Puldon, D; Purohit, M; Puzo, P; Qian, J; Qin, G; Qin, Y; Quadt, A; Quarrie, D R; Quayle, W B; Queitsch-Maitland, M; Quilty, D; Raddum, S; Radeka, V; Radescu, V; Radhakrishnan, S K; Radloff, P; Rados, P; Ragusa, F; Rahal, G; Rajagopalan, S; Rammensee, M; Rangel-Smith, C; Rauscher, F; Rave, S; Ravenscroft, T; Raymond, M; Read, A L; Readioff, N P; Rebuzzi, D M; Redelbach, A; Redlinger, G; Reece, R; Reeves, K; Rehnisch, L; Reichert, J; Reisin, H; Rembser, C; Ren, H; Renaud, A; Rescigno, M; Resconi, S; Rezanova, O L; Reznicek, P; Rezvani, R; Richter, R; Richter, S; Richter-Was, E; Ricken, O; Ridel, M; Rieck, P; Riegel, C J; Rieger, J; Rifki, O; Rijssenbeek, M; Rimoldi, A; Rinaldi, L; Ristić, B; Ritsch, E; Riu, I; Rizatdinova, F; Rizvi, E; Robertson, S H; Robichaud-Veronneau, A; Robinson, D; Robinson, J E M; Robson, A; Roda, C; Roe, S; Røhne, O; Romaniouk, A; Romano, M; Saez, S M Romano; Adam, E Romero; Rompotis, N; Ronzani, M; Roos, L; Ros, E; Rosati, S; Rosbach, K; Rose, P; Rosenthal, O; Rossetti, V; Rossi, E; Rossi, L P; Rosten, J H N; Rosten, R; Rotaru, M; Roth, I; Rothberg, J; Rousseau, D; Royon, C R; Rozanov, A; Rozen, Y; Ruan, X; Rubbo, F; Rubinskiy, I; Rud, V I; Rudolph, C; Rudolph, M S; Rühr, F; Ruiz-Martinez, A; Rurikova, Z; Rusakovich, N A; Ruschke, A; Russell, H L; Rutherfoord, J P; Ruthmann, N; Ryabov, Y F; Rybar, M; Rybkin, G; Ryder, N C; Ryzhov, A; Saavedra, A F; Sabato, G; Sacerdoti, S; Saddique, A; Sadrozinski, H F-W; Sadykov, R; Tehrani, F Safai; Saha, P; Sahinsoy, M; Saimpert, M; Saito, T; Sakamoto, H; Sakurai, Y; Salamanna, G; Salamon, A; Loyola, J E Salazar; Saleem, M; Salek, D; De Bruin, P H Sales; Salihagic, D; Salnikov, A; Salt, J; Salvatore, D; Salvatore, F; Salvucci, A; Salzburger, A; Sammel, D; Sampsonidis, D; Sanchez, A; Sánchez, J; Martinez, V Sanchez; Sandaker, H; Sandbach, R L; Sander, H G; Sanders, M P; Sandhoff, M; Sandoval, C; Sandstroem, R; Sankey, D P C; Sannino, M; Sansoni, A; Santoni, C; Santonico, R; Santos, H; Castillo, I Santoyo; Sapp, K; Sapronov, A; Saraiva, J G; Sarrazin, B; Sasaki, O; Sasaki, Y; Sato, K; Sauvage, G; Sauvan, E; Savage, G; Savard, P; Sawyer, C; Sawyer, L; Saxon, J; Sbarra, C; Sbrizzi, A; Scanlon, T; Scannicchio, D A; Scarcella, M; Scarfone, V; Schaarschmidt, J; Schacht, P; Schaefer, D; Schaefer, R; Schaeffer, J; Schaepe, S; Schaetzel, S; Schäfer, U; Schaffer, A C; Schaile, D; Schamberger, R D; Scharf, V; Schegelsky, V A; Scheirich, D; Schernau, M; Schiavi, C; Schillo, C; Schioppa, M; Schlenker, S; Schmieden, K; Schmitt, C; Schmitt, S; Schmitt, S; Schmitz, S; Schneider, B; Schnellbach, Y J; Schnoor, U; Schoeffel, L; Schoening, A; Schoenrock, B D; Schopf, E; Schorlemmer, A L S; Schott, M; Schouten, D; Schovancova, J; Schramm, S; Schreyer, M; Schuh, N; Schultens, M J; Schultz-Coulon, H-C; Schulz, H; Schumacher, M; Schumm, B A; Schune, Ph; Schwanenberger, C; Schwartzman, A; Schwarz, T A; Schwegler, Ph; Schweiger, H; Schwemling, Ph; Schwienhorst, R; Schwindling, J; Schwindt, T; Scifo, E; Sciolla, G; Scuri, F; Scutti, F; Searcy, J; Sedov, G; Sedykh, E; Seema, P; Seidel, S C; Seiden, A; Seifert, F; Seixas, J M; Sekhniaidze, G; Sekhon, K; Sekula, S J; Seliverstov, D M; Semprini-Cesari, N; Serfon, C; Serin, L; Serkin, L; Serre, T; Sessa, M; Seuster, R; Severini, H; Sfiligoj, T; Sforza, F; Sfyrla, A; Shabalina, E; Shamim, M; Shan, L Y; Shang, R; Shank, J T; Shapiro, M; Shatalov, P B; Shaw, K; Shaw, S M; Shcherbakova, A; Shehu, C Y; Sherwood, P; Shi, L; Shimizu, S; Shimmin, C O; Shimojima, M; Shiyakova, M; Shmeleva, A; Saadi, D Shoaleh; Shochet, M J; Shojaii, S; Shrestha, S; Shulga, E; Shupe, M A; Sicho, P; Sidebo, P E; Sidiropoulou, O; Sidorov, D; Sidoti, A; Siegert, F; Sijacki, Dj; Silva, J; Silver, Y; Silverstein, S B; Simak, V; Simard, O; Simic, Lj; Simion, S; Simioni, E; Simmons, B; Simon, D; Simon, M; Sinervo, P; Sinev, N B; Sioli, M; Siragusa, G; Sisakyan, A N; Sivoklokov, S Yu; Sjölin, J; Sjursen, T B; Skinner, M B; Skottowe, H P; Skubic, P; Slater, M; Slavicek, T; Slawinska, M; Sliwa, K; Smakhtin, V; Smart, B H; Smestad, L; Smirnov, S Yu; Smirnov, Y; Smirnova, L N; Smirnova, O; Smith, M N K; Smith, R W; Smizanska, M; Smolek, K; Snesarev, A A; Snidero, G; Snyder, S; Sobie, R; Socher, F; Soffer, A; Soh, D A; Sokhrannyi, G; Sanchez, C A Solans; Solar, M; Solc, J; Soldatov, E Yu; Soldevila, U; Solodkov, A A; Soloshenko, A; Solovyanov, O V; Solovyev, V; Sommer, P; Song, H Y; Soni, N; Sood, A; Sopczak, A; Sopko, B; Sopko, V; Sorin, V; Sosa, D; Sosebee, M; Sotiropoulou, C L; Soualah, R; Soukharev, A M; South, D; Sowden, B C; Spagnolo, S; Spalla, M; Spangenberg, M; Spanò, F; Spearman, W R; Sperlich, D; Spettel, F; Spighi, R; Spigo, G; Spiller, L A; Spousta, M; Denis, R D St; Stabile, A; Staerz, S; Stahlman, J; Stamen, R; Stamm, S; Stanecka, E; Stanek, R W; Stanescu, C; Stanescu-Bellu, M; Stanitzki, M M; Stapnes, S; Starchenko, E A; Stark, J; Staroba, P; Starovoitov, P; Staszewski, R; Steinberg, P; Stelzer, B; Stelzer, H J; Stelzer-Chilton, O; Stenzel, H; Stewart, G A; Stillings, J A; Stockton, M C; Stoebe, M; Stoicea, G; Stolte, P; Stonjek, S; Stradling, A R; Straessner, A; Stramaglia, M E; Strandberg, J; Strandberg, S; Strandlie, A; Strauss, E; Strauss, M; Strizenec, P; Ströhmer, R; Strom, D M; Stroynowski, R; Strubig, A; Stucci, S A; Stugu, B; Styles, N A; Su, D; Su, J; Subramaniam, R; Succurro, A; Suchek, S; Sugaya, Y; Suk, M; Sulin, V V; Sultansoy, S; Sumida, T; Sun, S; Sun, X; Sundermann, J E; Suruliz, K; Susinno, G; Sutton, M R; Suzuki, S; Svatos, M; Swiatlowski, M; Sykora, I; Sykora, T; Ta, D; Taccini, C; Tackmann, K; Taenzer, J; Taffard, A; Tafirout, R; Taiblum, N; Takai, H; Takashima, R; Takeda, H; Takeshita, T; Takubo, Y; Talby, M; Talyshev, A A; Tam, J Y C; Tan, K G; Tanaka, J; Tanaka, R; Tanaka, S; Tannenwald, B B; Araya, S Tapia; Tapprogge, S; Tarem, S; Tarrade, F; Tartarelli, G F; Tas, P; Tasevsky, M; Tashiro, T; Tassi, E; Delgado, A Tavares; Tayalati, Y; Taylor, A C; Taylor, F E; Taylor, G N; Taylor, P T E; Taylor, W; Teischinger, F A; Teixeira-Dias, P; Temming, K K; Temple, D; Kate, H Ten; Teng, P K; Teoh, J J; Tepel, F; Terada, S; Terashi, K; Terron, J; Terzo, S; Testa, M; Teuscher, R J; Theveneaux-Pelzer, T; Thomas, J P; Thomas-Wilsker, J; Thompson, E N; Thompson, P D; Thompson, R J; Thompson, A S; Thomsen, L A; Thomson, E; Thomson, M; Thun, R P; Tibbetts, M J; Torres, R E Ticse; Tikhomirov, V O; Tikhonov, Yu A; Timoshenko, S; Tiouchichine, E; Tipton, P; Tisserant, S; Todome, K; Todorov, T; Todorova-Nova, S; Tojo, J; Tokár, S; Tokushuku, K; Tollefson, K; Tolley, E; Tomlinson, L; Tomoto, M; Tompkins, L; Toms, K; Torrence, E; Torres, H; Pastor, E Torró; Toth, J; Touchard, F; Tovey, D R; Trefzger, T; Tremblet, L; Tricoli, A; Trigger, I M; Trincaz-Duvoid, S; Tripiana, M F; Trischuk, W; Trocmé, B; Troncon, C; Trottier-McDonald, M; Trovatelli, M; Truong, L; Trzebinski, M; Trzupek, A; Tsarouchas, C; Tseng, J C-L; Tsiareshka, P V; Tsionou, D; Tsipolitis, G; Tsirintanis, N; Tsiskaridze, S; Tsiskaridze, V; Tskhadadze, E G; Tsui, K M; Tsukerman, I I; Tsulaia, V; Tsuno, S; Tsybychev, D; Tudorache, A; Tudorache, V; Tuna, A N; Tupputi, S A; Turchikhin, S; Turecek, D; Turra, R; Turvey, A J; Tuts, P M; Tykhonov, A; Tylmad, M; Tyndel, M; Ueda, I; Ueno, R; Ughetto, M; Ukegawa, F; Unal, G; Undrus, A; Unel, G; Ungaro, F C; Unno, Y; Unverdorben, C; Urban, J; Urquijo, P; Urrejola, P; Usai, G; Usanova, A; Vacavant, L; Vacek, V; Vachon, B; Valderanis, C; Valencic, N; Valentinetti, S; Valero, A; Valery, L; Valkar, S; Vallecorsa, S; Ferrer, J A Valls; Van Den Wollenberg, W; Van Der Deijl, P C; van der Geer, R; van der Graaf, H; van Eldik, N; van Gemmeren, P; Van Nieuwkoop, J; van Vulpen, I; van Woerden, M C; Vanadia, M; Vandelli, W; Vanguri, R; Vaniachine, A; Vannucci, F; Vardanyan, G; Vari, R; Varnes, E W; Varol, T; Varouchas, D; Vartapetian, A; Varvell, K E; Vazeille, F; Schroeder, T Vazquez; Veatch, J; Veloce, L M; Veloso, F; Velz, T; Veneziano, S; Ventura, A; Ventura, D; Venturi, M; Venturi, N; Venturini, A; Vercesi, V; Verducci, M; Verkerke, W; Vermeulen, J C; Vest, A; Vetterli, M C; Viazlo, O; Vichou, I; Vickey, T; Boeriu, O E Vickey; Viehhauser, G H A; Viel, S; Vigne, R; Villa, M; Perez, M Villaplana; Vilucchi, E; Vincter, M G; Vinogradov, V B; Vivarelli, I; Vlachos, S; Vladoiu, D; Vlasak, M; Vogel, M; Vokac, P; Volpi, G; Volpi, M; von der Schmitt, H; von Radziewski, H; von Toerne, E; Vorobel, V; Vorobev, K; Vos, M; Voss, R; Vossebeld, J H; Vranjes, N; Milosavljevic, M Vranjes; Vrba, V; Vreeswijk, M; Vuillermet, R; Vukotic, I; Vykydal, Z; Wagner, P; Wagner, W; Wahlberg, H; Wahrmund, S; Wakabayashi, J; Walder, J; Walker, R; Walkowiak, W; Wang, C; Wang, F; Wang, H; Wang, H; Wang, J; Wang, J; Wang, K; Wang, R; Wang, S M; Wang, T; Wang, T; Wang, X; Wanotayaroj, C; Warburton, A; Ward, C P; Wardrope, D R; Washbrook, A; Wasicki, C; Watkins, P M; Watson, A T; Watson, I J; Watson, M F; Watts, G; Watts, S; Waugh, B M; Webb, S; Weber, M S; Weber, S W; Webster, J S; Weidberg, A R; Weinert, B; Weingarten, J; Weiser, C; Weits, H; Wells, P S; Wenaus, T; Wengler, T; Wenig, S; Wermes, N; Werner, M; Werner, P; Wessels, M; Wetter, J; Whalen, K; Wharton, A M; White, A; White, M J; White, R; White, S; Whiteson, D; Wickens, F J; Wiedenmann, W; Wielers, M; Wienemann, P; Wiglesworth, C; Wiik-Fuchs, L A M; Wildauer, A; Wilkens, H G; Williams, H H; Williams, S; Willis, C; Willocq, S; Wilson, A; Wilson, J A; Wingerter-Seez, I; Winklmeier, F; Winter, B T; Wittgen, M; Wittkowski, J; Wollstadt, S J; Wolter, M W; Wolters, H; Wosiek, B K; Wotschack, J; Woudstra, M J; Wozniak, K W; Wu, M; Wu, M; Wu, S L; Wu, X; Wu, Y; Wyatt, T R; Wynne, B M; Xella, S; Xu, D; Xu, L; Yabsley, B; Yacoob, S; Yakabe, R; Yamada, M; Yamaguchi, D; Yamaguchi, Y; Yamamoto, A; Yamamoto, S; Yamanaka, T; Yamauchi, K; Yamazaki, Y; Yan, Z; Yang, H; Yang, H; Yang, Y; Yao, W-M; Yap, Y C; Yasu, Y; Yatsenko, E; Wong, K H Yau; Ye, J; Ye, S; Yeletskikh, I; Yen, A L; Yildirim, E; Yorita, K; Yoshida, R; Yoshihara, K; Young, C; Young, C J S; Youssef, S; Yu, D R; Yu, J; Yu, J M; Yu, J; Yuan, L; Yuen, S P Y; Yurkewicz, A; Yusuff, I; Zabinski, B; Zaidan, R; Zaitsev, A M; Zalieckas, J; Zaman, A; Zambito, S; Zanello, L; Zanzi, D; Zeitnitz, C; Zeman, M; Zemla, A; Zeng, J C; Zeng, Q; Zengel, K; Zenin, O; Ženiš, T; Zerwas, D; Zhang, D; Zhang, F; Zhang, G; Zhang, H; Zhang, J; Zhang, L; Zhang, R; Zhang, X; Zhang, Z; Zhao, X; Zhao, Y; Zhao, Z; Zhemchugov, A; Zhong, J; Zhou, B; Zhou, C; Zhou, L; Zhou, L; Zhou, M; Zhou, N; Zhu, C G; Zhu, H; Zhu, J; Zhu, Y; Zhuang, X; Zhukov, K; Zibell, A; Zieminska, D; Zimine, N I; Zimmermann, C; Zimmermann, S; Zinonos, Z; Zinser, M; Ziolkowski, M; Živković, L; Zobernig, G; Zoccoli, A; Nedden, M Zur; Zurzolo, G; Zwalinski, L

    2017-01-01

    The reconstruction of the signal from hadrons and jets emerging from the proton-proton collisions at the Large Hadron Collider (LHC) and entering the ATLAS calorimeters is based on a three-dimensional topological clustering of individual calorimeter cell signals. The cluster formation follows cell signal-significance patterns generated by electromagnetic and hadronic showers. In this, the clustering algorithm implicitly performs a topological noise suppression by removing cells with insignificant signals which are not in close proximity to cells with significant signals. The resulting topological cell clusters have shape and location information, which is exploited to apply a local energy calibration and corrections depending on the nature of the cluster. Topological cell clustering is established as a well-performing calorimeter signal definition for jet and missing transverse momentum reconstruction in ATLAS.

  2. Cluster-cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

    NASA Astrophysics Data System (ADS)

    Alves, S. G.; Martins, M. L.

    2010-09-01

    Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and in vitro drug testing. In the present paper, a cluster-cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simulations reveal that in the absence of chemotaxy the mean cluster size and the total number of clusters scale in time as stretched exponentials dependent on the particle replication rate. Also, the dynamical cluster size distribution functions are represented by a scaling relation in which the scaling function involves a stretched exponential of the time. The introduction of chemoattraction among the particles leads to distribution functions decaying as power laws with exponents that decrease in time. The fractal dimensions and size distributions of the simulated clusters are qualitatively discussed in terms of those determined experimentally for several normal and tumoral cell lines growing in culture. It is shown that particle replication and chemotaxy account for the simplest cluster size distributions of cellular aggregates observed in culture.

  3. Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

    PubMed Central

    Pratt, Christopher P.; Kuljis, Dika A.; Homanics, Gregg E.; He, Jianjun; Kolodieznyi, Dmytro; Dudem, Srikanth; Hollywood, Mark A.; Barth, Alison L.; Bruchez, Marcel P.

    2017-01-01

    BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking. PMID:29163049

  4. DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

    PubMed

    Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

    2018-04-17

    Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and

  5. Topological cell clustering in the ATLAS calorimeters and its performance in LHC Run 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aad, G.; Abbott, B.; Abdallah, J.

    The reconstruction of the signal from hadrons and jets emerging from the proton–proton collisions at the Large Hadron Collider (LHC) and entering the ATLAS calorimeters is based on a three-dimensional topological clustering of individual calorimeter cell signals. The cluster formation follows cell signal-significance patterns generated by electromagnetic and hadronic showers. In this, the clustering algorithm implicitly performs a topological noise suppression by removing cells with insignificant signals which are not in close proximity to cells with significant signals. The resulting topological cell clusters have shape and location information, which is exploited to apply a local energy calibration and corrections dependingmore » on the nature of the cluster. Lastly, topological cell clustering is established as a well-performing calorimeter signal definition for jet and missing transverse momentum reconstruction in ATLAS.« less

  6. Topological cell clustering in the ATLAS calorimeters and its performance in LHC Run 1

    DOE PAGES

    Aad, G.; Abbott, B.; Abdallah, J.; ...

    2017-07-24

    The reconstruction of the signal from hadrons and jets emerging from the proton–proton collisions at the Large Hadron Collider (LHC) and entering the ATLAS calorimeters is based on a three-dimensional topological clustering of individual calorimeter cell signals. The cluster formation follows cell signal-significance patterns generated by electromagnetic and hadronic showers. In this, the clustering algorithm implicitly performs a topological noise suppression by removing cells with insignificant signals which are not in close proximity to cells with significant signals. The resulting topological cell clusters have shape and location information, which is exploited to apply a local energy calibration and corrections dependingmore » on the nature of the cluster. Lastly, topological cell clustering is established as a well-performing calorimeter signal definition for jet and missing transverse momentum reconstruction in ATLAS.« less

  7. Parallel hyperbolic PDE simulation on clusters: Cell versus GPU

    NASA Astrophysics Data System (ADS)

    Rostrup, Scott; De Sterck, Hans

    2010-12-01

    Increasingly, high-performance computing is looking towards data-parallel computational devices to enhance computational performance. Two technologies that have received significant attention are IBM's Cell Processor and NVIDIA's CUDA programming model for graphics processing unit (GPU) computing. In this paper we investigate the acceleration of parallel hyperbolic partial differential equation simulation on structured grids with explicit time integration on clusters with Cell and GPU backends. The message passing interface (MPI) is used for communication between nodes at the coarsest level of parallelism. Optimizations of the simulation code at the several finer levels of parallelism that the data-parallel devices provide are described in terms of data layout, data flow and data-parallel instructions. Optimized Cell and GPU performance are compared with reference code performance on a single x86 central processing unit (CPU) core in single and double precision. We further compare the CPU, Cell and GPU platforms on a chip-to-chip basis, and compare performance on single cluster nodes with two CPUs, two Cell processors or two GPUs in a shared memory configuration (without MPI). We finally compare performance on clusters with 32 CPUs, 32 Cell processors, and 32 GPUs using MPI. Our GPU cluster results use NVIDIA Tesla GPUs with GT200 architecture, but some preliminary results on recently introduced NVIDIA GPUs with the next-generation Fermi architecture are also included. This paper provides computational scientists and engineers who are considering porting their codes to accelerator environments with insight into how structured grid based explicit algorithms can be optimized for clusters with Cell and GPU accelerators. It also provides insight into the speed-up that may be gained on current and future accelerator architectures for this class of applications. Program summaryProgram title: SWsolver Catalogue identifier: AEGY_v1_0 Program summary URL

  8. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.

    2011-05-31

    T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. In this study, we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC contentmore » within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. In conclusion, this threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.« less

  9. Cerebellar cortex development in the weaver condition presents regional and age-dependent abnormalities without differences in Purkinje cells neurogenesis.

    PubMed

    Martí, Joaquín; Santa-Cruz, María C; Hervás, José P; Bayer, Shirley A; Villegas, Sandra

    2016-01-01

    Ataxias are neurological disorders associated with the degeneration of Purkinje cells (PCs). Homozygous weaver mice (wv/wv) have been proposed as a model for hereditary cerebellar ataxia because they present motor abnormalities and PC loss. To ascertain the physiopathology of the weaver condition, the development of the cerebellar cortex lobes was examined at postnatal day (P): P8, P20 and P90. Three approaches were used: 1) quantitative determination of several cerebellar features; 2) qualitative evaluation of the developmental changes occurring in the cortical lobes; and 3) autoradiographic analyses of PC generation and placement. Our results revealed a reduction in the size of the wv/wv cerebellum as a whole, confirming previous results. However, as distinguished from these reports, we observed that quantified parameters contribute differently to the abnormal growth of the wv/wv cerebellar lobes. Qualitative analysis showed anomalies in wv/wv cerebellar cytoarchitecture, depending on the age and lobe analyzed. Such abnormalities included the presence of the external granular layer after P20 and, at P90, ectopic cells located in the molecular layer following several placement patterns. Finally, we obtained autoradiographic evidence that wild-type and wv/wv PCs presented similar neurogenetic timetables, as reported. However, the innovative character of this current work lies in the fact that the neurogenetic gradients of wv/wv PCs were not modified from P8 to P90. A tendency for the accumulation of late-formed PCs in the anterior and posterior lobes was found, whereas early-generated PCs were concentrated in the central and inferior lobes. These data suggested that wv/wv PCs may migrate properly to their final destinations. The extrapolation of our results to patients affected with cerebellar ataxias suggests that all cerebellar cortex lobes are affected with several age-dependent alterations in cytoarchitectonics. We also propose that PC loss may be regionally

  10. ODE, RDE and SDE models of cell cycle dynamics and clustering in yeast.

    PubMed

    Boczko, Erik M; Gedeon, Tomas; Stowers, Chris C; Young, Todd R

    2010-07-01

    Biologists have long observed periodic-like oxygen consumption oscillations in yeast populations under certain conditions, and several unsatisfactory explanations for this phenomenon have been proposed. These ‘autonomous oscillations’ have often appeared with periods that are nearly integer divisors of the calculated doubling time of the culture. We hypothesize that these oscillations could be caused by a form of cell cycle synchronization that we call clustering. We develop some novel ordinary differential equation models of the cell cycle. For these models, and for random and stochastic perturbations, we give both rigorous proofs and simulations showing that both positive and negative growth rate feedback within the cell cycle are possible agents that can cause clustering of populations within the cell cycle. It occurs for a variety of models and for a broad selection of parameter values. These results suggest that the clustering phenomenon is robust and is likely to be observed in nature. Since there are necessarily an integer number of clusters, clustering would lead to periodic-like behaviour with periods that are nearly integer divisors of the period of the cell cycle. Related experiments have shown conclusively that cell cycle clustering occurs in some oscillating yeast cultures.

  11. Lgr4 Protein Deficiency Induces Ataxia-like Phenotype in Mice and Impairs Long Term Depression at Cerebellar Parallel Fiber-Purkinje Cell Synapses*

    PubMed Central

    Guan, Xin; Duan, Yanhong; Zeng, Qingwen; Pan, Hongjie; Qian, Yu; Li, Dali; Cao, Xiaohua; Liu, Mingyao

    2014-01-01

    Cerebellar dysfunction causes ataxia characterized by loss of balance and coordination. Until now, the molecular and neuronal mechanisms of several types of inherited cerebellar ataxia have not been completely clarified. Here, we report that leucine-rich G protein-coupled receptor 4 (Lgr4/Gpr48) is highly expressed in Purkinje cells (PCs) in the cerebellum. Deficiency of Lgr4 leads to an ataxia-like phenotype in mice. Histologically, no obvious morphological changes were observed in the cerebellum of Lgr4 mutant mice. However, the number of PCs was slightly but significantly reduced in Lgr4−/− mice. In addition, in vitro electrophysiological analysis showed an impaired long term depression (LTD) at parallel fiber-PC (PF-PC) synapses in Lgr4−/− mice. Consistently, immunostaining experiments showed that the level of phosphorylated cAMP-responsive element-binding protein (Creb) was significantly decreased in Lgr4−/− PCs. Furthermore, treatment with forskolin, an adenylyl cyclase agonist, rescued phospho-Creb in PCs and reversed the impairment in PF-PC LTD in Lgr4−/− cerebellar slices, indicating that Lgr4 is an upstream regulator of Creb signaling, which is underlying PF-PC LTD. Together, our findings demonstrate for first time an important role for Lgr4 in motor coordination and cerebellar synaptic plasticity and provide a potential therapeutic target for certain types of inherited cerebellar ataxia. PMID:25063812

  12. The miR-290-295 cluster as multi-faceted players in mouse embryonic stem cells.

    PubMed

    Yuan, Kai; Ai, Wen-Bing; Wan, Lin-Yan; Tan, Xiao; Wu, Jiang-Feng

    2017-01-01

    Increasing evidence indicates that embryonic stem cell specific microRNAs (miRNAs) play an essential role in the early development of embryo. Among them, the miR-290-295 cluster is the most highly expressed in the mouse embryonic stem cells and involved in various biological processes. In this paper, we reviewed the research progress of the function of the miR-290-295 cluster in embryonic stem cells. The miR-290-295 cluster is involved in regulating embryonic stem cell pluripotency maintenance, self-renewal, and reprogramming somatic cells to an embryonic stem cell-like state. Moreover, the miR-290-295 cluster has a latent pro-survival function in embryonic stem cells and involved in tumourigenesis and senescence with a great significance. Elucidating the interaction between the miR-290-295 cluster and other modes of gene regulation will provide us new ideas on the biology of pluripotent stem cells. In the near future, the broad prospects of the miRNA cluster will be shown in the stem cell field, such as altering cell identities with high efficiency through the transient introduction of tissue-specific miRNA cluster.

  13. Glutamate transporter EAAT4 in Purkinje cells controls intersynaptic diffusion of climbing fiber transmitter mediating inhibition of GABA release from interneurons.

    PubMed

    Satake, Shin'ichiro; Song, Si-Young; Konishi, Shiro; Imoto, Keiji

    2010-12-01

    Neurotransmitters diffuse out of the synaptic cleft and act on adjacent synapses to exert concerted control of the synaptic strength within neural pathways that converge on single target neurons. The excitatory transmitter released from climbing fibers (CFs), presumably glutamate, is shown to inhibit γ-aminobutyric acid (GABA) release at basket cell (BC)-Purkinje cell (PC) synapses in the rat cerebellar cortex through its extrasynaptic diffusion and activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on BC axon terminals. This study aimed at examining how the CF transmitter-diffusion-mediated presynaptic inhibition is controlled by glutamate transporters. Pharmacological blockade of the PC-selective neuronal transporter EAAT4 markedly enhanced CF-induced inhibition of GABAergic transmission. Tetanic CF-stimulation elicited long-term potentiation of glutamate transporters in PCs, and thereby attenuated the CF-induced inhibition. Combined use of electrophysiology and immunohistochemistry revealed a significant inverse relationship between the level of EAAT4 expression and the inhibitory action of CF-stimulation on the GABA release at different cerebellar lobules - the CF-induced inhibition was profound in lobule III, where the EAAT4 expression level was low, whereas it was minimal in lobule X, where EAAT4 was abundant. The findings clearly demonstrate that the neuronal glutamate transporter EAAT4 in PCs plays a critical role in the extrasynaptic diffusion of CF transmitter - it appears not only to retrogradely determine the degree of CF-mediated inhibition of GABAergic inputs to the PC by controlling the glutamate concentration for intersynaptic diffusion, but also regulate synaptic information processing in the cerebellar cortex depending on its differential regional distribution as well as use-dependent plasticity of uptake efficacy. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience

  14. DIMM-SC: a Dirichlet mixture model for clustering droplet-based single cell transcriptomic data.

    PubMed

    Sun, Zhe; Wang, Ting; Deng, Ke; Wang, Xiao-Feng; Lafyatis, Robert; Ding, Ying; Hu, Ming; Chen, Wei

    2018-01-01

    Single cell transcriptome sequencing (scRNA-Seq) has become a revolutionary tool to study cellular and molecular processes at single cell resolution. Among existing technologies, the recently developed droplet-based platform enables efficient parallel processing of thousands of single cells with direct counting of transcript copies using Unique Molecular Identifier (UMI). Despite the technology advances, statistical methods and computational tools are still lacking for analyzing droplet-based scRNA-Seq data. Particularly, model-based approaches for clustering large-scale single cell transcriptomic data are still under-explored. We developed DIMM-SC, a Dirichlet Mixture Model for clustering droplet-based Single Cell transcriptomic data. This approach explicitly models UMI count data from scRNA-Seq experiments and characterizes variations across different cell clusters via a Dirichlet mixture prior. We performed comprehensive simulations to evaluate DIMM-SC and compared it with existing clustering methods such as K-means, CellTree and Seurat. In addition, we analyzed public scRNA-Seq datasets with known cluster labels and in-house scRNA-Seq datasets from a study of systemic sclerosis with prior biological knowledge to benchmark and validate DIMM-SC. Both simulation studies and real data applications demonstrated that overall, DIMM-SC achieves substantially improved clustering accuracy and much lower clustering variability compared to other existing clustering methods. More importantly, as a model-based approach, DIMM-SC is able to quantify the clustering uncertainty for each single cell, facilitating rigorous statistical inference and biological interpretations, which are typically unavailable from existing clustering methods. DIMM-SC has been implemented in a user-friendly R package with a detailed tutorial available on www.pitt.edu/∼wec47/singlecell.html. wei.chen@chp.edu or hum@ccf.org. Supplementary data are available at Bioinformatics online. © The Author

  15. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  16. 'Medusa head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 3: Anti-Yo/CDR2, anti-Nb/AP3B2, PCA-2, anti-Tr/DNER, other antibodies, diagnostic pitfalls, summary and outlook.

    PubMed

    Jarius, S; Wildemann, B

    2015-09-17

    Serological testing for anti-neural autoantibodies is important in patients presenting with idiopathic cerebellar ataxia, since these autoantibodies may indicate cancer, determine treatment and predict prognosis. While some of them target nuclear antigens present in all or most CNS neurons (e.g. anti-Hu, anti-Ri), others more specifically target antigens present in the cytoplasm or plasma membrane of Purkinje cells (PC). In this series of articles, we provide a detailed review of the clinical and paraclinical features, oncological, therapeutic and prognostic implications, pathogenetic relevance, and differential laboratory diagnosis of the 12 most common PC autoantibodies (often referred to as 'Medusa head antibodies' due to their characteristic somatodendritic binding pattern when tested by immunohistochemistry). To assist immunologists and neurologists in diagnosing these disorders, typical high-resolution immunohistochemical images of all 12 reactivities are presented, diagnostic pitfalls discussed and all currently available assays reviewed. Of note, most of these antibodies target antigens involved in the mGluR1/calcium pathway essential for PC function and survival. Many of the antigens also play a role in spinocerebellar ataxia. Part 1 focuses on anti-metabotropic glutamate receptor 1-, anti-Homer protein homolog 3-, anti-Sj/inositol 1,4,5-trisphosphate receptor- and anti-carbonic anhydrase-related protein VIII-associated autoimmune cerebellar ataxia (ACA); part 2 covers anti-protein kinase C gamma-, anti-glutamate receptor delta-2-, anti-Ca/RhoGTPase-activating protein 26- and anti-voltage-gated calcium channel-associated ACA; and part 3 reviews the current knowledge on anti-Tr/delta notch-like epidermal growth factor-related receptor-, anti-Nb/AP3B2-, anti-Yo/cerebellar degeneration-related protein 2- and Purkinje cell antibody 2-associated ACA, discusses differential diagnostic aspects and provides a summary and outlook.

  17. Cerebellar Expression of Copper Chaperone for Superoxide, Cytosolic Cu/Zn-Superoxide Dismutase, 4-Hydroxy-2-Nonenal, Acrolein and Heat Shock Protein 32 in Patients with Menkes Kinky Hair Disease: Immunohistochemical Study

    PubMed Central

    Yokoyama, Atsushi; Ohno, Kousaku; Hirano, Asao; Shintaku, Masayuki; Kato, Masako; Hayashi, Kazuhiko; Kato, Shinsuke

    2014-01-01

    Background To clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky hair disease (MD), a disorder of copper absorption, we investigated the morphological and functional abnormalities of residual Purkinje cells in MD patients and the mechanism of cell death. Methods Seven MD patients and 39 neurologically normal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinje cells. In addition, we used immunohistochemistry to detect copper-dependent enzymes [cytosolic Cu/Zn-superoxide dismutase (SOD1) and copper chaperone for superoxide dismutase (CCS)], oxidative stress markers [4-hydroxy-2-nonenal (HNE) and acrolein] and heat shock protein 32 (hsp 32). Results The surviving MD Purkinje cells showed abnormal development, such as somatic sprouts and heterotopic location. Due to maldevelopment and degeneration, dendrites showed the cactus and weeping willow patterns. Axonal degeneration led to the formation of torpedoes. Quantitative analysis revealed loss of approximately 50% of the Purkinje cells in MD patients. Almost all of the normal Purkinje cells were positive for immunostaining by anti-CCS and anti-SOD1 antibodies, with staining of the cell bodies, dendrites and axons. Normal Purkinje cells were not stained by antibodies for HNE, acrolein or hsp 32. In MD patients, the majority of Purkinje cells were positive for CCS, but the positive rate for SOD1 was only about 23%. Approximately 56%, 42% and 40% of the Purkinje cells of MD patients were positive for HNE, acrolein and hsp 32, respectively. Conclusion In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment and degeneration. In the residual Purkinje cells, CCS expression seems to be nearly normal as a protective response to decreased SOD1 activity due to copper deficiency. Because oxidative stress is elevated secondary to decreased SOD1 activity, hsp 32 is induced as another protective mechanism. PMID:25067875

  18. Cerebellar expression of copper chaperone for superoxide, cytosolic cu/zn-superoxide dismutase, 4-hydroxy-2-nonenal, acrolein and heat shock protein 32 in patients with menkes kinky hair disease: immunohistochemical study.

    PubMed

    Yokoyama, Atsushi; Ohno, Kousaku; Hirano, Asao; Shintaku, Masayuki; Kato, Masako; Hayashi, Kazuhiko; Kato, Shinsuke

    2014-03-01

    To clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky hair disease (MD), a disorder of copper absorption, we investigated the morphological and functional abnormalities of residual Purkinje cells in MD patients and the mechanism of cell death. Seven MD patients and 39 neurologically normal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinje cells. In addition, we used immunohistochemistry to detect copper-dependent enzymes [cytosolic Cu/Zn-superoxide dismutase (SOD1) and copper chaperone for superoxide dismutase (CCS)], oxidative stress markers [4-hydroxy-2-nonenal (HNE) and acrolein] and heat shock protein 32 (hsp 32). The surviving MD Purkinje cells showed abnormal development, such as somatic sprouts and heterotopic location. Due to maldevelopment and degeneration, dendrites showed the cactus and weeping willow patterns. Axonal degeneration led to the formation of torpedoes. Quantitative analysis revealed loss of approximately 50% of the Purkinje cells in MD patients. Almost all of the normal Purkinje cells were positive for immunostaining by anti-CCS and anti-SOD1 antibodies, with staining of the cell bodies, dendrites and axons. Normal Purkinje cells were not stained by antibodies for HNE, acrolein or hsp 32. In MD patients, the majority of Purkinje cells were positive for CCS, but the positive rate for SOD1 was only about 23%. Approximately 56%, 42% and 40% of the Purkinje cells of MD patients were positive for HNE, acrolein and hsp 32, respectively. In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment and degeneration. In the residual Purkinje cells, CCS expression seems to be nearly normal as a protective response to decreased SOD1 activity due to copper deficiency. Because oxidative stress is elevated secondary to decreased SOD1 activity, hsp 32 is induced as another protective mechanism.

  19. Flow-Mediated Stem Cell Labeling with Superparamagnetic Iron Oxide Nanoparticle Clusters

    PubMed Central

    Shkumatov, Artem; Lai, Mei-Hsiu; Smith, Cartney E.; Rich, Max; Kong, Hyunjoon

    2013-01-01

    This study presents a strategy to enhance the uptake of superparamagnetic iron oxide nanoparticle (SPIO) clusters by manipulating the cellular mechanical environment. Specifically, stem cells exposed to an orbital flow ingested almost two-fold greater amount of SPIO clusters than those cultured statically. Improvements in MR contrast were subsequently achieved for labeled cells in collagen gels and a mouse model. Overall, this strategy will serve to improve the efficiency of cell tracking and therapies. PMID:24033276

  20. Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

    NASA Technical Reports Server (NTRS)

    Sutherland, Betsy M.; Bennett, Paula V.; Cintron-Torres, Nela; Hada, Megumi; Trunk, John; Monteleone, Denise; Sutherland, John C.; Laval, Jacques; Stanislaus, Marisha; Gewirtz, Alan

    2002-01-01

    Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

  1. FLOCK cluster analysis of mast cell event clustering by high-sensitivity flow cytometry predicts systemic mastocytosis.

    PubMed

    Dorfman, David M; LaPlante, Charlotte D; Pozdnyakova, Olga; Li, Betty

    2015-11-01

    In our high-sensitivity flow cytometric approach for systemic mastocytosis (SM), we identified mast cell event clustering as a new diagnostic criterion for the disease. To objectively characterize mast cell gated event distributions, we performed cluster analysis using FLOCK, a computational approach to identify cell subsets in multidimensional flow cytometry data in an unbiased, automated fashion. FLOCK identified discrete mast cell populations in most cases of SM (56/75 [75%]) but only a minority of non-SM cases (17/124 [14%]). FLOCK-identified mast cell populations accounted for 2.46% of total cells on average in SM cases and 0.09% of total cells on average in non-SM cases (P < .0001) and were predictive of SM, with a sensitivity of 75%, a specificity of 86%, a positive predictive value of 76%, and a negative predictive value of 85%. FLOCK analysis provides useful diagnostic information for evaluating patients with suspected SM, and may be useful for the analysis of other hematopoietic neoplasms. Copyright© by the American Society for Clinical Pathology.

  2. Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters

    PubMed Central

    Ng, Mei Rosa; Besser, Achim; Brugge, Joan S; Danuser, Gaudenz

    2014-01-01

    Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions. DOI: http://dx.doi.org/10.7554/eLife.03282.001 PMID:25479385

  3. Magnetic assembly of 3D cell clusters: visualizing the formation of an engineered tissue.

    PubMed

    Ghosh, S; Kumar, S R P; Puri, I K; Elankumaran, S

    2016-02-01

    Contactless magnetic assembly of cells into 3D clusters has been proposed as a novel means for 3D tissue culture that eliminates the need for artificial scaffolds. However, thus far its efficacy has only been studied by comparing expression levels of generic proteins. Here, it has been evaluated by visualizing the evolution of cell clusters assembled by magnetic forces, to examine their resemblance to in vivo tissues. Cells were labeled with magnetic nanoparticles, then assembled into 3D clusters using magnetic force. Scanning electron microscopy was used to image intercellular interactions and morphological features of the clusters. When cells were held together by magnetic forces for a single day, they formed intercellular contacts through extracellular fibers. These kept the clusters intact once the magnetic forces were removed, thus serving the primary function of scaffolds. The cells self-organized into constructs consistent with the corresponding tissues in vivo. Epithelial cells formed sheets while fibroblasts formed spheroids and exhibited position-dependent morphological heterogeneity. Cells on the periphery of a cluster were flattened while those within were spheroidal, a well-known characteristic of connective tissues in vivo. Cells assembled by magnetic forces presented visual features representative of their in vivo states but largely absent in monolayers. This established the efficacy of contactless assembly as a means to fabricate in vitro tissue models. © 2016 John Wiley & Sons Ltd.

  4. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    PubMed Central

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-01-01

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

  5. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    NASA Astrophysics Data System (ADS)

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-06-01

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

  6. RNA Polymerase II cluster dynamics predict mRNA output in living cells

    PubMed Central

    Cho, Won-Ki; Jayanth, Namrata; English, Brian P; Inoue, Takuma; Andrews, J Owen; Conway, William; Grimm, Jonathan B; Spille, Jan-Hendrik; Lavis, Luke D; Lionnet, Timothée; Cisse, Ibrahim I

    2016-01-01

    Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output. DOI: http://dx.doi.org/10.7554/eLife.13617.001 PMID:27138339

  7. Phase resetting in a model of cardiac Purkinje fiber.

    PubMed Central

    Guevara, M R; Shrier, A

    1987-01-01

    The phase-resetting response of a model of spontaneously active cardiac Purkinje fiber is investigated. The effect on the interbeat interval of injecting a 20-ms duration depolarizing current pulse is studied as a function of the phase in the cycle at which the pulse is delivered. At low current amplitudes, a triphasic response is recorded as the pulse is advanced through the cycle. At intermediate current amplitudes, the response becomes quinquephasic, due to the presence of supernormal excitability. At high current amplitudes, a triphasic response is seen once more. At low stimulus amplitudes, type 1 phase resetting occurs; at medium amplitudes, a type could not be ascribed to the phase resetting because of the presence of effectively all-or-none depolarization; at high amplitudes, type 0 phase resetting occurs. The modeling results closely correspond with published experimental data; in particular type 1 and type 0 phase resetting are seen. Implications for the induction of ventricular arrhythmias are considered. PMID:3663827

  8. Tomographic brain imaging with nucleolar detail and automatic cell counting

    NASA Astrophysics Data System (ADS)

    Hieber, Simone E.; Bikis, Christos; Khimchenko, Anna; Schweighauser, Gabriel; Hench, Jürgen; Chicherova, Natalia; Schulz, Georg; Müller, Bert

    2016-09-01

    Brain tissue evaluation is essential for gaining in-depth insight into its diseases and disorders. Imaging the human brain in three dimensions has always been a challenge on the cell level. In vivo methods lack spatial resolution, and optical microscopy has a limited penetration depth. Herein, we show that hard X-ray phase tomography can visualise a volume of up to 43 mm3 of human post mortem or biopsy brain samples, by demonstrating the method on the cerebellum. We automatically identified 5,000 Purkinje cells with an error of less than 5% at their layer and determined the local surface density to 165 cells per mm2 on average. Moreover, we highlight that three-dimensional data allows for the segmentation of sub-cellular structures, including dendritic tree and Purkinje cell nucleoli, without dedicated staining. The method suggests that automatic cell feature quantification of human tissues is feasible in phase tomograms obtained with isotropic resolution in a label-free manner.

  9. Reduced expression of the Ca(2+) transporter protein PMCA2 slows Ca(2+) dynamics in mouse cerebellar Purkinje neurones and alters the precision of motor coordination.

    PubMed

    Empson, Ruth M; Turner, Paul R; Nagaraja, Raghavendra Y; Beesley, Philip W; Knöpfel, Thomas

    2010-03-15

    Cerebellar Purkinje neurones (PNs) express high levels of the plasma membrane calcium ATPase, PMCA2, a transporter protein critical for the clearance of calcium from excitable cells. Genetic deletion of one PMCA2 encoding gene in heterozygous PMCA2 knock-out (PMCA2(+/-) mice enabled us to determine how PMCA2 influences PN calcium regulation without the complication of the severe morphological changes associated with complete PMCA2 knock-out (PMCA2(-/-) in these cells. The PMCA2(+/-) cerebellum expressed half the normal levels of PMCA2 and this nearly doubled the time taken for PN dendritic calcium transients to recover (mean fast and slow recovery times increased from 70 ms to 110 ms and from 600 ms to 1100 ms). The slower calcium recovery had distinct consequences for PMCA2(+/-) PN physiology. The PNs exhibited weaker climbing fibre responses, prolonged outward Ca(2+)-dependent K(+) current (mean fast and slow recovery times increased from 136 ms to 192 ms and from 595 ms to 1423 ms) and a slower mean frequency of action potential firing (7.4 Hz compared with 15.8 Hz). Our findings were consistent with prolonged calcium accumulation in the cytosol of PMCA2(+/-) Purkinje neurones. Although PMCA2(+/-) mice exhibited outwardly normal behaviour and little change in their gait pattern, when challenged to run on a narrow beam they exhibited clear deficits in hindlimb coordination. Training improved the motor performance of both PMCA2(+/-) and wild-type mice, although PMCA2(+/-) mice were always impaired. We conclude that reduced calcium clearance perturbs calcium dynamics in PN dendrites and that this is sufficient to disrupt the accuracy of cerebellar processing and motor coordination.

  10. Cell-targeted platinum nanoparticles and nanoparticle clusters.

    PubMed

    Papst, Stefanie; Brimble, Margaret A; Evans, Clive W; Verdon, Daniel J; Feisst, Vaughan; Dunbar, P Rod; Tilley, Richard D; Williams, David E

    2015-06-21

    Herein, we report the facile preparation of cell-targeted platinum nanoparticles (PtNPs), through the design of peptides that, as a single molecule added in small concentration during the synthesis, control the size of PtNP clusters during their growth, stabilise the PtNPs in aqueous suspension and enable the functionalisation of the PtNPs with a versatile range of cell-targeting ligands. Water-soluble PtNPs targeted respectively at blood group antigens and at integrin receptors are demonstrated.

  11. Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images

    PubMed Central

    Wang, Yuliang; Zhang, Zaicheng; Wang, Huimin; Bi, Shusheng

    2015-01-01

    Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells. PMID:26066315

  12. Hydroxyurea Treatment and Development of the Rat Cerebellum: Effects on the Neurogenetic Profiles and Settled Patterns of Purkinje Cells and Deep Cerebellar Nuclei Neurons.

    PubMed

    Martí, Joaquín; Santa-Cruz, M C; Serra, Roger; Hervás, José P

    2016-11-01

    The current paper analyzes the development of the male and female rat cerebellum exposed to hydroxyurea (HU) (300 or 600 mg/kg) as embryo and collected at postnatal day 90. Our study reveals that the administration of this drug compromises neither the cytoarchitecture of the cerebellar cortex nor deep nuclei (DCN). However, in comparison with the saline group, we observed that several cerebellar parameters were lower in the HU injected groups. These parameters included area of the cerebellum, cerebellar cortex length, molecular layer area, Purkinje cell number, granule cell counts, internal granular layer, white matter and cerebellar nuclei areas, and number of deep cerebellar nuclei neurons. These features were larger in the rats injected with saline, smaller in those exposed to 300 mg/kg of HU and smallest in the group receiving 600 mg/kg of this agent. No sex differences in the effect of the HU were observed. In addition, we infer the neurogenetic timetables and the neurogenetic gradients of PCs and DCN neurons in rats exposed to either saline or HU as embryos. For this purpose, 5-bromo-2'-deoxyuridine was injected into pregnant rats previously administered with saline or HU. This thymidine analog was administered following a progressively delayed cumulative labeling method. The data presented here show that systematic differences exist in the pattern of neurogenesis and in the spatial location of cerebellar neurons between rats injected with saline or HU. No sex differences in the effect of the HU were observed. These findings have implications for the administration of this compound to women in gestation as the effects of HU on the development of the cerebellum might persist throughout their offsprings' life.

  13. Visualization of T Cell-Regulated Monocyte Clusters Mediating Keratinocyte Death in Acquired Cutaneous Immunity.

    PubMed

    Liu, Zheng; Yang, Fei; Zheng, Hao; Fan, Zhan; Qiao, Sha; Liu, Lei; Tao, Juan; Luo, Qingming; Zhang, Zhihong

    2018-06-01

    It remains unclear how monocytes are mobilized to amplify inflammatory reactions in T cell-mediated adaptive immunity. Here, we investigate dynamic cellular events in the cascade of inflammatory responses through intravital imaging of a multicolor-labeled murine contact hypersensitivity model. We found that monocytes formed clusters around hair follicles in the contact hypersensitivity model. In this process, effector T cells encountered dendritic cells under regions of monocyte clusters and secreted IFN-γ, which mobilizes CCR2-dependent monocyte interstitial migration and CXCR2-dependent monocyte cluster formation. We showed that hair follicles shaped the inflammatory microenvironment for communication among the monocytes, keratinocytes, and effector T cells. After disrupting the T cell-mobilized monocyte clusters through CXCR2 antagonization, monocyte activation and keratinocyte apoptosis were significantly inhibited. Our study provides a new perspective on effector T cell-regulated monocyte behavior, which amplifies the inflammatory reaction in acquired cutaneous immunity. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Unique Cardiac Purkinje Fiber Transient Outward Current β-Subunit Composition

    PubMed Central

    Xiao, Ling; Koopmann, Tamara T.; Ördög, Balázs; Postema, Pieter G.; Verkerk, Arie O.; Iyer, Vivek; Sampson, Kevin J.; Boink, Gerard J.J.; Mamarbachi, Maya A.; Varro, Andras; Jordaens, Luc; Res, Jan; Kass, Robert S.; Wilde, Arthur A.; Bezzina, C.R.; Nattel, Stanley

    2015-01-01

    Rationale A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current (Ito) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF Ito and that its overexpression might specifically alter PF Ito properties and repolarization. Objective To assess the potential role of DPP6 in PF Ito. Methods and Results Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle Ito had similar density, but PF Ito differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, Ito density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K+-channel interacting β-subunit K+-channel interacting protein type-2, essential for normal expression of Ito in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small Ito; Ito amplitude was greatly enhanced by coexpression with K+-channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K+-channel interacting protein type-2 failed to alter Ito compared with Kv4.3/K+-channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF Ito composition) greatly enhanced Ito compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that Ito enhancement can greatly accelerate PF repolarization. Conclusions These results point to a previously unknown central role of DPP6 in PF Ito, with DPP6 gain of function

  15. Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

    PubMed

    Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

    2005-05-01

    Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

  16. Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Edwards, Joshua R.; Marty, M. Sue; Atchison, William D.

    2005-11-01

    The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 {mu}M MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 {mu}M MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in amore » proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca{sup 2+} and non-Ca{sup 2+} divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 {mu}M MeHg, 97.7% of Purkinje cells were viable. At 3 {mu}M MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 {mu}M MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca{sup 2+} divalent cation released by MeHg in Purkinje neurons.« less

  17. 3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

    PubMed

    Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

    2015-01-01

    Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

  18. Epigenetic repression of HOXB cluster in oral cancer cell lines.

    PubMed

    Xavier, Flávia Caló Aquino; Destro, Maria Fernanda de Souza Setubal; Duarte, Carina Magalhães Esteves; Nunes, Fabio Daumas

    2014-08-01

    Aberrant DNA methylation is a fundamental transcriptional control mechanism in carcinogenesis. The expression of homeobox genes is usually controlled by an epigenetic mechanism, such as the methylation of CpG islands in the promoter region. The aim of this study was to describe the differential methylation pattern of HOX genes in oral squamous cell carcinoma (OSCC) cell lines and transcript status in a group of hypermethylated and hypomethylated genes. Quantitative analysis of DNA methylation was performed on two OSCC cell lines (SCC4 and SCC9) using a method denominated Human Homeobox Genes EpiTect Methyl qPCR Arrays, which allowed fast, precise methylation detection of 24 HOX specific genes without bisulfite conversion. Methylation greater than 50% was detected in HOXA11, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXC8 and HOXD10. Both cell lines demonstrated similar hypermethylation status for eight HOX genes. A similar pattern of promoter hypermethylation and hypomethylation was demonstrated for the HOXB cluster and HOXA cluster, respectively. Moreover, the hypermethylation profile of the HOXB cluster, especially HOXB4, was correlated with decreased transcript expression, which was restored following treatment with 5-aza-2'-deoxycytidine. The homeobox methylation profile in OSCC cell lines is consistent with an epigenetic biomarker. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. A multitask clustering approach for single-cell RNA-seq analysis in Recessive Dystrophic Epidermolysis Bullosa

    PubMed Central

    Petegrosso, Raphael; Tolar, Jakub

    2018-01-01

    Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC. PMID:29630593

  20. Blue two-photon fluorescence metal cluster probe precisely marking cell nuclei of two cell lines.

    PubMed

    Wang, Yaling; Cui, Yanyan; Liu, Ru; Wei, Yueteng; Jiang, Xinglu; Zhu, Huarui; Gao, Liang; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun

    2013-11-25

    A bifunctional peptide was designed to in situ reduce Cu ions and anchor a Cu cluster. The peptide-Cu cluster probe, mainly composed of Cu14, emitted blue two-photon fluorescence under femtosecond laser excitation. Most important, the probe can specifically mark the nuclei of HeLa and A549 cells, respectively.

  1. Manipulation of visible-light polarization with dendritic cell-cluster metasurfaces.

    PubMed

    Fang, Zhen-Hua; Chen, Huan; An, Di; Luo, Chun-Rong; Zhao, Xiao-Peng

    2018-06-26

    Cross-polarization conversion plays an important role in visible light manipulation. Metasurface with asymmetric structure can be used to achieve polarization conversion of linearly polarized light. Based on this, we design a quasi-periodic dendritic metasurface model composed of asymmetric dendritic cells. The simulation indicates that the asymmetric dendritic structure can vertically rotate the polarization direction of the linear polarization wave in visible light. Silver dendritic cell-cluster metasurface samples were prepared by the bottom-up electrochemical deposition. It experimentally proved that they could realize the cross - polarization conversion in visible light. Cross-polarized propagating light is deflected into anomalous refraction channels. Dendritic cell-cluster metasurface with asymmetric quasi-periodic structure conveys significance in cross-polarization conversion research and features extensive practical application prospect and development potential.

  2. MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway.

    PubMed

    Pande, Hari Om; Tesfaye, Dawit; Hoelker, Michael; Gebremedhn, Samuel; Held, Eva; Neuhoff, Christiane; Tholen, Ernst; Schellander, Karl; Wondim, Dessie Salilew

    2018-05-01

    The granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches. The role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling

  3. Minimal disease detection of B-cell lymphoproliferative disorders by flow cytometry: multidimensional cluster analysis.

    PubMed

    Duque, Ricardo E

    2012-04-01

    Flow cytometric analysis of cell suspensions involves the sequential 'registration' of intrinsic and extrinsic parameters of thousands of cells in list mode files. Thus, it is almost irresistible to describe phenomena in numerical terms or by 'ratios' that have the appearance of 'accuracy' due to the presence of numbers obtained from thousands of cells. The concepts involved in the detection and characterization of B cell lymphoproliferative processes are revisited in this paper by identifying parameters that, when analyzed appropriately, are both necessary and sufficient. The neoplastic process (cluster) can be visualized easily because the parameters that distinguish it form a cluster in multidimensional space that is unique and distinguishable from neighboring clusters that are not of diagnostic interest but serve to provide a background. For B cell neoplasia it is operationally necessary to identify the multidimensional space occupied by a cluster whose kappa:lambda ratio is 100:0 or 0:100. Thus, the concept of kappa:lambda ratio is without meaning and would not detect B cell neoplasia in an unacceptably high number of cases.

  4. Inhibition by enflurane and methoxyflurane of postdrive hyperpolarization in canine Purkinje fibers.

    PubMed

    Pratila, M; Vogel, S; Sperelakis, N

    1984-05-01

    When a pacemaker cell is driven with a train of stimuli at a rate faster than its own, the termination of the drive is followed by a transient hyperpolarization, due to the activity of an electrogenic Na+-K+ pump. In this study, the effect of the halogenated ethers, enflurane and methoxyflurane, on postdrive hyperpolarization (PDH) was determined in cardiac Purkinje fibers. The fibers were removed from freshly excised canine hearts and superfused with a Tyrode's solution (containing 2.7 or 3.5 mM K+). The preparation was paced at 0.2 Hz before and after drives, and at 2 Hz during drives. Under control conditions, drives of 2 min produced a PDH of 5.5 +/- 0.2 mV. Enflurane (1.5-5%) significantly reduced the PDH. At 4 to 5%, enflurane reduced the PDH to a mean value of 42% of the control. Methoxyflurane was more potent than enflurane in affecting the PDH. At 0.5 to 0.75%, methoxyflurane reduced the PDH to 5% of the control. At higher (1-1.5%) concentrations of methoxyflurane, the PDH was converted to a depolarization, which varied between 0.5 and 8.0 mV. The PDH was restored to control levels within 10 to 20 min after washout of either anesthetic agent. Methoxyflurane (0.5 or 1%) enhanced the automaticity of spontaneously firing cells (2.35 mM K+ Tyrode's solution used). This positive chronotropic action coincided with a depolarization of 2 to 8 mV. Enflurane, at concentrations of 3 to 5%, gave similar results. On the action potential, methoxyflurane, at 1%, reduced the amplitude and duration (measured at 50% repolarization) of the plateau, and also the maximal upstroke velocity (+Vmax) of the rising phase.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Developmental Hypothyroxinemia and Hypothyroidism Reduce Parallel Fiber-Purkinje Cell Synapses in Rat Offspring by Downregulation of Neurexin1/Cbln1/GluD2 Tripartite Complex.

    PubMed

    Wang, Yuan; Dong, Jing; Wang, Yi; Wei, Wei; Song, Binbin; Shan, Zhongyan; Teng, Weiping; Chen, Jie

    2016-10-01

    Iodine is a significant micronutrient. Iodine deficiency (ID)-induced hypothyroxinemia and hypothyroidism during developmental period can cause cerebellar dysfunction. However, mechanisms are still unclear. Therefore, the present research aims to study effects of developmental hypothyroxinemia caused by mild ID and hypothyroidism caused by severe ID or methimazole (MMZ) on parallel fiber-Purkinje cell (PF-PC) synapses in filial cerebellum. Maternal hypothyroxinemia and hypothyroidism models were established in Wistar rats using ID diet and deionized water supplemented with different concentrations of potassium iodide or MMZ water. Birth weight and cerebellum weight were measured. We also examined PF-PC synapses using immunofluorescence, and western blot analysis was conducted to investigate the activity of Neurexin1/cerebellin1 (Cbln1)/glutamate receptor d2 (GluD2) tripartite complex. Our results showed that hypothyroxinemia and hypothyroidism decreased birth weight and cerebellum weight and reduced the PF-PC synapses on postnatal day (PN) 14 and PN21. Accordingly, the mean intensity of vesicular glutamate transporter (VGluT1) and Calbindin immunofluorescence was reduced in mild ID, severe ID, and MMZ groups. Moreover, maternal hypothyroxinemia and hypothyroidism reduced expression of Neurexin1/Cbln1/GluD2 tripartite complex. Our study supports the hypothesis that developmental hypothyroxinemia and hypothyroidism reduce PF-PC synapses, which may be attributed to the downregulation of Neurexin1/Cbln1/GluD2 tripartite complex.

  6. Effects of radiation quality and oxygen on clustered DNA lesions and cell death.

    PubMed

    Stewart, Robert D; Yu, Victor K; Georgakilas, Alexandros G; Koumenis, Constantinos; Park, Joo Han; Carlson, David J

    2011-11-01

    Radiation quality and cellular oxygen concentration have a substantial impact on DNA damage, reproductive cell death and, ultimately, the potential efficacy of radiation therapy for the treatment of cancer. To better understand and quantify the effects of radiation quality and oxygen on the induction of clustered DNA lesions, we have now extended the Monte Carlo Damage Simulation (MCDS) to account for reductions in the initial lesion yield arising from enhanced chemical repair of DNA radicals under hypoxic conditions. The kinetic energy range and types of particles considered in the MCDS have also been expanded to include charged particles up to and including (56)Fe ions. The induction of individual and clustered DNA lesions for arbitrary mixtures of different types of radiation can now be directly simulated. For low-linear energy transfer (LET) radiations, cells irradiated under normoxic conditions sustain about 2.9 times as many double-strand breaks (DSBs) as cells irradiated under anoxic conditions. New experiments performed by us demonstrate similar trends in the yields of non-DSB (Fpg and Endo III) clusters in HeLa cells irradiated by γ rays under aerobic and hypoxic conditions. The good agreement among measured and predicted DSBs, Fpg and Endo III cluster yields suggests that, for the first time, it may be possible to determine nucleotide-level maps of the multitude of different types of clustered DNA lesions formed in cells under reduced oxygen conditions. As particle LET increases, the MCDS predicts that the ratio of DSBs formed under normoxic to hypoxic conditions by the same type of radiation decreases monotonically toward unity. However, the relative biological effectiveness (RBE) of higher-LET radiations compared to (60)Co γ rays (0.24 keV/μm) tends to increase with decreasing oxygen concentration. The predicted RBE of a 1 MeV proton (26.9 keV/μm) relative to (60)Co γ rays for DSB induction increases from 1.9 to 2.3 as oxygen concentration

  7. p53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

    PubMed Central

    Yi, Qiyi; Zhao, Xiaoyu; Huang, Yun; Ma, Tieliang; Zhang, Yingyin; Hou, Heli; Cooke, Howard J.; Yang, Da-Qing; Wu, Mian; Shi, Qinghua

    2011-01-01

    Background p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Principal Findings Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Conclusions p53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. PMID:22076149

  8. Repeated inhalation of sevoflurane inhibits the information transmission of Purkinje cells and delays motor development via the GABAA receptor ε subunit in neonatal mice.

    PubMed

    Fang, Hong; Wang, Ze-Hua; Bu, Ying-Jiang; Yuan, Zhi-Jun; Wang, Guo-Qiang; Guo, Yan; Cheng, Xiao-Yun; Qiu, Wen-Jie

    2018-01-01

    General anesthesia is widely used in pediatric surgery, although the influence of general anesthesia on cerebellar information transmission and motor function is unclear. In the present study, neonatal mice received repeated inhalation of sevoflurane, and electrophysiological alterations in Purkinje cells (PCs) and the development of motor functions were detected. In addition, γ‑aminobutyric acidA receptor ε (GABAA‑R ε) subunit knockout mice were used to investigate the mechanism of action of sevoflurane on cerebellar function. In the neonatal mice, the field potential response of PCs induced by sensory stimulation and the motor function indices were markedly inhibited by sevoflurane, and the inhibitory effect was positively associated with the number of repetitions of anesthesia. In additional the GABAA‑R ε subunit level of PCs was promoted by sevoflurane in a dose‑dependent manner, and the inhibitory effects of sevoflurane on PC field potential response and motor function were alleviated in GABAA‑R ε subunit knockout mice. The GABAA‑R ε subunit was activated by sevoflurane, leading to inhibition of sensory information transmission in the cerebellar cortex, field potential responses of PCs and the development of cerebellar motor function. The present study provided experimental evidence for the safe usage of sevoflurane in clinical anesthesia, and suggested that GABAA‑R ε subunit antagonists may be considered for combined application with general anesthesia with repeated inhalation of sevoflurane, for adverse effect prevention in the clinic.

  9. Gray-level co-occurrence matrix analysis of several cell types in mouse brain using resolution-enhanced photothermal microscopy

    NASA Astrophysics Data System (ADS)

    Kobayashi, Takayoshi; Sundaram, Durga; Nakata, Kazuaki; Tsurui, Hiromichi

    2017-03-01

    Qualifications of intracellular structure were performed for the first time using the gray-level co-occurrence matrix (GLCM) method for images of cells obtained by resolution-enhanced photothermal imaging. The GLCM method has been used to extract five parameters of texture features for five different types of cells in mouse brain; pyramidal neurons and glial cells in the basal nucleus (BGl), dentate gyrus granule cells, cerebellar Purkinje cells, and cerebellar granule cells. The parameters are correlation, contrast, angular second moment (ASM), inverse difference moment (IDM), and entropy for the images of cells of interest in a mouse brain. The parameters vary depending on the pixel distance taken in the analysis method. Based on the obtained results, we identified that the most suitable GLCM parameter is IDM for pyramidal neurons and BGI, granule cells in the dentate gyrus, Purkinje cells and granule cells in the cerebellum. It was also found that the ASM is the most appropriate for neurons in the basal nucleus.

  10. Expression of key ion channels in the rat cardiac conduction system by laser capture microdissection and quantitative real-time PCR.

    PubMed

    Ou, Yan; Niu, Xiao-lin; Ren, Fu-xian

    2010-09-01

    The objective of this study was to investigate the molecular basis of the inferior nodal extension (INE) in the atrioventricular junctional area that accounts for arrhythmias. The INE was separated from the adult rat heart by laser capture microdissection. The mRNA expression of ion channels was detected by quantitative real-time PCR. Hierarchical clustering was used to demonstrate clustering of expression of genes in sections. The mRNA expression of HCN4, Ca(v)3.1 and Ca(v)3.2 was high in the INE, atrioventricular node and sino-atrial node, and that of Ca(v)3.2 high in Purkinje fibres. Although the expression of HCN1 and Ca(v)1.3 was low in the rat heart, it was relatively higher in the INE, atrioventricular node and sino-atrial node than in right atrial and right ventricular (working) myocytes. Both HCN2 and Ca(v)1.2 were expressed at higher levels in working myocytes than in nodal tissues and in the INE. Hierarchical clustering analysis demonstrated that the expression of the HCN and calcium channels in INE was similar to that in the slow-response automatic cells and different from that in working myocytes and Purkinje fibres. The expression of HCN and calcium channels in the INE of the adult rat heart is similar to that of slow-response automatic cells and provides a substrate for automatic phase 4 depolarization in cells.

  11. Aberrant c-erbB2 expression in cell clusters overlying focally disrupted breast myoepithelial cell layers: a trigger or sign for emergence of more aggressive cell clones?

    PubMed Central

    Zhang, Xichen; Hashemi, Shahreyar Shar; Yousefi, Morvarid; Ni, Jinsong; Wang, Qiuyue; Gao, Ling; Gong, Pengtao; Gao, Chunling; Sheng, Joy; Mason, Jeffrey; Man, Yan-gao

    2008-01-01

    Our recent studies revealed that cell clusters overlying focal myoepithelial cell layer disruption (FMCLD) had a significantly higher frequency of genetic instabilities and expression of invasion-related genes than their adjacent counterparts within the same duct. Our current study attempted to assess whether these cell clusters would also have elevated c-erbB2 expression. Human breast tumors (n=50) with a high frequency of FMCLD were analyzed with double immunohistochemistry, real-time RT-PCR, and chromogenic in situ hybridization for c-erbB2 protein and gene expression. Of 448 FMCLD detected, 404 (90.2%) were associated with cell clusters that had intense c-erbB2 immunoreactivities primarily in their cytoplasm, in contrast to their adjacent counterparts within the same duct, which had no or barely detectable c-erbB2 expression. These c-erbB2 positive cells were arranged as tongue-like projections, “puncturing” into the stroma, and about 20% of them were in direct continuity with tube-like structures that resembled blood vessels. Aberrant c-erbB2 expression was also seen in clusters of architecturally normal-appearing ducts that had distinct cytological abnormalities in both ME and epithelial cells, whereas not in their clear-cut normal counterparts. Molecular assays detected markedly higher c-erbB2 mRNA and gene amplification in cell clusters associated with FMCLD than in those associated with non-disrupted ME cell layers. Our findings suggest that cell clusters overlying FMCLD may represent the precursors of pending invasive lesions, and that aberrant cerbB2 expression may trigger or signify the emergence of biologically more aggressive cell clones. PMID:18726004

  12. Clustered localization of STAT3 during the cell cycle detected by super-resolution fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Gao, Jing; Chen, Junling; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tong, Ti; Wang, Hongda

    2017-06-01

    Signal transducer and activator of transcription 3 (STAT3) plays a key role in various cellular processes such as cell proliferation, differentiation, apoptosis and immune responses. In particular, STAT3 has emerged as a potential molecular target for cancer therapy. The functional role and standard activation mechanism of STAT3 have been well studied, however, the spatial distribution of STAT3 during the cell cycle is poorly known. Therefore, it is indispensable to study STAT3 spatial arrangement and nuclear-cytoplasimic localization at the different phase of cell cycle in cancer cells. By direct stochastic optical reconstruction microscopy imaging, we find that STAT3 forms various number and size of clusters at the different cell-cycle stage, which could not be clearly observed by conventional fluorescent microscopy. STAT3 clusters get more and larger gradually from G1 to G2 phase, during which time transcription and other related activities goes on consistently. The results suggest that there is an intimate relationship between the clustered characteristic of STAT3 and the cell-cycle behavior. Meanwhile, clustering would facilitate STAT3 rapid response to activating signals due to short distances between molecules. Our data might open a new door to develop an antitumor drug for inhibiting STAT3 signaling pathway by destroying its clusters.

  13. Altered differentiation and clustering of Sertoli cells in transgenic mice showing a Sertoli cell specific knockout of the connexin 43 gene.

    PubMed

    Weider, Karola; Bergmann, Martin; Giese, Sarah; Guillou, Florian; Failing, Klaus; Brehm, Ralph

    2011-07-01

    Histological analysis revealed that Sertoli cell specific knockout of the predominant testicular gap junction protein connexin 43 results in a spermatogenic arrest at the level of spermatogonia or Sertoli cell-only syndrome, intratubular cell clusters and still proliferating adult Sertoli cells, implying an important role for connexin 43 in the Sertoli and germ cell development. This study aimed to determine the (1) Sertoli cell maturation state, (2) time of occurrence and (3) composition, differentiation and fate of clustered cells in knockout mice. Using immunohistochemistry connexin 43 deficient Sertoli cells showed an accurate start of the mature markers androgen receptor and GATA-1 during puberty and a vimentin expression from neonatal to adult. Expression of anti-Muellerian hormone, as a marker of Sertoli cell immaturity, was finally down-regulated during puberty, but its disappearance was delayed. This observed extended anti-Müllerian hormone synthesis during puberty was confirmed by western blot and Real-Time PCR and suggests a partial alteration in the Sertoli cell differentiation program. Additionally, Sertoli cells of adult knockouts showed a permanent and uniform expression of GATA-1 at protein and mRNA level, maybe caused by the lack of maturing germ cells and missing negative feedback signals. At ultrastructural level, basally located adult Sertoli cells obtained their mature appearance, demonstrated by the tripartite nucleolus as a typical feature of differentiated Sertoli cells. Intratubular clustered cells were mainly formed by abnormal Sertoli cells and single attached apoptotic germ cells, verified by immunohistochemistry, TUNEL staining and transmission electron microscopy. Clusters first appeared during puberty and became more numerous in adulthood with increasing cell numbers per cluster suggesting an age-related process. In conclusion, adult connexin 43 deficient Sertoli cells seem to proliferate while maintaining expression of mature

  14. Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis

    PubMed Central

    Matsui, Takaaki; Thitamadee, Siripong; Murata, Tomoko; Kakinuma, Hisaya; Nabetani, Takuji; Hirabayashi, Yoshio; Hirate, Yoshikazu; Okamoto, Hitoshi; Bessho, Yasumasa

    2011-01-01

    The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer's vesicle (KV), a key organ required for the left–right asymmetric body plan in zebrafish, is generated from a cluster of ∼20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering. Cnpy1 positively regulates FGF signals within DFCs, which in turn promote Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left–right asymmetric body plan. PMID:21628557

  15. Photothermolysis by laser-induced microbubbles generated around gold nanorod clusters selectively formed in leukemia cells

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitri; Lukianova-Hleb, Ekaterina; Zhdanok, Sergei; Rostro, Betty; Simonette, Rebecca; Hafner, Jason; Konopleva, Marina; Andreeff, Michael; Conjusteau, Andre; Oraevsky, Alexander

    2008-02-01

    In an effort of developing clinical LANTCET (laser-activated nano-thermolysis as cell elimination technology) we achieved selective destruction of individual tumor cells through laser generation of vapor microbubbles around clusters of light absorbing gold nanorods (GNR) selectively formed in target tumor cells. Among all gold nanoparticles, nanorods offer the highest optical absorption in the near-infrared. We applied covalent conjugates of gold nanorods with targeting vectors such as monoclonal antibodies CD33 (specific for Acute Myeloid Leukemia), while GNR conjugates with polyethylene-glycol (PEG) were used as nonspecific targeting control. GNR clusters were formed inside the tumor cells at 37 °C due to endocytosis of large concentration of nanorods accumulated on the surface of tumor cells targeted at 4 °C. Formation of GNR clusters significantly reduces the threshold of tumor cell damage making LANTCET safe for normal cells. Appearance of GNR clusters was verified directly with optical resonance scattering microscopy. LANTCET was performed in vitro with living cells of (1) model myeloid K562 cells (CD33 positive), (2) primary human bone marrow CD33-positive blast cells from patients diagnosed with acute myeloid leukemia. Laser-induced microbubbles were generated and detected with a photothermal microscope equipped with a tunable Ti-Sa pulsed laser. GNT cluster formation caused a 100-fold decrease in the threshold optical fluence for laser microbubble generation in tumor cells compared with that in normal cells under the same targeting and irradiation conditions. Combining imaging based on resonance optical scattering with photothermal imaging of microbubbles, we developed a method for detection, image-guided treatment and monitoring of LANTCET. Pilot experiments were performed in flow mode bringing LANTCET closer to reality of clinical procedure of purging tumor cells from bone marrow grafts.

  16. Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region.

    PubMed

    Harada, Kaho; Nobuhisa, Ikuo; Anani, Maha; Saito, Kiyoka; Taga, Tetsuya

    2017-07-01

    In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45 low c-Kit high AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45 low c-Kit high cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters by linear-array-based photoacoustic tomography

    NASA Astrophysics Data System (ADS)

    Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Shao, Jin-Yu; Wang, Lihong V.

    2017-04-01

    Circulating tumor cell (CTC) clusters, arising from multicellular groupings in a primary tumor, greatly elevate the metastatic potential of cancer compared with single CTCs. High-throughput detection and quantification of CTC clusters are important for understanding the tumor metastatic process and improving cancer therapy. Here, we applied a linear-array-based photoacoustic tomography (LA-PAT) system and improved the image reconstruction for label-free high-throughput CTC cluster detection and quantification in vivo. The feasibility was first demonstrated by imaging CTC cluster ex vivo. The relationship between the contrast-to-noise ratios (CNRs) and the number of cells in melanoma tumor cell clusters was investigated and verified. Melanoma CTC clusters with a minimum of four cells could be detected, and the number of cells could be computed from the CNR. Finally, we demonstrated imaging of injected melanoma CTC clusters in rats in vivo. Similarly, the number of cells in the melanoma CTC clusters could be quantified. The data showed that larger CTC clusters had faster clearance rates in the bloodstream, which agreed with the literature. The results demonstrated the capability of LA-PAT to detect and quantify melanoma CTC clusters in vivo and showed its potential for tumor metastasis study and cancer therapy.

  18. Clustering single cells: a review of approaches on high-and low-depth single-cell RNA-seq data.

    PubMed

    Menon, Vilas

    2017-12-11

    Advances in single-cell RNA-sequencing technology have resulted in a wealth of studies aiming to identify transcriptomic cell types in various biological systems. There are multiple experimental approaches to isolate and profile single cells, which provide different levels of cellular and tissue coverage. In addition, multiple computational strategies have been proposed to identify putative cell types from single-cell data. From a data generation perspective, recent single-cell studies can be classified into two groups: those that distribute reads shallowly over large numbers of cells and those that distribute reads more deeply over a smaller cell population. Although there are advantages to both approaches in terms of cellular and tissue coverage, it is unclear whether different computational cell type identification methods are better suited to one or the other experimental paradigm. This study reviews three cell type clustering algorithms, each representing one of three broad approaches, and finds that PCA-based algorithms appear most suited to low read depth data sets, whereas gene clustering-based and biclustering algorithms perform better on high read depth data sets. In addition, highly related cell classes are better distinguished by higher-depth data, given the same total number of reads; however, simultaneous discovery of distinct and similar types is better served by lower-depth, higher cell number data. Overall, this study suggests that the depth of profiling should be determined by initial assumptions about the diversity of cells in the population, and that the selection of clustering algorithm(s) is subsequently based on the depth of profiling will allow for better identification of putative transcriptomic cell types. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. Spatially Restricted and Developmentally Dynamic Expression of Engrailed Genes in Multiple Cerebellar Cell Types

    PubMed Central

    Wilson, Sandra L.; Kalinovsky, Anna; Orvis, Grant D.

    2011-01-01

    The cerebellum is a highly organized structure partitioned into lobules along the anterior–posterior (A-P) axis and into striped molecular domains along the medial–lateral (M-L) axis. The Engrailed (En) homeobox genes are required for patterning the morphological and molecular domains along both axes, as well as for the establishment of the normal afferent topography required to generate a fully functional cerebellum. As a means to understand how the En genes regulate multiple levels of cerebellum construction, we characterized En1 and En2 expression around birth and at postnatal day (P)21 during the period when the cerebellum undergoes a remarkable transformation from a smooth ovoid structure to a highly foliated structure. We show that both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21. En1 and En2 expression, however, undergoes profound changes in their cellular and spatial distributions between embryonic stages and P21, and their expression domains become largely distinct. Comparison of the distribution of En-expressing Purkinje cells relative to early- and late-onset Purkinje cell M-L stripe proteins revealed that although En1- and En2-expressing Purkinje cell domains do not strictly align with those of ZEBRINII at P21, a clear pattern exists that is most evident at E17.5 by an inverse correlation between the level of En2 expression and PLCβ4 and EPHA4. PMID:21431469

  20. Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining.

    PubMed

    Sharp, Christopher A; Roberts, Sally; Evans, Helena; Brown, Sharon J

    2009-11-01

    Intervertebral disc (IVD) cells within the annulus fibrosus (AF) and nucleus pulposus (NP) maintain distinct functional extracellular matrices and operate within a potentially noxious and stressful environment. How disc cells respond to stress and whether stress is responsible for triggering degeneration is unknown. Disc cell proliferation and cluster formation are most marked in degenerate IVDs, possibly indicating attempts at matrix repair. In other tissues, stress proteins increase rapidly after stress protecting cell function and, although implicated in degeneration of articular cartilage, have received little attention in degenerative IVD pathologies. We have compared the distribution of stress protein immunolocalization in pathological and control IVDs. Disc tissues were obtained at surgery from 43 patients with degenerative disc disease (DDD) and herniation, and 12 controls at postmortem. Tissues were immunostained with a polyclonal antibody for heat shock factor 1 (HSF-1) and monoclonal antibodies for the heat shock proteins, Hsp27 and Hsp72, using an indirect immunoperoxidase method. Positively stained cells were expressed as a percentage of the total. Cell cluster formation was also assessed. The proportion of cells in clusters was similar in the AF (both 2%) and NP (8 and 9%) of control and DDD samples, whereas in herniated tissues this was increased (AF 12%, NP 14%). Stress antigen staining tended to be more frequent in clustered rather than in single/doublet cells, and this was significant (P < 0.005) in both the AF and NP of herniated discs. Clustered cells, which are most common in herniated discs, may be mounting a protective response to abnormal environmental factors associated with disc degeneration. A better understanding of the stress response in IVD cells may allow its utilization in disc cell therapies.

  1. Real-time dynamics of RNA Polymerase II clustering in live human cells

    NASA Astrophysics Data System (ADS)

    Cisse, Ibrahim

    2014-03-01

    Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

  2. Zebrin II Is Expressed in Sagittal Stripes in the Cerebellum of Dragon Lizards (Ctenophorus sp.).

    PubMed

    Wylie, Douglas R; Hoops, Daniel; Aspden, Joel W; Iwaniuk, Andrew N

    2016-01-01

    Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). In contrast, in snakes and turtles, ZII is not expressed heterogeneously; rather all Purkinje cells are ZII+. Here, we examined the expression of ZII in the cerebellum of lizards to elucidate the evolutionary origins of ZII stripes in Sauropsida. We focused on the central netted dragon (Ctenophorus nuchalis) but also examined cerebellar ZII expression in 5 other dragon species (Ctenophorus spp.). In contrast to what has been observed in snakes and turtles, we found that in these lizards, ZII is heterogeneously expressed. In the posterior part of the cerebellum, on each side of the midline, there were 3 sagittal stripes consisting of Purkinje cells with high ZII expression (ZII+) alternating with 2 sagittal stripes with weaker ZII expression (ZIIw). More anteriorly, most of the Purkinje cells were ZII+, except laterally, where the Purkinje cells did not express ZII (ZII-). Finally, all Purkinje cells in the auricle (flocculus) were ZII-. Overall, the parasagittal heterogeneous expression of ZII in the cerebellum of lizards is similar to that in mammals and birds, and contrasts with the homogenous ZII+ expression seen in snakes and turtles. We suggest that a sagittal heterogeneous expression of ZII represents the ancestral condition in stem reptiles which was lost in snakes and turtles. © 2017 S. Karger AG, Basel.

  3. Germ cell cluster organization and oogenesis in the tardigrade Dactylobiotus parthenogeneticus Bertolani, 1982 (Eutardigrada, Murrayidae).

    PubMed

    Poprawa, Izabela; Hyra, Marta; Rost-Roszkowska, Magdalena Maria

    2015-07-01

    Germ cell cluster organization and the process of oogenesis in Dactylobiotus parthenogeneticus have been described using transmission electron microscopy and light microscopy. The reproductive system of D. parthenogeneticus is composed of a single, sac-like, meroistic ovary and a single oviduct that opens into the cloaca. Two zones can be distinguished in the ovary: a small germarium that is filled with oogonia and a vitellarium that is filled with germ cell clusters. The germ cell cluster, which has the form of a modified rosette, consists of eight cells that are interconnected by stable cytoplasmic bridges. The cell that has the highest number of stable cytoplasmic bridges (four bridges) finally develops into the oocyte, while the remaining cells become trophocytes. Vitellogenesis of a mixed type occurs in D. parthenogeneticus. One part of the yolk material is produced inside the oocyte (autosynthesis), while the second part is synthesized in the trophocytes and transported to the oocyte through the cytoplasmic bridges. The eggs are covered with two envelopes: a thin vitelline envelope and a three-layered chorion. The surface of the chorion forms small conical processes, the shape of which is characteristic for the species that was examined. In our paper, we present the first report on the rosette type of germ cell clusters in Parachela.

  4. Self-Organized Cerebellar Tissue from Human Pluripotent Stem Cells and Disease Modeling with Patient-Derived iPSCs.

    PubMed

    Muguruma, Keiko

    2018-02-01

    Recent advances in the techniques that differentiate induced pluripotent stem cells (iPSCs) into specific types of cells enabled us to establish in vitro cell-based models as a platform for drug discovery. iPSC-derived disease models are advantageous to generation of a large number of cells required for high-throughput screening. Furthermore, disease-relevant cells differentiated from patient-derived iPSCs are expected to recapitulate the disorder-specific pathogenesis and physiology in vitro. Such disease-relevant cells will be useful for developing effective therapies. We demonstrated that cerebellar tissues are generated from human PSCs (hPSCs) in 3D culture systems that recapitulate the in vivo microenvironments associated with the isthmic organizer. Recently, we have succeeded in generation of spinocerebellar ataxia (SCA) patient-derived Purkinje cells by combining the iPSC technology and the self-organizing stem cell 3D culture technology. We demonstrated that SCA6-derived Purkinje cells exhibit vulnerability to triiodothyronine depletion, which is suppressed by treatment with thyrotropin-releasing hormone and Riluzole. We further discuss applications of patient-specific iPSCs to intractable cerebellar disease.

  5. Loss of MyD88 alters neuroinflammatory response and attenuates early Purkinje cell loss in a spinocerebellar ataxia type 6 mouse model

    PubMed Central

    Aikawa, Tomonori; Mogushi, Kaoru; Iijima-Tsutsui, Kumiko; Ishikawa, Kinya; Sakurai, Miyano; Tanaka, Hiroshi; Mizusawa, Hidehiro; Watase, Kei

    2015-01-01

    Spinocerebellar ataxia type 6 (SCA6) is dominantly inherited neurodegenerative disease, caused by an expansion of CAG repeat encoding a polyglutamine (PolyQ) tract in the Cav2.1 voltage-gated calcium channel. Its key pathological features include selective degeneration of the cerebellar Purkinje cells (PCs), a common target for PolyQ-induced toxicity in various SCAs. Mutant Cav2.1 confers toxicity primarily through a toxic gain-of-function mechanism; however, its molecular basis remains elusive. Here, we studied the cerebellar gene expression patterns of young Sca6-MPI118Q/118Q knockin (KI) mice, which expressed mutant Cav2.1 from an endogenous locus and recapitulated many phenotypic features of human SCA6. Transcriptional signatures in the MPI118Q/118Q mice were distinct from those in the Sca1154Q/2Q mice, a faithful SCA1 KI mouse model. Temporal expression profiles of the candidate genes revealed that the up-regulation of genes associated with microglial activation was initiated before PC degeneration and was augmented as the disease progressed. Histological analysis of the MPI118Q/118Q cerebellum showed the predominance of M1-like pro-inflammatory microglia and it was concomitant with elevated expression levels of tumor necrosis factor, interleukin-6, Toll-like receptor (TLR) 2 and 7. Genetic ablation of MyD88, a major adaptor protein conveying TLR signaling, altered expression patterns of M1/M2 microglial phenotypic markers in the MPI118Q/118Q cerebellum. More importantly, it ameliorated PC loss and partially rescued motor impairments in the early disease phase. These results suggest that early neuroinflammatory response may play an important role in the pathogenesis of SCA6 and its modulation could pave the way for slowing the disease progression during the early stage of the disease. PMID:26034136

  6. Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

    PubMed Central

    Aittaleb, Mohamed; Chen, Po-Ju; Akaaboune, Mohammed

    2015-01-01

    ABSTRACT Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn−/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity. PMID:26330529

  7. Cerebellar granule cells encode the expectation of reward

    PubMed Central

    Wagner, Mark J; Kim, Tony Hyun; Savall, Joan; Schnitzer, Mark J; Luo, Liqun

    2017-01-01

    The human brain contains ~60 billion cerebellar granule cells1, which outnumber all other neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes2–6. Although evidence suggests a role for cerebellum in cognition7–10, granule cells are known to encode only sensory11–13 and motor14 context. Using two-photon calcium imaging in behaving mice, here we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from current understanding of these neurons and dramatically enriches contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum. PMID:28321129

  8. GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells

    PubMed Central

    Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe

    2017-01-01

    Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. PMID:29046391

  9. Biosynthesis and biological action of pineal allopregnanolone

    PubMed Central

    Tsutsui, Kazuyoshi; Haraguchi, Shogo

    2014-01-01

    The pineal gland transduces photoperiodic changes to the neuroendocrine system by rhythmic secretion of melatonin. We recently provided new evidence that the pineal gland is a major neurosteroidogenic organ and actively produces a variety of neurosteroids de novo from cholesterol in birds. Notably, allopregnanolone is a major pineal neurosteroid that is far more actively produced in the pineal gland than the brain and secreted by the pineal gland in juvenile birds. Subsequently, we have demonstrated the biological action of pineal allopregnanolone on Purkinje cells in the cerebellum during development in juvenile birds. Pinealectomy (Px) induces apoptosis of Purkinje cells, whereas allopregnanolone administration to Px chicks prevents cell death. Furthermore, Px increases the number of Purkinje cells that express active caspase-3, a crucial mediator of apoptosis, and allopregnanolone administration to Px chicks decreases the number of Purkinje cells expressing active caspase-3. It thus appears that pineal allopregnanolone prevents cell death of Purkinje cells by suppressing the activity of caspase-3 during development. This paper highlights new aspects of the biosynthesis and biological action of pineal allopregnanolone. PMID:24834027

  10. A phenanthrene derived PARP inhibitor is an extra-centrosomes de-clustering agent exclusively eradicating human cancer cells

    PubMed Central

    2011-01-01

    Background Cells of most human cancers have supernumerary centrosomes. To enable an accurate chromosome segregation and cell division, these cells developed a yet unresolved molecular mechanism, clustering their extra centrosomes at two poles, thereby mimicking mitosis in normal cells. Failure of this bipolar centrosome clustering causes multipolar spindle structures and aberrant chromosomes segregation that prevent normal cell division and lead to 'mitotic catastrophe cell death'. Methods We used cell biology and biochemical methods, including flow cytometry, immunocytochemistry and live confocal imaging. Results We identified a phenanthrene derived PARP inhibitor, known for its activity in neuroprotection under stress conditions, which exclusively eradicated multi-centrosomal human cancer cells (mammary, colon, lung, pancreas, ovarian) while acting as extra-centrosomes de-clustering agent in mitosis. Normal human proliferating cells (endothelial, epithelial and mesenchymal cells) were not impaired. Despite acting as PARP inhibitor, the cytotoxic activity of this molecule in cancer cells was not attributed to PARP inhibition alone. Conclusion We identified a water soluble phenanthridine that exclusively targets the unique dependence of most human cancer cells on their supernumerary centrosomes bi-polar clustering for their survival. This paves the way for a new selective cancer-targeting therapy, efficient in a wide range of human cancers. PMID:21943092

  11. Under a nonadherent state, bone marrow mesenchymal stem cells can be efficiently induced into functional islet-like cell clusters to normalize hyperglycemia in mice: a control study.

    PubMed

    Zhang, Yihua; Dou, Zhongying

    2014-05-08

    Bone marrow mesenchymal stem cells (BMSCs) possess low immunogenicity and immunosuppression as an allograft, can differentiate into insulin-producing cells (IPCs) by in vitro induction, and may be a valuable cell source to regenerate pancreatic islets. However, the very low differentiation efficiency of BMSCs towards IPCs under adherent induction has thus far hindered the clinical exploitation of these cells. The aim of this study is to explore a new way to efficiently induce BMSCs into IPCs and lay the groundwork for their clinical exploitation. In comparison with adherent induction, BMSCs of human first-trimester abortus (hfBMSCs) under a nonadherent state were induced towards IPCs in noncoated plastic dishes using a three-stage induction procedure developed by the authors. Induction effects were evaluated by statistics of the cell clustering rate of induced cells, and ultrastructural observation, dithizone staining, quantitative polymerase chain reaction and immunofluorescence assay, insulin and c-peptide release under glucose stimulus of cell clusters, as well as transplantation test of the cell clusters in diabetic model mice. With (6.175 ± 0.263) × 105 cells in 508.5 ± 24.5 cell clusters, (3.303 ± 0.331) × 105 single cells and (9.478 ± 0.208) × 105 total cell count on average, 65.08 ± 2.98% hfBMSCs differentiated into pancreatic islet-like cell clusters after nonadherent induction. With (3.993 ± 0.344) × 105 cells in 332.3 ± 41.6 cell clusters, (5.437 ± 0.434) × 105 single cells and (9.430 ± 0.340) × 105 total cell count on average, 42.37 ± 3.70% hfBMSCs differentiated into pancreatic islet-like cell clusters after adherent induction (P < 0.01, n = 10). The former is significantly higher than the latter. Calculated according to the cell clustering rate and IPC percentage in the cell clusters, 29.80 ± 3.95% hfBMSCs differentiated into IPCs after nonadherent induction

  12. Linear-array-based photoacoustic tomography for label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters

    NASA Astrophysics Data System (ADS)

    Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Wang, Lihong V.

    2017-03-01

    Circulating tumor cell (CTC) clusters arise from multicellular grouping in the primary tumor and elevate the metastatic potential by 23 to 50 fold compared to single CTCs. High throughout detection and quantification of CTC clusters is critical for understanding the tumor metastasis process and improving cancer therapy. In this work, we report a linear-array-based photoacoustic tomography (LA-PAT) system capable of label-free high-throughput CTC cluster detection and quantification in vivo. LA-PAT detects CTC clusters and quantifies the number of cells in them based on the contrast-to-noise ratios (CNRs) of photoacoustic signals. The feasibility of LA-PAT was first demonstrated by imaging CTC clusters ex vivo. LA-PAT detected CTC clusters in the blood-filled microtubes and computed the number of cells in the clusters. The size distribution of the CTC clusters measured by LA-PAT agreed well with that obtained by optical microscopy. We demonstrated the ability of LA-PAT to detect and quantify CTC clusters in vivo by imaging injected CTC clusters in rat tail veins. LA-PAT detected CTC clusters immediately after injection as well as when they were circulating in the rat bloodstreams. Similarly, the numbers of cells in the clusters were computed based on the CNRs of the photoacoustic signals. The data showed that larger CTC clusters disappear faster than the smaller ones. The results prove the potential of LA-PAT as a promising tool for both preclinical tumor metastasis studies and clinical cancer therapy evaluation.

  13. Effects of mild hyperthyroidism on levels of amino acids in the developing Lurcher cerebellum.

    PubMed

    Messer, A; Eisenberg, B; Martin, D L

    1989-01-01

    This study examines the question of whether intrinsically defective mutant Lurcher Purkinje cells, which degenerate during postnatal weeks two to five, followed by later loss of granule cells are competent to respond to neonatal hyperthyroidism, which is known to cause premature differentiation of Purkinje cells and an acceleration of the peak of proliferation in granule cells in normal rodent cerebellum. Both total amounts and concentrations (per mg wet weight) of Tau, Glu, Asp and GABA were assayed as markers of cell function in Lurcher and wild-type mice made very mildly hyperthyroid by feeding nursing dams high-thyroxine food. Tau, which is present in relatively high concentrations in Purkinje cells, was affected by hyperthyroid treatment in the Lurcher in a manner that is most consistent with an acceleration of the degenerative process in Purkinje cells. The acidic amino acids Glu and Asp show later changes and response to hormone which seem to be a reaction to the Purkinje cell pattern, probably in the granule cells. We conclude that the Lurcher cerebellum is particularly sensitive to thyroid hormone, and that it responds to low levels of hyperthyroidism in a distinct way.

  14. GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

    PubMed

    Paladino, Simona; Lebreton, Stéphanie; Lelek, Mickaël; Riccio, Patrizia; De Nicola, Sergio; Zimmer, Christophe; Zurzolo, Chiara

    2017-12-01

    Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model. © 2017 The Author(s).

  15. Combination of automated high throughput platforms, flow cytometry, and hierarchical clustering to detect cell state.

    PubMed

    Kitsos, Christine M; Bhamidipati, Phani; Melnikova, Irena; Cash, Ethan P; McNulty, Chris; Furman, Julia; Cima, Michael J; Levinson, Douglas

    2007-01-01

    This study examined whether hierarchical clustering could be used to detect cell states induced by treatment combinations that were generated through automation and high-throughput (HT) technology. Data-mining techniques were used to analyze the large experimental data sets to determine whether nonlinear, non-obvious responses could be extracted from the data. Unary, binary, and ternary combinations of pharmacological factors (examples of stimuli) were used to induce differentiation of HL-60 cells using a HT automated approach. Cell profiles were analyzed by incorporating hierarchical clustering methods on data collected by flow cytometry. Data-mining techniques were used to explore the combinatorial space for nonlinear, unexpected events. Additional small-scale, follow-up experiments were performed on cellular profiles of interest. Multiple, distinct cellular profiles were detected using hierarchical clustering of expressed cell-surface antigens. Data-mining of this large, complex data set retrieved cases of both factor dominance and cooperativity, as well as atypical cellular profiles. Follow-up experiments found that treatment combinations producing "atypical cell types" made those cells more susceptible to apoptosis. CONCLUSIONS Hierarchical clustering and other data-mining techniques were applied to analyze large data sets from HT flow cytometry. From each sample, the data set was filtered and used to define discrete, usable states that were then related back to their original formulations. Analysis of resultant cell populations induced by a multitude of treatments identified unexpected phenotypes and nonlinear response profiles.

  16. Poor Prognosis Indicated by Venous Circulating Tumor Cell Clusters in Early-Stage Lung Cancers.

    PubMed

    Murlidhar, Vasudha; Reddy, Rishindra M; Fouladdel, Shamileh; Zhao, Lili; Ishikawa, Martin K; Grabauskiene, Svetlana; Zhang, Zhuo; Lin, Jules; Chang, Andrew C; Carrott, Philip; Lynch, William R; Orringer, Mark B; Kumar-Sinha, Chandan; Palanisamy, Nallasivam; Beer, David G; Wicha, Max S; Ramnath, Nithya; Azizi, Ebrahim; Nagrath, Sunitha

    2017-09-15

    Early detection of metastasis can be aided by circulating tumor cells (CTC), which also show potential to predict early relapse. Because of the limited CTC numbers in peripheral blood in early stages, we investigated CTCs in pulmonary vein blood accessed during surgical resection of tumors. Pulmonary vein (PV) and peripheral vein (Pe) blood specimens from patients with lung cancer were drawn during the perioperative period and assessed for CTC burden using a microfluidic device. From 108 blood samples analyzed from 36 patients, PV had significantly higher number of CTCs compared with preoperative Pe ( P < 0.0001) and intraoperative Pe ( P < 0.001) blood. CTC clusters with large number of CTCs were observed in 50% of patients, with PV often revealing larger clusters. Long-term surveillance indicated that presence of clusters in preoperative Pe blood predicted a trend toward poor prognosis. Gene expression analysis by RT-qPCR revealed enrichment of p53 signaling and extracellular matrix involvement in PV and Pe samples. Ki67 expression was detected in 62.5% of PV samples and 59.2% of Pe samples, with the majority (72.7%) of patients positive for Ki67 expression in PV having single CTCs as opposed to clusters. Gene ontology analysis revealed enrichment of cell migration and immune-related pathways in CTC clusters, suggesting survival advantage of clusters in circulation. Clusters display characteristics of therapeutic resistance, indicating the aggressive nature of these cells. Thus, CTCs isolated from early stages of lung cancer are predictive of poor prognosis and can be interrogated to determine biomarkers predictive of recurrence. Cancer Res; 77(18); 5194-206. ©2017 AACR . ©2017 American Association for Cancer Research.

  17. Dystonia and Cerebellar Degeneration in the Leaner Mouse Mutant

    PubMed Central

    Raike, Robert S.; Hess, Ellen J.; Jinnah, H.A.

    2015-01-01

    Cerebellar degeneration is traditionally associated with ataxia. Yet, there are examples of both ataxia and dystonia occurring in individuals with cerebellar degeneration. There is also substantial evidence suggesting that cerebellar dysfunction alone may cause dystonia. The types of cerebellar defects that may cause ataxia, dystonia, or both have not been delineated. In the current study, we explored the relationship between cerebellar degeneration and dystonia using the leaner mouse mutant. Leaner mice have severe dystonia that is associated with dysfunctional and degenerating cerebellar Purkinje cells. Whereas the density of Purkinje cells was not significantly reduced in 4 week-old leaner mice, approximately 50% of the neurons were lost by 34 weeks of age. On the other hand, the dystonia and associated functional disability became significantly less severe during this same interval. In other words, dystonia improved as Purkinje cells were lost, suggesting that dysfunctional Purkinje cells, rather than Purkinje cell loss, contribute to the dystonia. These results provide evidence that distorted cerebellar function may cause dystonia and support the concept that different types of cerebellar defects can have different functional consequences. PMID:25791619

  18. Insulin Regulates Glut4 Confinement in Plasma Membrane Clusters in Adipose Cells

    PubMed Central

    Lizunov, Vladimir A.; Stenkula, Karin; Troy, Aaron; Cushman, Samuel W.; Zimmerberg, Joshua

    2013-01-01

    Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin’s effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60–240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters ∼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis. PMID:23520472

  19. Insulin regulates Glut4 confinement in plasma membrane clusters in adipose cells.

    PubMed

    Lizunov, Vladimir A; Stenkula, Karin; Troy, Aaron; Cushman, Samuel W; Zimmerberg, Joshua

    2013-01-01

    Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin's effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60-240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters ∼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.

  20. Positive feedback can lead to dynamic nanometer-scale clustering on cell membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wehrens, Martijn; Rein ten Wolde, Pieter; Mugler, Andrew, E-mail: amugler@purdue.edu

    2014-11-28

    Clustering of molecules on biological membranes is a widely observed phenomenon. A key example is the clustering of the oncoprotein Ras, which is known to be important for signal transduction in mammalian cells. Yet, the mechanism by which Ras clusters form and are maintained remains unclear. Recently, it has been discovered that activated Ras promotes further Ras activation. Here we show using particle-based simulation that this positive feedback is sufficient to produce persistent clusters of active Ras molecules at the nanometer scale via a dynamic nucleation mechanism. Furthermore, we find that our cluster statistics are consistent with experimental observations ofmore » the Ras system. Interestingly, we show that our model does not support a Turing regime of macroscopic reaction-diffusion patterning, and therefore that the clustering we observe is a purely stochastic effect, arising from the coupling of positive feedback with the discrete nature of individual molecules. These results underscore the importance of stochastic and dynamic properties of reaction diffusion systems for biological behavior.« less

  1. The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans

    PubMed Central

    Striegel, Deborah A.; Hara, Manami; Periwal, Vipul

    2015-01-01

    Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets. PMID:26266953

  2. The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans.

    PubMed

    Striegel, Deborah A; Hara, Manami; Periwal, Vipul

    2015-08-01

    Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets.

  3. Neuronal Atrophy Early in Degenerative Ataxia Is a Compensatory Mechanism to Regulate Membrane Excitability

    PubMed Central

    Dell'Orco, James M.; Wasserman, Aaron H.; Chopra, Ravi; Ingram, Melissa A. C.; Hu, Yuan-Shih; Singh, Vikrant; Wulff, Heike; Opal, Puneet; Orr, Harry T.

    2015-01-01

    Neuronal atrophy in neurodegenerative diseases is commonly viewed as an early event in a continuum that ultimately results in neuronal loss. In a mouse model of the polyglutamine disorder spinocerebellar ataxia type 1 (SCA1), we tested the hypothesis that cerebellar Purkinje neuron atrophy serves an adaptive role rather than being simply a nonspecific response to injury. In acute cerebellar slices from SCA1 mice, we find that Purkinje neuron pacemaker firing is initially normal but, with the onset of motor dysfunction, becomes disrupted, accompanied by abnormal depolarization. Remarkably, subsequent Purkinje cell atrophy is associated with a restoration of pacemaker firing. The early inability of Purkinje neurons to support repetitive spiking is due to unopposed calcium currents resulting from a reduction in large-conductance calcium-activated potassium (BK) and subthreshold-activated potassium channels. The subsequent restoration of SCA1 Purkinje neuron firing correlates with the recovery of the density of these potassium channels that accompanies cell atrophy. Supporting a critical role for BK channels, viral-mediated increases in BK channel expression in SCA1 Purkinje neurons improves motor dysfunction and partially restores Purkinje neuron morphology. Cerebellar perfusion of flufenamic acid, an agent that restores the depolarized membrane potential of SCA1 Purkinje neurons by activating potassium channels, prevents Purkinje neuron dendritic atrophy. These results suggest that Purkinje neuron dendritic remodeling in ataxia is an adaptive response to increases in intrinsic membrane excitability. Similar adaptive remodeling could apply to other vulnerable neuronal populations in neurodegenerative disease. SIGNIFICANCE STATEMENT In neurodegenerative disease, neuronal atrophy has long been assumed to be an early nonspecific event preceding neuronal loss. However, in a mouse model of spinocerebellar ataxia type 1 (SCA1), we identify a previously unappreciated

  4. Biocompatibility of cluster-assembled nanostructured TiO2 with primary and cancer cells.

    PubMed

    Carbone, Roberta; Marangi, Ida; Zanardi, Andrea; Giorgetti, Luca; Chierici, Elisabetta; Berlanda, Giuseppe; Podestà, Alessandro; Fiorentini, Francesca; Bongiorno, Gero; Piseri, Paolo; Pelicci, Pier Giuseppe; Milani, Paolo

    2006-06-01

    We have characterized the biocompatibility of nanostructured TiO2 films produced by the deposition of a supersonic beam of TiOx clusters. Physical analysis shows that these films possess, at the nanoscale, a granularity and porosity mimicking those of typical extracellular matrix structures and adsorption properties that could allow surface functionalization with different macromolecules such as DNA, proteins, and peptides. To explore the biocompatibility of this novel nanostructured surface, different cancer and primary cells were analyzed in terms of morphological appearance (by bright field microscopy and immunofluorescence) and growth properties, with the aim to evaluate cluster-assembled TiO2 films as substrates for cell-based and tissue-based applications. Our results strongly suggest that this new biomaterial supports normal growth and adhesion of primary and cancer cells with no need for coating with ECM proteins; we thus propose this new material as an optimal substrate for different applications in cell-based assays, biosensors or microfabricated medical devices.

  5. A high fat diet containing saturated but not unsaturated fatty acids enhances T cell receptor clustering on the nanoscale.

    PubMed

    Shaikh, Saame Raza; Boyle, Sarah; Edidin, Michael

    2015-09-01

    Cell culture studies show that the nanoscale lateral organization of surface receptors, their clustering or dispersion, can be altered by changing the lipid composition of the membrane bilayer. However, little is known about similar changes in vivo, which can be effected by changing dietary lipids. We describe the use of a newly developed method, k-space image correlation spectroscopy, kICS, for analysis of quantum dot fluorescence to show that a high fat diet can alter the nanometer-scale clustering of the murine T cell receptor, TCR, on the surface of naive CD4(+) T cells. We found that diets enriched primarily in saturated fatty acids increased TCR nanoscale clustering to a level usually seen only on activated cells. Diets enriched in monounsaturated or n-3 polyunsaturated fatty acids had no effect on TCR clustering. Also none of the high fat diets affected TCR clustering on the micrometer scale. Furthermore, the effect of the diets was similar in young and middle aged mice. Our data establish proof-of-principle that TCR nanoscale clustering is sensitive to the composition of dietary fat. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Cntnap2 expression in the cerebellum of Foxp2(R552H) mice, with a mutation related to speech-language disorder.

    PubMed

    Fujita, Eriko; Tanabe, Yuko; Momoi, Mariko Y; Momoi, Takashi

    2012-01-11

    Foxp2(R552H) knock-in (KI) mice carrying a mutation related to human speech-language disorder exhibit impaired ultrasonic vocalization and poor Purkinje cell development. Foxp2 is a forkhead domain-containing transcriptional repressor that associates with its co-repressor CtBP; Foxp2(R552H) displays reduced DNA binding activity. A genetic connection between FOXP2 and CNTNAP2 has been demonstrated in vitro, but not in vivo. Here we show that Cntnap2 mRNA levels significantly increased in the cerebellum of Foxp2(R552H) KI pups, although the cerebellar population of Foxp2-positive Purkinje cells was very small. Furthermore, Cntnap2 immunofluorescence did not decrease in the poorly developed Purkinje cells of Foxp2(R552H) KI pups, although synaptophysin immunofluorescence decreased. Cntnap2 and CtBP were ubiquitously expressed, while Foxp2 co-localized with CtBP only in Purkinje cells. Taken together, these observations suggest that Foxp2 may regulate ultrasonic vocalization by associating with CtBP in Purkinje cells; Cntnap2 may be a target of this co-repressor. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  7. A cluster pattern algorithm for the analysis of multiparametric cell assays.

    PubMed

    Kaufman, Menachem; Bloch, David; Zurgil, Naomi; Shafran, Yana; Deutsch, Mordechai

    2005-09-01

    The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.

  8. Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

    PubMed

    Maruyama, C L M; Leigh, N J; Nelson, J W; McCall, A D; Mellas, R E; Lei, P; Andreadis, S T; Baker, O J

    2015-11-01

    Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features. © International & American Associations for Dental Research 2015.

  9. Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

    PubMed Central

    Salmond, G P; Lutkenhaus, J F; Donachie, W D

    1980-01-01

    We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. Images PMID:6998962

  10. Ablation of glutamate receptor GluRδ2 in adult Purkinje cells causes multiple innervation of climbing fibers by inducing aberrant invasion to parallel fiber innervation territory.

    PubMed

    Miyazaki, Taisuke; Yamasaki, Miwako; Takeuchi, Tomonori; Sakimura, Kenji; Mishina, Masayoshi; Watanabe, Masahiko

    2010-11-10

    Glutamate receptor GluRδ2 is exclusively expressed in Purkinje cells (PCs) from early development and plays key roles in parallel fiber (PF) synapse formation, elimination of surplus climbing fibers (CFs), long-term depression, motor coordination, and motor learning. To address its role in adulthood, we previously developed a mouse model of drug-induced GluRδ2 ablation in adult PCs (Takeuchi et al., 2005). In that study, we demonstrated an essential role to maintain the connectivity of PF-PC synapses, based on the observation that both mismatching of presynaptic and postsynaptic specializations and disconnection of PF-PC synapses are progressively increased after GluRδ2 ablation. Here, we pursued its role for CF wiring in adult cerebellum. In parallel with the disconnection of PF-PC synapses, ascending CF branches exhibited distal extension to innervate distal dendrites of the target and neighboring PCs. Furthermore, transverse CF branches, a short motile collateral rarely forming synapses in wild-type animals, displayed aberrant mediolateral extension to innervate distal dendrites of neighboring and remote PCs. Consequently, many PCs were wired by single main CF and other surplus CFs innervating a small part of distal dendrites. Electrophysiological recording further revealed that surplus CF-EPSCs characterized with slow rise time and small amplitude emerged after GluRδ2 ablation, and increased progressively both in number and amplitude. Therefore, GluRδ2 is essential for maintaining CF monoinnervation in adult cerebellum by suppressing aberrant invasion of CF branches to the territory of PF innervation. Thus, GluRδ2 fuels heterosynaptic competition and gives PFs the competitive advantages over CFs throughout the animal's life.

  11. Dendritic excitation–inhibition balance shapes cerebellar output during motor behaviour

    PubMed Central

    Jelitai, Marta; Puggioni, Paolo; Ishikawa, Taro; Rinaldi, Arianna; Duguid, Ian

    2016-01-01

    Feedforward excitatory and inhibitory circuits regulate cerebellar output, but how these circuits interact to shape the somatodendritic excitability of Purkinje cells during motor behaviour remains unresolved. Here we perform dendritic and somatic patch-clamp recordings in vivo combined with optogenetic silencing of interneurons to investigate how dendritic excitation and inhibition generates bidirectional (that is, increased or decreased) Purkinje cell output during self-paced locomotion. We find that granule cells generate a sustained depolarization of Purkinje cell dendrites during movement, which is counterbalanced by variable levels of feedforward inhibition from local interneurons. Subtle differences in the dendritic excitation–inhibition balance generate robust, bidirectional changes in simple spike (SSp) output. Disrupting this balance by selectively silencing molecular layer interneurons results in unidirectional firing rate changes, increased SSp regularity and disrupted locomotor behaviour. Our findings provide a mechanistic understanding of how feedforward excitatory and inhibitory circuits shape Purkinje cell output during motor behaviour. PMID:27976716

  12. A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

    PubMed

    Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

    2014-05-21

    Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions. Copyright © 2014 the authors 0270-6474/14/347203-13$15.00/0.

  13. Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes.

    PubMed

    Zhang, Yihua; Shen, Wenzheng; Hua, Jinlian; Lei, Anmin; Lv, Changrong; Wang, Huayan; Yang, Chunrong; Gao, Zhimin; Dou, Zhongying

    2010-12-01

    Bone marrow mesenchymal stem cells (BMSCs) have been reported to possess low immunogenicity and cause immunosuppression of recipients when allografted. They can differentiate into insulin-producing cells and may be a valuable source for islet formation. However, the extremely low differentiating rate of adult BMSCs toward insulin-producing cells and the insufficient insulin secretion of the differentiated BMSCs in vitro prevent their clinical use in diabetes treatment. Little is known about the potential of cell replacement therapy with human BMSCs. Previously, we isolated and identified human first-trimester fetal BMSCs (hfBMSCs). Under a novel four-step induction procedure established in this study, the hfBMSCs effectively differentiated into functional pancreatic islet-like cell clusters that contained 62 ± 14% insulin-producing cells, expressed a broad gene profile related to pancreatic islet β-cell development, and released high levels of insulin (2.245 ± 0.222 pmol/100 clusters per 30 min) and C-peptide (2.200 ± 0.468 pmol/100 clusters per 30 min) in response to 25 mmol/L glucose stimulus in vitro. The pancreatic islet-like cell clusters normalized the blood glucose level of diabetic model mice for at least 9 weeks when xenografted; blood glucose levels in these mice rose abnormally again when the grafts were removed. Examination of the grafts indicated that the transplanted cells survived in recipients and produced human insulin and C-peptide in situ. These results demonstrate that hfBMSCs derived from a human first-trimester abortus can differentiate into pancreatic islet-like cell clusters following an established four-step induction. The insulin-producing clusters present advantages in cell replacement therapy of type 1 diabetic model mice.

  14. Changes in complex spike activity during classical conditioning

    PubMed Central

    Rasmussen, Anders; Jirenhed, Dan-Anders; Wetmore, Daniel Z.; Hesslow, Germund

    2014-01-01

    The cerebellar cortex is necessary for adaptively timed conditioned responses (CRs) in eyeblink conditioning. During conditioning, Purkinje cells acquire pause responses or “Purkinje cell CRs” to the conditioned stimuli (CS), resulting in disinhibition of the cerebellar nuclei (CN), allowing them to activate motor nuclei that control eyeblinks. This disinhibition also causes inhibition of the inferior olive (IO), via the nucleo-olivary pathway (N-O). Activation of the IO, which relays the unconditional stimulus (US) to the cortex, elicits characteristic complex spikes in Purkinje cells. Although Purkinje cell activity, as well as stimulation of the CN, is known to influence IO activity, much remains to be learned about the way that learned changes in simple spike firing affects the IO. In the present study, we analyzed changes in simple and complex spike firing, in extracellular Purkinje cell records, from the C3 zone, in decerebrate ferrets undergoing training in a conditioning paradigm. In agreement with the N-O feedback hypothesis, acquisition resulted in a gradual decrease in complex spike activity during the conditioned stimulus, with a delay that is consistent with the long N-O latency. Also supporting the feedback hypothesis, training with a short interstimulus interval (ISI), which does not lead to acquisition of a Purkinje cell CR, did not cause a suppression of complex spike activity. In contrast, observations that extinction did not lead to a recovery in complex spike activity and the irregular patterns of simple and complex spike activity after the conditioned stimulus are less conclusive. PMID:25140129

  15. Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

    PubMed Central

    Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

    2016-01-01

    Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect. PMID:27257076

  16. Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

    PubMed

    Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

    2013-08-01

    Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Sertoli cell-specific ablation of miR-17-92 cluster significantly alters whole testis transcriptome without apparent phenotypic effects.

    PubMed

    Hurtado, Alicia; Real, Francisca M; Palomino, Rogelio; Carmona, Francisco David; Burgos, Miguel; Jiménez, Rafael; Barrionuevo, Francisco J

    2018-01-01

    MicroRNAs are frequently organized into polycistronic clusters whose transcription is controlled by a single promoter. The miR-17-92 cluster is expressed in most embryonic and postnatal organs. It is a potent oncogene associated to several types of cancer and it is involved in several important developmental processes. In the testis, expression of the miR-17-92 cluster in the germ cells is necessary to maintain normal spermatogenesis. This cluster is also expressed in Sertoli cells (the somatic cells of the seminiferous tubules), which require miRNAs for correct cell development and survival. To study the possible role of miR-17-92 in Sertoli cell development and function and, in order to overcome the postnatal lethality of miR-17-92-/ mice, we conditionally deleted it in embryonic Sertoli cells shortly after the sex determination stage using an Amh-Cre allele. Mutant mice developed apparently normal testes and were fertile, but their testis transcriptomes contained hundreds of moderately deregulated genes, indicating that testis homeostasis is tightly controlled in mammals and that miR-17-92 expression in Sertoli cells contribute to maintain normal gene expression levels, but is unnecessary for testis development and function. Our results show that significant deregulation of hundreds of genes might have no functional consequences.

  18. Control of clustered action potential firing in a mathematical model of entorhinal cortex stellate cells.

    PubMed

    Tait, Luke; Wedgwood, Kyle; Tsaneva-Atanasova, Krasimira; Brown, Jon T; Goodfellow, Marc

    2018-07-14

    The entorhinal cortex is a crucial component of our memory and spatial navigation systems and is one of the first areas to be affected in dementias featuring tau pathology, such as Alzheimer's disease and frontotemporal dementia. Electrophysiological recordings from principle cells of medial entorhinal cortex (layer II stellate cells, mEC-SCs) demonstrate a number of key identifying properties including subthreshold oscillations in the theta (4-12 Hz) range and clustered action potential firing. These single cell properties are correlated with network activity such as grid firing and coupling between theta and gamma rhythms, suggesting they are important for spatial memory. As such, experimental models of dementia have revealed disruption of organised dorsoventral gradients in clustered action potential firing. To better understand the mechanisms underpinning these different dynamics, we study a conductance based model of mEC-SCs. We demonstrate that the model, driven by extrinsic noise, can capture quantitative differences in clustered action potential firing patterns recorded from experimental models of tau pathology and healthy animals. The differential equation formulation of our model allows us to perform numerical bifurcation analyses in order to uncover the dynamic mechanisms underlying these patterns. We show that clustered dynamics can be understood as subcritical Hopf/homoclinic bursting in a fast-slow system where the slow sub-system is governed by activation of the persistent sodium current and inactivation of the slow A-type potassium current. In the full system, we demonstrate that clustered firing arises via flip bifurcations as conductance parameters are varied. Our model analyses confirm the experimentally suggested hypothesis that the breakdown of clustered dynamics in disease occurs via increases in AHP conductance. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Plasma protein induced clustering of red blood cells in micro capillaries

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Brust, Mathias; Aouane, Othmane; Flormann, Daniel; Thiebaud, Marine; Verdier, Claude; Coupier, Gwennou; Podgorski, Thomas; Misbah, Chaouqi; Selmi, Hassib

    2013-11-01

    The plasma molecule fibrinogen induces aggregation of RBCs to clusters, the so called rouleaux. Higher shear rates in bulk flow can break them up which results in the pronounced shear thinning of blood. This led to the assumption that rouleaux formation does not take place in the microcapillaries of the vascular network where high shear rates are present. However, the question is of high medical relevance. Cardio vascular disorders are still the main cause of death in the western world and cardiac patients have often higher fibrinogen level. We performed AFM based single cell force spectroscopy to determine the work of separation. Measurements at low hematocrit in a microfluidic channel show that the number of size of clusters is determined by the adhesion strength and we found that cluster formation is strongly enhanced by fibrinogen at physiological concentrations, even at shear rate as high as 1000 1/s. Numerical simulations based on a boundary integral method confirm our findings and the clustering transition takes place both in the experiments and in the simulations at the same interaction energies. In vivo measurements with intravital fluorescence microscopy in a dorsal skin fold chamber in a mouse reveal that RBCs indeed form clusters in the micrcapillary flow. This work was supported by the German Science Foundation research imitative SFB1027.

  20. Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines.

    PubMed

    Eryilmaz, Marion; Schmitt, Eberhard; Krufczik, Matthias; Theda, Franziska; Lee, Jin-Ho; Cremer, Christoph; Bestvater, Felix; Schaufler, Wladimir; Hausmann, Michael; Hildenbrand, Georg

    2018-01-22

    In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell's decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

  1. Geographic Clusters of Basal Cell Carcinoma in a Northern California Health Plan Population.

    PubMed

    Ray, G Thomas; Kulldorff, Martin; Asgari, Maryam M

    2016-11-01

    Rates of skin cancer, including basal cell carcinoma (BCC), the most common cancer, have been increasing over the past 3 decades. A better understanding of geographic clustering of BCCs can help target screening and prevention efforts. Present a methodology to identify spatial clusters of BCC and identify such clusters in a northern California population. This retrospective study used a BCC registry to determine rates of BCC by census block group, and used spatial scan statistics to identify statistically significant geographic clusters of BCCs, adjusting for age, sex, and socioeconomic status. The study population consisted of white, non-Hispanic members of Kaiser Permanente Northern California during years 2011 and 2012. Statistically significant geographic clusters of BCC as determined by spatial scan statistics. Spatial analysis of 28 408 individuals who received a diagnosis of at least 1 BCC in 2011 or 2012 revealed distinct geographic areas with elevated BCC rates. Among the 14 counties studied, BCC incidence ranged from 661 to 1598 per 100 000 person-years. After adjustment for age, sex, and neighborhood socioeconomic status, a pattern of 5 discrete geographic clusters emerged, with a relative risk ranging from 1.12 (95% CI, 1.03-1.21; P = .006) for a cluster in eastern Sonoma and northern Napa Counties to 1.40 (95% CI, 1.15-1.71; P < .001) for a cluster in east Contra Costa and west San Joaquin Counties, compared with persons residing outside that cluster. In this study of a northern California population, we identified several geographic clusters with modestly elevated incidence of BCC. Knowledge of geographic clusters can help inform future research on the underlying etiology of the clustering including factors related to the environment, health care access, or other characteristics of the resident population, and can help target screening efforts to areas of highest yield.

  2. FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.

    PubMed

    Kong, Hyun Joon; Polte, Thomas R; Alsberg, Eben; Mooney, David J

    2005-03-22

    The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

  3. Uruguay Roselli 1938 and Rosellichnus, N. Ichnogenus: Two ichnogenera for clusters of fossil bee cells

    USGS Publications Warehouse

    Genise, Jorge F.; Bown, Thomas M.

    1996-01-01

    The systematics of the fossil bee nest ichnogenus Uruguay from the Uruguayan Cretaceous or lower Tertiary is reviewed and two new ichnospecies of a new ichnogenus, Rosellichnus, are proposed for fossil bee nests from the Miocene of the United Arab Emirates and Patagonian Argentina. Uruguay contains U. auroranormae(the ich‐notype) and U. rivasi, which differs from the type ichnospecies in having clustered cells arranged in three rows, with one central and two marginal rows of subparallel cells. No bee nest architecture known to us unequivocally fits with any ichnospecies described herein. Uruguay was constructed in the subsoil and may be of halictid origin; however, its large cell size, thick cell walls, and the presence of a vestibular cell do not fit the architecture of nests of extant halictids. Rosellichnus arabicus lacks the complete cavity surrounding the cell cluster that is typical in nests of Old World halictids, but it may, nonetheless, still be of halictid origin. R. pa‐tagonicus has an architecture more similar to nests of anthophorine bees than to those of Neotropical Halictidae. Therefore, the ichnogenus Rosellichnus may include both halictid and anthophorine constructions. Although the smooth inner cell walls, spiral cell caps, and clustering of cells indicate that both Uruguay and Rosellichnus are trace fossils of bee origin, it is likely that both represent nest architectures that are no longer employed by extant bees, or those that are as yet unknown. The paleoenvironments of both the Argentine and Emirates species of Rosellichnus indicate that the trace‐makers of this ichnogenus constructed their nests in open ground sandy soils.

  4. Modeling tensional homeostasis in multicellular clusters.

    PubMed

    Tam, Sze Nok; Smith, Michael L; Stamenović, Dimitrije

    2017-03-01

    Homeostasis of mechanical stress in cells, or tensional homeostasis, is essential for normal physiological function of tissues and organs and is protective against disease progression, including atherosclerosis and cancer. Recent experimental studies have shown that isolated cells are not capable of maintaining tensional homeostasis, whereas multicellular clusters are, with stability increasing with the size of the clusters. Here, we proposed simple mathematical models to interpret experimental results and to obtain insight into factors that determine homeostasis. Multicellular clusters were modeled as one-dimensional arrays of linearly elastic blocks that were either jointed or disjointed. Fluctuating forces that mimicked experimentally measured cell-substrate tractions were obtained from Monte Carlo simulations. These forces were applied to the cluster models, and the corresponding stress field in the cluster was calculated by solving the equilibrium equation. It was found that temporal fluctuations of the cluster stress field became attenuated with increasing cluster size, indicating that the cluster approached tensional homeostasis. These results were consistent with previously reported experimental data. Furthermore, the models revealed that key determinants of tensional homeostasis in multicellular clusters included the cluster size, the distribution of traction forces, and mechanical coupling between adjacent cells. Based on these findings, we concluded that tensional homeostasis was a multicellular phenomenon. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Large nerve cells with long axons in the granular layer and white matter of the murine cerebellum.

    PubMed Central

    Müller, T

    1994-01-01

    The murine cerebellum was investigated by light microscopy using an improved modification of Ehrlich's methylene blue supravital staining technique. The dye exhibited a special affinity for the perikarya as well as the axons of Purkinje cells. In addition, large fusiform or stellate nerve cells which were characterised by long descending axons were seen to be distributed diffusely within the granular layer and the subcortical white matter. These findings indicate the existence of a 2nd type of projection neuron besides the Purkinje cells and are therefore in full accordance with older neuroanatomical observations based on silver impregnation. When correlated with recent studies on the occurrence of different calcium-binding proteins, the results show that the large perikarya demonstrated immunohistochemically within the granular layer seem to belong to the group of methylene blue positive neurons. Nevertheless, the definitive association of a single neuron with a nerve cell class is only possible if the axon is stained and clearly identifiable. Because of its selectivity for a special type of nerve cell, including its axon, the histological method used in this study may therefore also be suitable for investigating other parts of the brain and the spinal cord. Images Fig. 1 Fig. 2 PMID:7516932

  6. Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

    PubMed

    Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

    2014-07-01

    Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Potential and tension changes induced by sodium removal in dog Purkinje fibres: role of an electrogenic sodium-calcium exchange.

    PubMed Central

    Croaboeuf, E; Gautier, P; Giuraudou, P

    1981-01-01

    1. Isolated dog Purkinje fibres were bathed in K-free media or in the presence of ouabain 10(-4) M in order to depress the electrogenic sodium pump activity. Membrane potential and mechanical tension were recorded in the presence of normal external sodium concentration and during lowering or removal of external Na. 2. Lowering or removal of external Na (Na being replaced by choline, Tris, sucrose or Li) induced a hyperpolarization and a contracture which reached a maximum after 1 or 2 min and then decreased progressively. Using Tris, Em increased from -40 +/- 3 to -72 +/- 10 mV (n = 39). The Na-free contracture and hyperpolarization did not occur in the absence of Na pump depression. 3. Tetrodotoxin (1.2 x 10(-5)M), Mn (4 mM), verapamil (1-4 x 10(-5) M) tetraethylammonium (5 mM), 4-aminopyridine (5 mM) and Cs (20 mM, in the presence of ouabain) did not alter the Na-free contracture and hyperpolarization. On the other hand Mn (20 mM), acid media (external pH less than 6.0) and low temperatures depressed or suppressed both the hyperpolarization and contracture. Lanthanum (0.4 mM) did not suppress the hyperpolarization and the contracture. On the contrary the Na-free contracture was generally increased in the presence of La. 4. Caffeine (10 mM) induced strong contractures with no changes in Em, thus demonstrating the possibility for the Purkinje fibers of developing contractures without concomitant hyperpolarizations. 5. It can be concluded that the Na-free contracture and hyperpolarization are not due to changes in passive conductances but are related to the functioning of an electrogenic Na-Ca exchange mechanism which carries inwardly 1 Ca and outwardly 3 or more Na. Images Fig. 1 PMID:7264984

  8. Phosphatase CD45 Both Positively and Negatively Regulates T Cell Receptor Phosphorylation in Reconstituted Membrane Protein Clusters*♦

    PubMed Central

    Furlan, Gabriela; Minowa, Takashi; Hanagata, Nobutaka; Kataoka-Hamai, Chiho; Kaizuka, Yoshihisa

    2014-01-01

    T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation. PMID:25128530

  9. Clustering of Ca2+ transients in interstitial cells of Cajal defines slow wave duration

    PubMed Central

    Drumm, Bernard T.; Hennig, Grant W.; Battersby, Matthew J.; Sung, Tae Sik

    2017-01-01

    Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca2+-activated Cl− channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca2+ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca2+ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca2+ sensor, GCaMP3, in ICC. Ca2+ transients in ICC-MY display a complex firing pattern caused by localized Ca2+ release events arising from multiple sites in cell somata and processes. Ca2+ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca2+ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca2+ transients. Summation of CTCs results in relatively uniform Ca2+ responses from one slow wave to another. These Ca2+ transients are caused by Ca2+ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP3 receptors. Reduced extracellular Ca2+ concentrations and T-type Ca2+ channel blockers decreased the number of firing sites and firing probability of Ca2+ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca2+ transients from multiple intracellular release sites. These events are organized into clusters by Ca2+ influx through T-type Ca2+ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves. PMID:28592421

  10. Diverse functions of miR-17-92 cluster microRNAs in T helper cells.

    PubMed

    Baumjohann, Dirk

    2018-06-01

    T helper (Th) cells are critically involved in adaptive immune responses against various pathogens. In contrast, dysregulated T helper cell responses are associated with a variety of diseases, including autoimmunity, allergies, and cancer. Differentiation of naïve CD4 + T cells into effector T helper cell subsets, including Th1, Th2, Th17, Treg, and T follicular helper (Tfh), requires precise dosing of signaling molecules and transcription factors. MicroRNAs (miRNAs), which are small endogenously expressed RNAs that regulate gene expression, play important roles in these processes. The miR-17-92 cluster, a miRNA polycistron also known as oncomiR-1, has emerged as a central integrator of gene expression events that govern T helper cell differentiation pathways. The complexity of miR-17-92-mediated gene regulation lies in the nature of this miRNA cluster, which consists of six different miRNAs. Individual miR-17-92 miRNAs, albeit initially transcribed as one transcript, can have cooperative or opposing effects on biological processes. Therefore, a better understanding of the molecular regulation of miR-17-92 and its downstream networks will provide important insights into T helper cell differentiation and diversity that may be harnessed for the design of advanced T cell-targeting therapies. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Contribution of the Pmra Promoter to Expression of Genes in the Escherichia coli mra Cluster of Cell Envelope Biosynthesis and Cell Division Genes

    PubMed Central

    Mengin-Lecreulx, Dominique; Ayala, Juan; Bouhss, Ahmed; van Heijenoort, Jean; Parquet, Claudine; Hara, Hiroshi

    1998-01-01

    Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (Pmra) on the chromosome and its replacement by the lac promoter (Pmra::Plac) led to isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from Pmra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which Pmra::Plac promoter expression was repressed or fully induced. It was confirmed that the Pmra promoter is required for expression of the first nine genes of the mra cluster: mraZ (orfC), mraW (orfB), ftsL (mraR), ftsI, murE, murF, mraY, murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced Pmra::Plac cells. This was correlated with an accumulation of the nucleotide precursors UDP–N-acetylglucosamine and UDP–N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when Pmra expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ, is also mainly (but not exclusively) dependent on the Pmra promoter. PMID:9721276

  12. Structure-related clustering of gene expression fingerprints of thp-1 cells exposed to smaller polycyclic aromatic hydrocarbons.

    PubMed

    Wan, B; Yarbrough, J W; Schultz, T W

    2008-01-01

    This study was undertaken to test the hypothesis that structurally similar PAHs induce similar gene expression profiles. THP-1 cells were exposed to a series of 12 selected PAHs at 50 microM for 24 hours and gene expressions profiles were analyzed using both unsupervised and supervised methods. Clustering analysis of gene expression profiles revealed that the 12 tested chemicals were grouped into five clusters. Within each cluster, the gene expression profiles are more similar to each other than to the ones outside the cluster. One-methylanthracene and 1-methylfluorene were found to have the most similar profiles; dibenzothiophene and dibenzofuran were found to share common profiles with fluorine. As expression pattern comparisons were expanded, similarity in genomic fingerprint dropped off dramatically. Prediction analysis of microarrays (PAM) based on the clustering pattern generated 49 predictor genes that can be used for sample discrimination. Moreover, a significant analysis of Microarrays (SAM) identified 598 genes being modulated by tested chemicals with a variety of biological processes, such as cell cycle, metabolism, and protein binding and KEGG pathways being significantly (p < 0.05) affected. It is feasible to distinguish structurally different PAHs based on their genomic fingerprints, which are mechanism based.

  13. The Formation of Tight Tumor Clusters Affects the Efficacy of Cell Cycle Inhibitors: A Hybrid Model Study

    PubMed Central

    Kim, MunJu; Reed, Damon; Rejniak, Katarzyna A.

    2014-01-01

    Cyclin-dependent kinases (CDKs) are vital in regulating cell cycle progression, and, thus, in highly proliferating tumor cells CDK inhibitors are gaining interest as potential anticancer agents. Clonogenic assay experiments are frequently used to determine drug efficacy against the survival and proliferation of cancer cells. While the anticancer mechanisms of drugs are usually described at the intracellular single-cell level, the experimental measurements are sampled from the entire cancer cell population. This approach may lead to discrepancies between the experimental observations and theoretical explanations of anticipated drug mechanisms. To determine how individual cell responses to drugs that inhibit CDKs affect the growth of cancer cell populations, we developed a spatially explicit hybrid agent-based model. In this model, each cell is equipped with internal cell cycle regulation mechanisms, but it is also able to interact physically with its neighbors. We model cell cycle progression, focusing on the G1 and G2/M cell cycle checkpoints, as well as on related essential components, such as CDK1, CDK2, cell size, and DNA damage. We present detailed studies of how the emergent properties (e.g., cluster formation) of an entire cell population depend on altered physical and physiological parameters. We analyze the effects of CDK1 and CKD2 inhibitors on population growth, time-dependent changes in cell cycle distributions, and the dynamic evolution of spatial cell patterns. We show that cell cycle inhibitors that cause cell arrest at different cell cycle phases are not necessarily synergistically super-additive. Finally, we demonstrate that the physical aspects of cell population growth, such as the formation of tight cell clusters versus dispersed colonies, alter the efficacy of cell cycle inhibitors, both in 2D and 3D simulations. This finding may have implications for interpreting the treatment efficacy results of in vitro experiments, in which treatment is

  14. Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

    NASA Astrophysics Data System (ADS)

    Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

    2018-05-01

    Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

  15. Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines

    PubMed Central

    Eryilmaz, Marion; Schmitt, Eberhard; Krufczik, Matthias; Theda, Franziska; Lee, Jin-Ho; Cremer, Christoph; Bestvater, Felix; Schaufler, Wladimir; Hildenbrand, Georg

    2018-01-01

    In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair. PMID:29361783

  16. Mesenchymal stem cells and their conditioned medium improve integration of purified induced pluripotent stem cell-derived cardiomyocyte clusters into myocardial tissue.

    PubMed

    Rubach, Martin; Adelmann, Roland; Haustein, Moritz; Drey, Florian; Pfannkuche, Kurt; Xiao, Bing; Koester, Annette; Udink ten Cate, Floris E A; Choi, Yeong-Hoon; Neef, Klaus; Fatima, Azra; Hannes, Tobias; Pillekamp, Frank; Hescheler, Juergen; Šarić, Tomo; Brockmeier, Konrad; Khalil, Markus

    2014-03-15

    Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) might become therapeutically relevant to regenerate myocardial damage. Purified iPS-CMs exhibit poor functional integration into myocardial tissue. The aim of this study was to investigate whether murine mesenchymal stem cells (MSCs) or their conditioned medium (MScond) improves the integration of murine iPS-CMs into myocardial tissue. Vital or nonvital embryonic murine ventricular tissue slices were cocultured with purified clusters of iPS-CMs in combination with murine embryonic fibroblasts (MEFs), MSCs, or MScond. Morphological integration was assessed by visual scoring and functional integration by isometric force and field potential measurements. We observed a moderate morphological integration of iPS-CM clusters into vital, but a poor integration into nonvital, slices. MEFs and MSCs but not MScond improved morphological integration of CMs into nonvital slices and enabled purified iPS-CMs to confer force. Coculture of vital slices with iPS-CMs and MEFs or MSCs resulted in an improved electrical integration. A comparable improvement of electrical coupling was achieved with the cell-free MScond, indicating that soluble factors secreted by MSCs were involved in electrical coupling. We conclude that cells such as MSCs support the engraftment and adhesion of CMs, and confer force to noncontractile tissue. Furthermore, soluble factors secreted by MSCs mediate electrical coupling of purified iPS-CM clusters to myocardial tissue. These data suggest that MSCs may increase the functional engraftment and therapeutic efficacy of transplanted iPS-CMs into infarcted myocardium.

  17. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

    NASA Technical Reports Server (NTRS)

    Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

    2000-01-01

    Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

  18. Absence of the BRAF mutation in HBME1+ and CK19+ atypical cell clusters in Hashimoto thyroiditis: supportive evidence against preneoplastic change.

    PubMed

    Nasr, Michel R; Mukhopadhyay, Sanjay; Zhang, Shengle; Katzenstein, Anna-Luise A

    2009-12-01

    An association between Hashimoto thyroiditis and papillary thyroid carcinoma has been postulated for decades. We undertook this study to identify potential precursors of papillary thyroid carcinoma in Hashimoto thyroiditis using a combination of morphologic, immunohistochemical, and molecular techniques. For the study, samples from 59 cases of Hashimoto thyroiditis were stained with antibodies to HBME1 and cytokeratin (CK)19. Tiny HBME1+ and CK19+ atypical cell clusters were identified and analyzed for the BRAF mutation by the colorimetric Mutector assay and allele-specific polymerase chain reaction. HBME1+ and CK19+ atypical cell clusters were identified in 12 (20%) of 59 cases. The minute size (<1 mm) of the clusters and the incomplete nuclear changes precluded a diagnosis of papillary microcarcinoma. The atypical cell clusters from all 12 cases were negative for BRAF. The absence of the BRAF mutation in these atypical cell clusters suggests that they may not be preneoplastic. Caution should be exercised in interpreting positive HBME1 or CK19 staining in Hashimoto thyroiditis.

  19. Bioeffects of Radiofrequency Radiation: A Review Pertinent to Air Force Operations.

    DTIC Science & Technology

    1983-03-01

    medium, opacities and assocjited globular degeneration of the lens cells developed in 1 day. Similar effects were observed when lenses were warmed to...of microglial~ cells , with microgli l processes show ng initial signs f degene ation. Many of these "low-intensity" effects are similar to those...assess effects of the exposure on cerebellar Purkinje cells . Because of the immaturity of the neonates, the Purkinje cell layer was not clearly displayed

  20. Expression of the alpha and beta subunits of Ca2+/calmodulin kinase II in the cerebellum of jaundiced Gunn rats during development: a quantitative light microscopic analysis.

    PubMed

    Conlee, J W; Shapiro, S M; Churn, S B

    2000-04-01

    The homozygous (jj) jaundiced Gunn rat model for hyperbilirubinemia displays pronounced cerebellar hypoplasia. To examine the cellular mechanisms involved in bilirubin toxicity, this study focused on the effect of hyperbilirubinemia on calcium/calmodulin-dependent kinase II (CaM kinase II). CaM kinase II is a neuronally enriched enzyme which performs several important functions. Immunohistochemical analysis of alternating serial sections were performed using monoclonal antibodies for the alpha and beta subunits of CaM kinase II. Measurements were made of the total numbers of stained cells in each of the deep cerebellar nuclei and of Purkinje and granule cell densities in cerebellar lobules II, VI, and IX. The beta subunit was present in Purkinje cells and deep cerebellar nuclei of both groups at all ages, but only granule cells which had migrated through the Purkinje cell layer showed staining for beta subunit; external granule cells were completely negative. Many Purkinje cells had degenerated in the older animals, and the percent of granule cells stained for beta subunit was significantly reduced. The alpha subunit was found exclusively in Purkinje cells, although its appearance was delayed in the jaundiced animals. Sulfadimethoxine was administered to some jj rats 24 h or 15 days prior to sacrifice to increase brain bilirubin concentration. Results showed that bilirubin exposure modulated both alpha and beta CaM kinase II subunit expression in selective neuronal populations, but sulfadimethoxine had no acute effect on enzyme immunoreactivity. Thus, developmental expression of the alpha and beta subunits of CaM kinase II was affected by chronic bilirubin exposure during early postnatal development of jaundiced Gunn rats.

  1. Myeloid Clusters Are Associated with a Pro-Metastatic Environment and Poor Prognosis in Smoking-Related Early Stage Non-Small Cell Lung Cancer

    PubMed Central

    Zhang, Wang; Pal, Sumanta K.; Liu, Xueli; Yang, Chunmei; Allahabadi, Sachin; Bhanji, Shaira; Figlin, Robert A.; Yu, Hua; Reckamp, Karen L.

    2013-01-01

    Background This study aimed to understand the role of myeloid cell clusters in uninvolved regional lymph nodes from early stage non-small cell lung cancer patients. Methods Uninvolved regional lymph node sections from 67 patients with stage I–III resected non-small cell lung cancer were immunostained to detect myeloid clusters, STAT3 activity and occult metastasis. Anthracosis intensity, myeloid cluster infiltration associated with anthracosis and pSTAT3 level were scored and correlated with patient survival. Multivariate Cox regression analysis was performed with prognostic variables. Human macrophages were used for in vitro nicotine treatment. Results CD68+ myeloid clusters associated with anthracosis and with an immunosuppressive and metastasis-promoting phenotype and elevated overall STAT3 activity were observed in uninvolved lymph nodes. In patients with a smoking history, myeloid cluster score significantly correlated with anthracosis intensity and pSTAT3 level (P<0.01). Nicotine activated STAT3 in macrophages in long-term culture. CD68+ myeloid clusters correlated and colocalized with occult metastasis. Myeloid cluster score was an independent prognostic factor (P = 0.049) and was associated with survival by Kaplan-Maier estimate in patients with a history of smoking (P = 0.055). The combination of myeloid cluster score with either lymph node stage or pSTAT3 level defined two populations with a significant difference in survival (P = 0.024 and P = 0.004, respectively). Conclusions Myeloid clusters facilitate a pro-metastatic microenvironment in uninvolved regional lymph nodes and associate with occult metastasis in early stage non-small cell lung cancer. Myeloid cluster score is an independent prognostic factor for survival in patients with a history of smoking, and may present a novel method to inform therapy choices in the adjuvant setting. Further validation studies are warranted. PMID:23717691

  2. Diametrical clustering for identifying anti-correlated gene clusters.

    PubMed

    Dhillon, Inderjit S; Marcotte, Edward M; Roshan, Usman

    2003-09-01

    Clustering genes based upon their expression patterns allows us to predict gene function. Most existing clustering algorithms cluster genes together when their expression patterns show high positive correlation. However, it has been observed that genes whose expression patterns are strongly anti-correlated can also be functionally similar. Biologically, this is not unintuitive-genes responding to the same stimuli, regardless of the nature of the response, are more likely to operate in the same pathways. We present a new diametrical clustering algorithm that explicitly identifies anti-correlated clusters of genes. Our algorithm proceeds by iteratively (i). re-partitioning the genes and (ii). computing the dominant singular vector of each gene cluster; each singular vector serving as the prototype of a 'diametric' cluster. We empirically show the effectiveness of the algorithm in identifying diametrical or anti-correlated clusters. Testing the algorithm on yeast cell cycle data, fibroblast gene expression data, and DNA microarray data from yeast mutants reveals that opposed cellular pathways can be discovered with this method. We present systems whose mRNA expression patterns, and likely their functions, oppose the yeast ribosome and proteosome, along with evidence for the inverse transcriptional regulation of a number of cellular systems.

  3. Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single- and dual-species cultures.

    PubMed

    Behnke, Sabrina; Parker, Albert E; Woodall, Dawn; Camper, Anne K

    2011-10-01

    Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells. Burkholderia cepacia and Pseudomonas aeruginosa were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species B. cepacia, B. cepacia in coculture, and P. aeruginosa in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture.

  4. DNA-PKcs deficiency leads to persistence of oxidatively-induced clustered DNA lesions in human tumor cells

    PubMed Central

    Peddi, Prakash; Loftin, Charles W.; Dickey, Jennifer S.; Hair, Jessica M.; Burns, Kara J.; Aziz, Khaled; Francisco, Dave C.; Panayiotidis, Mihalis I.; Sedelnikova, Olga A.; Bonner, William M.; Winters, Thomas A.; Georgakilas, Alexandros G.

    2010-01-01

    DNA-dependent protein kinase (DNA-PK) is a key non-homologous end joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. In order to examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three different models of DNA-PK deficiency i.e. chemical inactivation of its kinase activity by novel inhibitors IC86621 and NU7026, knock-down and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). Compromised DNA-PK repair pathway has lead to accumulation of clustered DNA lesions induced by γ-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively-induced non-DSB clustered DNA lesions measured using a modified version of pulsed field gel electrophoresis or single cell gel electrophoresis (Comet assay). In all cases, DNA-PK inactivation lead to a higher level of lesion persistence even after 24–72 hrs of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters by first compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions due to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important not only for understating cancer etiology in the presence of a NHEJ deficiency but also lead to a better understanding of cancer treatments based on the induction of oxidative stress and inhibition of cluster repair. PMID:20193758

  5. Architecture of Eph receptor clusters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Himanen, Juha P.; Yermekbayeva, Laila; Janes, Peter W.

    2010-10-04

    Eph receptor tyrosine kinases and their ephrin ligands regulate cell navigation during normal and oncogenic development. Signaling of Ephs is initiated in a multistep process leading to the assembly of higher-order signaling clusters that set off bidirectional signaling in interacting cells. However, the structural and mechanistic details of this assembly remained undefined. Here we present high-resolution structures of the complete EphA2 ectodomain and complexes with ephrin-A1 and A5 as the base unit of an Eph cluster. The structures reveal an elongated architecture with novel Eph/Eph interactions, both within and outside of the Eph ligand-binding domain, that suggest the molecular mechanismmore » underlying Eph/ephrin clustering. Structure-function analysis, by using site-directed mutagenesis and cell-based signaling assays, confirms the importance of the identified oligomerization interfaces for Eph clustering.« less

  6. Ablation of the MiR-17-92 MicroRNA Cluster in Germ Cells Causes Subfertility in Female Mice.

    PubMed

    Wang, Jian; Xu, Bo; Tian, Geng G; Sun, Tao; Wu, Ji

    2018-01-01

    Oogenesis is a highly complex process that is intricately regulated by interactions of multiple genes and signaling molecules. However, the underlying molecular mechanisms are poorly understood. There is emerging evidence that microRNAs contribute to oogenesis. Here, we aimed to investigate the role of miR-17-92 cluster in regulating oogenesis. The miR-17-92 cluster was genetically ablated in germ cells of female mice by applying the Cre-loxp system for conditional gene knockout. Mating experiment, superovulation and histological analysis were used to assess the fertility of the model female mice. TUNEL assay was used to identify apoptotic cells in ovaries. The expression level of apoptosis- and follicular atresia- related genes was evaluated by qRT-PCR. Western blotting was performed to detect protein expression. Bioinformatics software and dual luciferase reporter assay were applied to predict and verify the target of miR-17-92 cluster. Deletion of miR-17-92 cluster in germ cells of female mice caused increased oocyte degradation and follicular atresia, perturbed oogenesis, and ultimately led to subfertility. Genes involved in follicular atresia and the mitochondrial apoptotic pathway were obviously up-regulated. Furthermore, we verified that miR-19a regulated oogenesis at the post-transcriptional level by targeting Bmf in the ovaries of miR-17-92 cluster conditional knockout female mice. The miR-17-92 cluster is an important regulator of oogenesis. These findings will assist in better understanding the etiology of disorders in oogenesis and in developing new therapeutic targets for female infertility. © 2018 The Author(s). Published by S. Karger AG, Basel.

  7. A cluster analysis method for identification of subpopulations of cells in flow cytometric list-mode arrays

    NASA Technical Reports Server (NTRS)

    Li, Z. K.

    1985-01-01

    A specialized program was developed for flow cytometric list-mode data using an heirarchical tree method for identifying and enumerating individual subpopulations, the method of principal components for a two-dimensional display of 6-parameter data array, and a standard sorting algorithm for characterizing subpopulations. The program was tested against a published data set subjected to cluster analysis and experimental data sets from controlled flow cytometry experiments using a Coulter Electronics EPICS V Cell Sorter. A version of the program in compiled BASIC is usable on a 16-bit microcomputer with the MS-DOS operating system. It is specialized for 6 parameters and up to 20,000 cells. Its two-dimensional display of Euclidean distances reveals clusters clearly, as does its 1-dimensional display. The identified subpopulations can, in suitable experiments, be related to functional subpopulations of cells.

  8. Neurokinin B-producing projection neurons in the lateral stripe of the striatum and cell clusters of the accumbens nucleus in the rat.

    PubMed

    Zhou, Ligang; Furuta, Takahiro; Kaneko, Takeshi

    2004-12-06

    Neurons producing preprotachykinin B (PPTB), the precursor of neurokinin B, constitute 5% of neurons in the dorsal striatum and project to the substantia innominata (SI) selectively. In the ventral striatum, PPTB-producing neurons are collected mainly in the lateral stripe of the striatum (LSS) and cell clusters of the accumbens nucleus (Acb). In the present study, we first examined the distribution of PPTB-immunoreactive neurons in rat ventral striatum and found that a large part of the PPTB-immunoreactive cell clusters was continuous to the LSS, but a smaller part was not. Thus, we divided the PPTB-immunoreactive cell clusters into the LSS-associated and non-LSS-associated ones. We next investigated the projection targets of the PPTB-producing ventral striatal neurons by combining immunofluorescence labeling and retrograde tracing. After injection of Fluoro-Gold into the basal component of the SI (SIb) and medial part of the interstitial nucleus of posterior limb of the anterior commissure, many PPTB-immunoreactive neurons were retrogradely labeled in the LSS-associated cell clusters and LSS, respectively. When the injection site included the ventral part of the sublenticular component of the SI(SIsl), retrogradely labeled neurons showed PPTB-immunoreactivity frequently in non-LSS-associated cell clusters. Furthermore, these PPTB-immunoreactive projections were confirmed by the double-fluorescence method after anterograde tracer injection into the ventral striatum containing the cell clusters. Since the dorsalmost part of the SIsl is known to receive strong inputs from PPTB-producing dorsal striatal neurons, the present results indicate that PPTB-producing ventral striatal neurons project to basal forebrain target regions in parallel with dorsal striatal neurons without significant convergence. 2004 Wiley-Liss, Inc.

  9. Distributed Cerebellar Motor Learning: A Spike-Timing-Dependent Plasticity Model

    PubMed Central

    Luque, Niceto R.; Garrido, Jesús A.; Naveros, Francisco; Carrillo, Richard R.; D'Angelo, Egidio; Ros, Eduardo

    2016-01-01

    Deep cerebellar nuclei neurons receive both inhibitory (GABAergic) synaptic currents from Purkinje cells (within the cerebellar cortex) and excitatory (glutamatergic) synaptic currents from mossy fibers. Those two deep cerebellar nucleus inputs are thought to be also adaptive, embedding interesting properties in the framework of accurate movements. We show that distributed spike-timing-dependent plasticity mechanisms (STDP) located at different cerebellar sites (parallel fibers to Purkinje cells, mossy fibers to deep cerebellar nucleus cells, and Purkinje cells to deep cerebellar nucleus cells) in close-loop simulations provide an explanation for the complex learning properties of the cerebellum in motor learning. Concretely, we propose a new mechanistic cerebellar spiking model. In this new model, deep cerebellar nuclei embed a dual functionality: deep cerebellar nuclei acting as a gain adaptation mechanism and as a facilitator for the slow memory consolidation at mossy fibers to deep cerebellar nucleus synapses. Equipping the cerebellum with excitatory (e-STDP) and inhibitory (i-STDP) mechanisms at deep cerebellar nuclei afferents allows the accommodation of synaptic memories that were formed at parallel fibers to Purkinje cells synapses and then transferred to mossy fibers to deep cerebellar nucleus synapses. These adaptive mechanisms also contribute to modulate the deep-cerebellar-nucleus-output firing rate (output gain modulation toward optimizing its working range). PMID:26973504

  10. [Subacute paraneoplastic cerebellar degeneration with anti-neuronal antibodies and gynecological cancers. A case report of a bilateral ovarian cancer where the classic "anti-YO" antibody is associated with another antineuronal antibody].

    PubMed

    Tremouilhac, C; Hamy, A; De Ferron, E; Paineau, J; Visset, J

    1996-01-01

    Subacute paraneoplastic cerebellar degeneration (SPCD) is a cerebellar syndrome associated with an identifiable or occult carcinoma without direct involvement of the nervous system by the cancer. This subacute syndrome is due to an extensive Purkinje cell destruction by anti-Purkinje cells autoantibodies. Some of them are specific for example "anti-YO" antibodies in gynecologic cancer situations. We report the case of a 50-year-old woman who presented an ovarien carcinoma revealed by a SPCD associated to an anti-Purkinje cell autoantibody "anti-YO" and to another unidentified autoantibody. Despite the treatment of the carcinoma, the invaliding SPCD did not regress. The diagnosis of SPCD requires identification and early treatment of the carcinoma, giving the patient the best chances for cure and avoiding major neurologic effects.

  11. Effect on Membrane Potential and Electrical Activity of Adding Sodium to Sodium-Depleted Cardiac Purkinje Fibers

    PubMed Central

    Wiggins, Jay R.; Cranefield, Paul F.

    1974-01-01

    Canine cardiac Purkinje fibers exposed to Na-free solutions containing 128 mM TEA and 16 mM Ca show resting potentials in the range -50 to -90 mV; if the concentration of Na in the perfusate is raised from 0 to 4 to 24 mM, hyperpolarization follows. If the initial resting potential is low, the hyperpolarization tends to be greater; the average increase in the presence of 8 mM Na is 14 mV. Such hyperpolarization is not induced by adding Na to K-free solutions, is not seen in cooled fibers, or in fibers exposed to 10-3 M ouabain, nor is it induced by adding Li and thus may result from electrogenic sodium extrusion. Fibers exposed to Na-free solutions are often spontaneously active; if they are quiescent they often show repetitive activity during depolarizing pulses. Such spontaneous or repetitive activity is suppressed by the addition of Na. This suppression may or may not be related to the hyperpolarization. PMID:4418558

  12. Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells.

    PubMed

    Silverman, Michael A; Shoag, Jonathan; Wu, Jennifer; Koretzky, Gary A

    2006-03-01

    We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal FcepsilonRI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon FcepsilonRI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the FcepsilonRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following FcepsilonRI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcepsilonRI-induced mast cell responses.

  13. Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells.

    PubMed

    Neves, Rui; Scheel, Christina; Weinhold, Sandra; Honisch, Ellen; Iwaniuk, Katharina M; Trompeter, Hans-Ingo; Niederacher, Dieter; Wernet, Peter; Santourlidis, Simeon; Uhrberg, Markus

    2010-08-03

    The miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT) process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors. We show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE) was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus. The present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.

  14. Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects

    PubMed Central

    Peng, Shiwen; Trimble, Cornelia; Alvarez, Ronald D.; Huh, Warner K.; Lin, Zhenhua; Monie, Archana; Hung, Chien-Fu; Wu, T.-C.

    2010-01-01

    Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal timeframe. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation. PMID:18401437

  15. Cytological Study of Grade 3 Endometrioid Adenocarcinoma of Endometrial Origin: Cytoarchitecture and Features of Cell Clusters Assessed With Endometrial Brushing Cytology--Focusing on a comparison with endometrioid adenocarcinoma Grade 1, 2.

    PubMed

    Matsui, Naruaki; Kajiwara, Hiroshi; Morishita, Akihiro; Tsukada, Hitomi; Nakazawa, Kazumi; Miyazawa, Masaki; Mikami, Mikio; Nakamura, Naoya; Sato, Shinkichi

    2015-06-20

    Aim of study was to clarify the cytological characteristics of grade 3 endometrioid adenocarcinoma of endometrial origin (G3 EA) by endometrial brushing cytology. The subjects were 11 patients in whom G3 EA was diagnosed by review of preoperative cytological specimens obtained at our hospital and related institutions between 2000 and 2010. These patients were investigated with respect to the preoperative cytological diagnosis, background changes, cell cluster patterns, and individual cellular findings. Background changes were classified as inflammatory or tumorous, while cell clusters were classified as overlapping cell cluster, sheet-like cell cluster, clump of high dense gland, papillary, or other cell cluster. Cellular findings were investigated by comparing the incidence of squamous and clear cell metaplasia, the nuclear rounding rate, and the nuclear area with the findings in a control group (35 patients with G1-2 EA). Background changes were classified as inflammatory in 63.6% and necrotic in 36.4%. The cell clusters were classified as overlapping cell cluster in 44.8%, cell cluster in 21.7%, clump of high dense gland in 10.0%, papillary in 4.0%, and other cell cluster in 19.5%. The incidence of squamous and clear cell metaplasia was 27.2% and 18.1%, respectively. The mean nuclear rounding rate was 0.97, and the mean nuclear area was 55.98 µm2. Investigation of the cytoarchitecture of G3 EA with endometrial brushing cytology revealed overlapping cell cluster and tumor cells of a relatively uniform size. These findings suggest that it is necessary to recognize that there are differences between the cytological findings of G3 EA and the usual features of G1-2 EA.

  16. Characteristics of the early immune response following transplantation of mouse ES cell derived insulin-producing cell clusters.

    PubMed

    Boyd, Ashleigh S; Wood, Kathryn J

    2010-06-04

    The fully differentiated progeny of ES cells (ESC) may eventually be used for cell replacement therapy (CRT). However, elements of the innate immune system may contribute to damage or destruction of these tissues when transplanted. Herein, we assessed the hitherto ill-defined contribution of the early innate immune response in CRT after transplantation of either ESC derived insulin producing cell clusters (IPCCs) or adult pancreatic islets. Ingress of neutrophil or macrophage cells was noted immediately at the site of IPCC transplantation, but this infiltration was attenuated by day three. Gene profiling identified specific inflammatory cytokines and chemokines that were either absent or sharply reduced by three days after IPCC transplantation. Thus, IPCC transplantation provoked less of an early immune response than pancreatic islet transplantation. Our study offers insights into the characteristics of the immune response of an ESC derived tissue in the incipient stages following transplantation and suggests potential strategies to inhibit cell damage to ensure their long-term perpetuation and functionality in CRT.

  17. A discrete in continuous mathematical model of cardiac progenitor cells formation and growth as spheroid clusters (Cardiospheres).

    PubMed

    Di Costanzo, Ezio; Giacomello, Alessandro; Messina, Elisa; Natalini, Roberto; Pontrelli, Giuseppe; Rossi, Fabrizio; Smits, Robert; Twarogowska, Monika

    2018-03-14

    We propose a discrete in continuous mathematical model describing the in vitro growth process of biophsy-derived mammalian cardiac progenitor cells growing as clusters in the form of spheres (Cardiospheres). The approach is hybrid: discrete at cellular scale and continuous at molecular level. In the present model, cells are subject to the self-organizing collective dynamics mechanism and, additionally, they can proliferate and differentiate, also depending on stochastic processes. The two latter processes are triggered and regulated by chemical signals present in the environment. Numerical simulations show the structure and the development of the clustered progenitors and are in a good agreement with the results obtained from in vitro experiments.

  18. Role of Plasticity at Different Sites across the Time Course of Cerebellar Motor Learning

    PubMed Central

    Lisberger, Stephen G.

    2014-01-01

    Learning comprises multiple components that probably involve cellular and synaptic plasticity at multiple sites. Different neural sites may play their largest roles at different times during behavioral learning. We have used motor learning in smooth pursuit eye movements of monkeys to determine how and when different components of learning occur in a known cerebellar circuit. The earliest learning occurs when one climbing-fiber response to a learning instruction causes simple-spike firing rate of Purkinje cells in the floccular complex of the cerebellum to be depressed transiently at the time of the instruction on the next trial. Trial-over-trial depression and the associated learning in eye movement are forgotten in <6 s, but facilitate long-term behavioral learning over a time scale of ∼5 min. During 100 repetitions of a learning instruction, simple-spike firing rate becomes progressively depressed in Purkinje cells that receive climbing-fiber inputs from the instruction. In Purkinje cells that prefer the opposite direction of pursuit and therefore do not receive climbing-fiber inputs related to the instruction, simple-spike responses undergo potentiation, but more weakly and more slowly. Analysis of the relationship between the learned changes in simple-spike firing and learning in eye velocity suggests an orderly progression of plasticity: first on Purkinje cells with complex-spike (CS) responses to the instruction, later on Purkinje cells with CS responses to the opposite direction of instruction, and last in sites outside the cerebellar cortex. Climbing-fiber inputs appear to play a fast and primary, but nonexclusive, role in pursuit learning. PMID:24849344

  19. Cell fusion in the brain: two cells forward, one cell back.

    PubMed

    Kemp, Kevin; Wilkins, Alastair; Scolding, Neil

    2014-11-01

    Adult stem cell populations, notably those which reside in the bone marrow, have been shown to contribute to several neuronal cell types in the rodent and human brain. The observation that circulating bone marrow cells can migrate into the central nervous system and fuse with, in particular, cerebellar Purkinje cells has suggested, at least in part, a potential mechanism behind this process. Experimentally, the incidence of cell fusion in the brain is enhanced with age, radiation exposure, inflammation, chemotherapeutic drugs and even selective damage to the neurons themselves. The presence of cell fusion, shown by detection of increased bi-nucleated neurons, has also been described in a variety of human central nervous system diseases, including both multiple sclerosis and Alzheimer's disease. Accumulating evidence is therefore raising new questions into the biological significance of cell fusion, with the possibility that it represents an important means of cell-mediated neuroprotection or rescue of highly complex neurons that cannot be replaced in adult life. Here, we discuss the evidence behind this phenomenon in the rodent and human brain, with a focus on the subsequent research investigating the physiological mechanisms of cell fusion underlying this process. We also highlight how these studies offer new insights into endogenous neuronal repair, opening new exciting avenues for potential therapeutic interventions against neurodegeneration and brain injury.

  20. The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection

    PubMed Central

    Arvin, Ann M.; Oliver, Stefan L.

    2016-01-01

    ABSTRACT The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (−1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a −0.5-log10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt. IMPORTANCE Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic

  1. Clustered carbohydrates as a target for natural killer cells: a model system.

    PubMed

    Kovalenko, Elena I; Abakushina, Elena; Telford, William; Kapoor, Veena; Korchagina, Elena; Khaidukov, Sergei; Molotkovskaya, Irina; Sapozhnikov, Alexander; Vlaskin, Pavel; Bovin, Nicolai

    2007-03-01

    Membrane-associated oligosaccharides are known to take part in interactions between natural killer (NK) cells and their targets and modulate NK cell activity. A model system was therefore developed using synthetic glycoconjugates as tools to modify the carbohydrate pattern on NK target cell surfaces. NK cells were then assessed for function in response to synthetic glycoconjugates, using both cytolysis-associated caspase 6 activation measured by flow cytometry and IFN-gamma production. Lipophilic neoglycoconjugates were synthesized to provide their easy incorporation into the target cell membranes and to make carbohydrate residues available for cell-cell interactions. While incorporation was successful based on fluorescence monitoring, glycoconjugate incorporation did not evoke artifactual changes in surface antigen expression, and had no negative effect on cell viability. Glycoconjugates contained Le(x), sulfated Le(x), and Le(y) sharing the common structure motif trisaccharide Le(x) were revealed to enhance cytotoxicity mediated specifically by CD16 +CD56+NK cells. The glycoconjugate effects were dependent on saccharide presentation in a polymeric form. Only polymeric, or clustered, but not monomeric glycoconjugates resulted in alteration of cytotoxicity in our system, suggesting that appropriate presentation is critical for carbohydrate recognition and subsequent biological effects.

  2. The cell adhesion molecule CHL1 interacts with patched-1 to regulate apoptosis during postnatal cerebellar development.

    PubMed

    Katic, Jelena; Loers, Gabriele; Tosic, Jelena; Schachner, Melitta; Kleene, Ralf

    2017-08-01

    The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as a novel CHL1-binding protein and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Colocalization and co-immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans -interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer, smoothened (SMO), and inhibitors of RhoA and Rho-associated kinase (ROCK) 1 and 2 prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice, enhanced apoptosis of granule, but not Purkinje, cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events during mouse cerebellar development. © 2017. Published by The Company of Biologists Ltd.

  3. Synchrony and neural coding in cerebellar circuits

    PubMed Central

    Person, Abigail L.; Raman, Indira M.

    2012-01-01

    The cerebellum regulates complex movements and is also implicated in cognitive tasks, and cerebellar dysfunction is consequently associated not only with movement disorders, but also with conditions like autism and dyslexia. How information is encoded by specific cerebellar firing patterns remains debated, however. A central question is how the cerebellar cortex transmits its integrated output to the cerebellar nuclei via GABAergic synapses from Purkinje neurons. Possible answers come from accumulating evidence that subsets of Purkinje cells synchronize their firing during behaviors that require the cerebellum. Consistent with models predicting that coherent activity of inhibitory networks has the capacity to dictate firing patterns of target neurons, recent experimental work supports the idea that inhibitory synchrony may regulate the response of cerebellar nuclear cells to Purkinje inputs, owing to the interplay between unusually fast inhibitory synaptic responses and high rates of intrinsic activity. Data from multiple laboratories lead to a working hypothesis that synchronous inhibitory input from Purkinje cells can set the timing and rate of action potentials produced by cerebellar nuclear cells, thereby relaying information out of the cerebellum. If so, then changing spatiotemporal patterns of Purkinje activity would allow different subsets of inhibitory neurons to control cerebellar output at different times. Here we explore the evidence for and against the idea that a synchrony code defines, at least in part, the input–output function between the cerebellar cortex and nuclei. We consider the literature on the existence of simple spike synchrony, convergence of Purkinje neurons onto nuclear neurons, and intrinsic properties of nuclear neurons that contribute to responses to inhibition. Finally, we discuss factors that may disrupt or modulate a synchrony code and describe the potential contributions of inhibitory synchrony to other motor circuits. PMID

  4. Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum.

    PubMed

    Delbaere, Joke; Van Herck, Stijn L J; Bourgeois, Nele M A; Vancamp, Pieter; Yang, Shuo; Wingate, Richard J T; Darras, Veerle M

    2016-12-01

    The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T 3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2 weeks of embryonic development points to a potential role of this transporter in the uptake of T 3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may

  5. Dysregulation and functional roles of miR-183-96-182 cluster in cancer cell proliferation, invasion and metastasis

    PubMed Central

    Ma, Yi; Liang, A-Juan; Fan, Yu-Ping; Huang, Yi-Ran; Zhao, Xiao-Ming; Sun, Yun; Chen, Xiang-Feng

    2016-01-01

    Previous studies have reported aberrant expression of the miR-183-96-182 cluster in a variety of tumors, which indicates its' diagnostic or prognostic value. However, a key characteristic of the miR-183-96-182 cluster is its varied expression levels, and pleomorphic functional roles in different tumors or under different conditions. In most tumor types, the cluster is highly expressed and promotes tumorigenesis, cancer progression and metastasis; yet tumor suppressive effects have also been reported in some tumors. In the present study, we discuss the upstream regulators and the downstream target genes of miR-183-96-182 cluster, and highlight the dysregulation and functional roles of this cluster in various tumor cells. Newer insights summarized in this review will help readers understand the different facets of the miR-183-96-182 cluster in cancer development and progression. PMID:27081087

  6. A proximity-based graph clustering method for the identification and application of transcription factor clusters.

    PubMed

    Spadafore, Maxwell; Najarian, Kayvan; Boyle, Alan P

    2017-11-29

    Transcription factors (TFs) form a complex regulatory network within the cell that is crucial to cell functioning and human health. While methods to establish where a TF binds to DNA are well established, these methods provide no information describing how TFs interact with one another when they do bind. TFs tend to bind the genome in clusters, and current methods to identify these clusters are either limited in scope, unable to detect relationships beyond motif similarity, or not applied to TF-TF interactions. Here, we present a proximity-based graph clustering approach to identify TF clusters using either ChIP-seq or motif search data. We use TF co-occurrence to construct a filtered, normalized adjacency matrix and use the Markov Clustering Algorithm to partition the graph while maintaining TF-cluster and cluster-cluster interactions. We then apply our graph structure beyond clustering, using it to increase the accuracy of motif-based TFBS searching for an example TF. We show that our method produces small, manageable clusters that encapsulate many known, experimentally validated transcription factor interactions and that our method is capable of capturing interactions that motif similarity methods might miss. Our graph structure is able to significantly increase the accuracy of motif TFBS searching, demonstrating that the TF-TF connections within the graph correlate with biological TF-TF interactions. The interactions identified by our method correspond to biological reality and allow for fast exploration of TF clustering and regulatory dynamics.

  7. Comparing the Chlorine Disinfection of Detached Biofilm Clusters with Those of Sessile Biofilms and Planktonic Cells in Single- and Dual-Species Cultures ▿ †

    PubMed Central

    Behnke, Sabrina; Parker, Albert E.; Woodall, Dawn; Camper, Anne K.

    2011-01-01

    Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells. Burkholderia cepacia and Pseudomonas aeruginosa were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species B. cepacia, B. cepacia in coculture, and P. aeruginosa in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture. PMID:21856824

  8. Linking Essential Tremor to the Cerebellum: Neuropathological Evidence.

    PubMed

    Louis, Elan D

    2016-06-01

    A fundamental question about essential tremor (ET) is whether its associated pathological changes and disease mechanisms are linkable to a specific brain region. To that end, recent tissue-based studies have made significant strides in elucidating changes in the ET brain. Emerging from these studies is increasing neuropathological evidence linking ET to the cerebellum. These studies have systematically identified a broad range of structural, degenerative changes in the ET cerebellum, spanning across all Purkinje cell compartments. These include the dendritic compartment (where there is an increase in number of Purkinje cell dendritic swellings, a pruning of the dendritic arbor, and a reduction in spine density), the cell body (where, aside from reductions in Purkinje cell linear density in some studies, there is an increase in the number of heterotopic Purkinje cell soma), and the axonal compartment (where a plethora of changes in axonal morphology have been observed, including an increase in the number of thickened axonal profiles, torpedoes, axonal recurrent collaterals, axonal branching, and terminal axonal sprouting). Additional changes, possibly due to secondary remodeling, have been observed in neighboring neuronal populations. These include a hypertrophy of basket cell axonal processes and changes in the distribution of climbing fiber-Purkinje cell synapses. These changes all distinguish ET from normal control brains. Initial studies further indicate that the profile (i.e., constellation) of these changes may separate ET from other diseases of the cerebellum, thereby serving as a disease signature. With the discovery of these changes, a new model of ET has arisen, which posits that it may be a neurodegenerative disorder centered in the cerebellar cortex. These newly emerging neuropathological studies pave the way for anatomically focused, hypothesis-driven, molecular mechanistic studies of disease pathogenesis.

  9. Transcription factor clusters regulate genes in eukaryotic cells

    PubMed Central

    Hedlund, Erik G; Friemann, Rosmarie; Hohmann, Stefan

    2017-01-01

    Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression. PMID:28841133

  10. Control of voluntary and optogenetically perturbed locomotion by spike rate and timing of neurons of the mouse cerebellar nuclei

    PubMed Central

    Sarnaik, Rashmi

    2018-01-01

    Neurons of the cerebellar nuclei (CbN), which generate cerebellar output, are inhibited by Purkinje cells. With extracellular recordings during voluntary locomotion in head-fixed mice, we tested how the rate and coherence of inhibition influence CbN cell firing and well-practiced movements. Firing rates of Purkinje and CbN cells were modulated systematically through the stride cycle (~200–300 ms). Optogenetically stimulating ChR2-expressing Purkinje cells with light steps or trains evoked either asynchronous or synchronous inhibition of CbN cells. Steps slowed CbN firing. Trains suppressed CbN cell firing less effectively, but consistently altered millisecond-scale spike timing. Steps or trains that perturbed stride-related modulation of CbN cell firing rates correlated well with irregularities of movement, suggesting that ongoing locomotion is sensitive to alterations in modulated CbN cell firing. Unperturbed locomotion continued more often during trains than steps, however, suggesting that stride-related modulation of CbN spiking is less readily disrupted by synchronous than asynchronous inhibition. PMID:29659351

  11. Desmoplastic small round cell tumor with sphere-like clusters mimicking adenocarcinoma.

    PubMed

    Hattori, Yukinori; Yoshida, Akihiko; Sasaki, Naoshi; Shibuki, Yasuo; Tamura, Kenji; Tsuta, Koji

    2015-03-01

    Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive neoplasm that predominantly affects young men. DSRCT often presents as multiple nodules on the serosal surface and is histologically categorized as a small round cell tumor. However, the cytological spectrum of DSRCT is not fully understood because of its rarity. Here, we report an unusual case of DSRCT that showed spheres of cells without stromal cores in pleural fluid cytology material, a finding that is typically associated with metastatic adenocarcinoma and mesothelioma. The specimen from a simultaneous needle biopsy showed the classic histology of DSRCT, comprising nests of small round cells set in desmoplasia. The diagnosis of DSRCT was further supported by immunohistochemical coexpression of cytokeratin and desmin, as well as Ewing sarcoma breakpoint region 1 gene rearrangement, which was determined by fluorescence in situ hybridization. The unusual cytological finding in this case illustrates a potential pitfall of the cytological diagnosis of pleural fluid or ascites. DSRCT should not be excluded from the differential diagnosis when sphere-like round cell clusters are observed in pleural or abdominal effusion, particularly in young male patients. © 2014 Wiley Periodicals, Inc.

  12. Creutzfeldt-Jakob disease with severe involvement of cerebral white matter and cerebellum.

    PubMed

    Berciano, J; Berciano, M T; Polo, J M; Figols, J; Ciudad, J; Lafarga, M

    1990-01-01

    We describe a patient with Creutzfeldt-Jakob disease (CJD) of the ataxic and panencephalopathic type. Postmortem examination revealed the characteristic lesions of CJD in the grey matter and profound white matter involvement was seen with immunocytochemical techniques. Ultrastructural white matter lesions were identical to those described in experimentally transmitted CJD. There was marked loss of cerebellar granule cells with virtual disappearance of parallel fibres, but Purkinje cells were only slightly reduced. Electron microscopic studies revealed extensive degenerative changes including cytoplasmic vacuoles in both cell types. Silver methods disclosed massive impregnation of white matter and striking abnormalities of Purkinje cells consisting of hypertrophy and flattening of thick dendritic branches, reduction in the number of terminal branchlets, segmentary loss of spines and polymorphic spines. These findings show the extensive involvement of all three cerebellar cortical layers and the reactive plasticity of Purkinje cells to deafferentiation. They favour the hypothesis that demyelination represents a primary lesion of the white matter.

  13. miR-17-92 Cluster Promotes Cholangiocarcinoma Growth

    PubMed Central

    Zhu, Hanqing; Han, Chang; Lu, Dongdong; Wu, Tong

    2015-01-01

    miR-17-92 is an oncogenic miRNA cluster implicated in the development of several cancers; however, it remains unknown whether the miR-17-92 cluster is able to regulate cholangiocarcinogenesis. This study was designed to investigate the biological functions and molecular mechanisms of the miR-17-92 cluster in cholangiocarcinoma. In situ hybridization and quantitative RT-PCR analysis showed that the miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared with the nonneoplastic biliary epithelial cells. Forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation, and invasiveness, in vitro. Overexpression of the miR-17-92 cluster or miR-92a also enhanced cholangiocarcinoma growth in vivo in hairless outbred mice with severe combined immunodeficiency (SHO-PrkdcscidHrhr). The tumor-suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells via sequence prediction, 3′ untranslated region luciferase activity assay, and Western blot analysis. Accordingly, overexpression of the PTEN open reading frame protein (devoid of 3′ untranslated region) prevented miR-92a– or miR-19a–induced cholangiocarcinoma cell growth. Microarray analysis revealed additional targets of the miR-17-92 cluster in human cholangiocarcinoma cells, including APAF-1 and PRDM2. Moreover, we observed that the expression of the miR-17-92 cluster is regulated by IL-6/Stat3, a key oncogenic signaling pathway pivotal in cholangiocarcinogenesis. Taken together, our findings disclose a novel IL-6/Stat3–miR-17-92 cluster–PTEN signaling axis that is crucial for cholangiocarcinogenesis and tumor progression. PMID:25239565

  14. WordCluster: detecting clusters of DNA words and genomic elements

    PubMed Central

    2011-01-01

    Background Many k-mers (or DNA words) and genomic elements are known to be spatially clustered in the genome. Well established examples are the genes, TFBSs, CpG dinucleotides, microRNA genes and ultra-conserved non-coding regions. Currently, no algorithm exists to find these clusters in a statistically comprehensible way. The detection of clustering often relies on densities and sliding-window approaches or arbitrarily chosen distance thresholds. Results We introduce here an algorithm to detect clusters of DNA words (k-mers), or any other genomic element, based on the distance between consecutive copies and an assigned statistical significance. We implemented the method into a web server connected to a MySQL backend, which also determines the co-localization with gene annotations. We demonstrate the usefulness of this approach by detecting the clusters of CAG/CTG (cytosine contexts that can be methylated in undifferentiated cells), showing that the degree of methylation vary drastically between inside and outside of the clusters. As another example, we used WordCluster to search for statistically significant clusters of olfactory receptor (OR) genes in the human genome. Conclusions WordCluster seems to predict biological meaningful clusters of DNA words (k-mers) and genomic entities. The implementation of the method into a web server is available at http://bioinfo2.ugr.es/wordCluster/wordCluster.php including additional features like the detection of co-localization with gene regions or the annotation enrichment tool for functional analysis of overlapped genes. PMID:21261981

  15. Cluster stability in the analysis of mass cytometry data.

    PubMed

    Melchiotti, Rossella; Gracio, Filipe; Kordasti, Shahram; Todd, Alan K; de Rinaldis, Emanuele

    2017-01-01

    Manual gating has been traditionally applied to cytometry data sets to identify cells based on protein expression. The advent of mass cytometry allows for a higher number of proteins to be simultaneously measured on cells, therefore providing a means to define cell clusters in a high dimensional expression space. This enhancement, whilst opening unprecedented opportunities for single cell-level analyses, makes the incremental replacement of manual gating with automated clustering a compelling need. To this aim many methods have been implemented and their successful applications demonstrated in different settings. However, the reproducibility of automatically generated clusters is proving challenging and an analytical framework to distinguish spurious clusters from more stable entities, and presumably more biologically relevant ones, is still missing. One way to estimate cell clusters' stability is the evaluation of their consistent re-occurrence within- and between-algorithms, a metric that is commonly used to evaluate results from gene expression. Herein we report the usage and importance of cluster stability evaluations, when applied to results generated from three popular clustering algorithms - SPADE, FLOCK and PhenoGraph - run on four different data sets. These algorithms were shown to generate clusters with various degrees of statistical stability, many of them being unstable. By comparing the results of automated clustering with manually gated populations, we illustrate how information on cluster stability can assist towards a more rigorous and informed interpretation of clustering results. We also explore the relationships between statistical stability and other properties such as clusters' compactness and isolation, demonstrating that whilst cluster stability is linked to other properties it cannot be reliably predicted by any of them. Our study proposes the introduction of cluster stability as a necessary checkpoint for cluster interpretation and

  16. Gap-junction coupling and ATP-sensitive potassium channels in human β -cell clusters: Effects on emergent dynamics

    NASA Astrophysics Data System (ADS)

    Loppini, A.; Pedersen, M. G.; Braun, M.; Filippi, S.

    2017-09-01

    The importance of gap-junction coupling between β cells in pancreatic islets is well established in mouse. Such ultrastructural connections synchronize cellular activity, confine biological heterogeneity, and enhance insulin pulsatility. Dysfunction of coupling has been associated with diabetes and altered β -cell function. However, the role of gap junctions between human β cells is still largely unexplored. By using patch-clamp recordings of β cells from human donors, we previously estimated electrical properties of these channels by mathematical modeling of pairs of human β cells. In this work we revise our estimate by modeling triplet configurations and larger heterogeneous clusters. We find that a coupling conductance in the range 0.005 -0.020 nS/pF can reproduce experiments in almost all the simulated arrangements. We finally explore the consequence of gap-junction coupling of this magnitude between β cells with mutant variants of the ATP-sensitive potassium channels involved in some metabolic disorders and diabetic conditions, translating studies performed on rodents to the human case. Our results are finally discussed from the perspective of therapeutic strategies. In summary, modeling of more realistic clusters with more than two β cells slightly lowers our previous estimate of gap-junction conductance and gives rise to patterns that more closely resemble experimental traces.

  17. Characteristics of the Early Immune Response Following Transplantation of Mouse ES Cell Derived Insulin-Producing Cell Clusters

    PubMed Central

    Boyd, Ashleigh S.; Wood, Kathryn J.

    2010-01-01

    Background The fully differentiated progeny of ES cells (ESC) may eventually be used for cell replacement therapy (CRT). However, elements of the innate immune system may contribute to damage or destruction of these tissues when transplanted. Methodology/Principal Findings Herein, we assessed the hitherto ill-defined contribution of the early innate immune response in CRT after transplantation of either ESC derived insulin producing cell clusters (IPCCs) or adult pancreatic islets. Ingress of neutrophil or macrophage cells was noted immediately at the site of IPCC transplantation, but this infiltration was attenuated by day three. Gene profiling identified specific inflammatory cytokines and chemokines that were either absent or sharply reduced by three days after IPCC transplantation. Thus, IPCC transplantation provoked less of an early immune response than pancreatic islet transplantation. Conclusions/Significance Our study offers insights into the characteristics of the immune response of an ESC derived tissue in the incipient stages following transplantation and suggests potential strategies to inhibit cell damage to ensure their long-term perpetuation and functionality in CRT. PMID:20532031

  18. Paired high-content analysis of prostate cancer cells in bone marrow and blood characterizes increased androgen receptor expression in tumor cell clusters

    PubMed Central

    Carlsson, Anders; Kuhn, Peter; Luttgen, Madelyn S.; Dizon, Kevin K.; Troncoso, Patricia; Corn, Paul G.; Kolatkar, Anand; Hicks, James B.; Logothetis, Christopher J.; Zurita., Amado J.

    2017-01-01

    Purpose Recent studies demonstrate that prostate cancer clones from different metastatic sites are dynamically represented in the blood of patients over time, suggesting that the paired evaluation of tumor cells in circulation and bone marrow, the primary target for prostate cancer metastasis, may provide complementary information. Experimental Design We adapted our single-cell high-content liquid biopsy platform to bone marrow aspirates (BMA), to concurrently identify and characterize prostate cancer cells in patients’ blood and bone and thus discern features associated to tumorigenicity and dynamics of metastatic progression. Results The incidence of tumor cells in BMAs increased as the disease advanced: 0% in biochemically-recurrent (n=52), 26% in newly diagnosed metastatic hormone-naïve (n=26), and 39% in metastatic castration-resistant (mCRPC; n=63) patients, and their number was often higher than in paired blood. Tumor cell detection in metastatic patients’ BMAs was concordant but 45% more sensitive than using traditional histopathologic interpretation of core bone marrow biopsies. Tumor cell clusters were more prevalent and bigger in BMAs than in blood, expressed higher levels of the androgen receptor protein per tumor cell and were prognostic in mCRPC. Moreover, the patterns of genomic copy number variation in single tumor cells in paired blood and BMAs showed significant inter and intrapatient heterogeneity. Conclusions Paired analysis of single prostate cancer cells in blood and bone shows promise for clinical application and provides complementary information. The high prevalence and prognostic significance of tumor cell clusters particularly in BMAs, suggest that these structures are key mediators of prostate cancer’s metastatic progression. PMID:27702818

  19. Effects of nicotine exposure during prenatal or perinatal period on cell numbers in adult rat hippocampus and cerebellum: a stereology study.

    PubMed

    Chen, Wei-Jung A; King, Karen A; Lee, Ruby E; Sedtal, Christopher S; Smith, Andrew M

    2006-11-02

    Smoking during pregnancy poses a potential risk to unborn children. The present study examined the long-term effects of early nicotine exposure on the number of pyramidal and granule cells in the hippocampus, and Purkinje cells in the cerebellar vermis. The loss of neurons is the most severe form of brain injury with significant functional implications. In this study, rats were exposed to nicotine during either the prenatal (PRE) period or both the prenatal and early postnatal (PERI) period. It was hypothesized that nicotine treatment would result in long-term decreases in neuronal numbers, and that PERI treatment would be more detrimental to these cell populations than the PRE treatment. The results showed that neither PRE nor PERI nicotine exposure reduces the numbers of pyramidal, granule or Purkinje cells. Neither the regions where these cells reside, nor the cell densities were affected by nicotine. Although no significant cell loss was observed, the current nicotine exposure regimens may lead to alterations in cellular functions or cytoarchitectures. The present results in conjunction with previous reports showing significant cell loss from nicotine exposure during the brain growth spurt suggest that "patch-like" nicotine exposure during prenatal period may alter the sensitivity or the responsiveness of the developing brain to the injurious effects of nicotine during the most vulnerable stage of brain development - the brain growth spurt. Furthermore, the current stereology cell counting results are not in agreement with some reports in the literature, and this discrepancy may simply be a function of different cell counting techniques used.

  20. LDL receptor-related protein mediates cell-surface clustering and hepatic sequestration of chylomicron remnants in LDLR-deficient mice.

    PubMed

    Yu, K C; Chen, W; Cooper, A D

    2001-06-01

    It has been proposed that in the liver, chylomicron remnants (lipoproteins carrying dietary lipid) may be sequestered before being internalized by hepatocytes. To study this, chylomicron remnants labeled with a fluorescent dye were perfused into isolated livers of LDL receptor-deficient (LDLR-deficient) mice (Ldlr(-/-)) and examined by confocal microscopy. In contrast to livers from normal mice, there was clustering of the chylomicron remnants on the cell surface in the space of DISSE: These remnant clusters colocalized with clusters of LDLR-related protein (LRP) and could be eliminated by low concentrations of receptor-associated protein, an inhibitor of LRP. When competed with ligands of heparan sulfate proteoglycans (HSPGs), the remnant clusters still appeared but were fewer in number, although syndecans (membrane HSPGs) colocalized with the remnant clusters. This suggests that the clustering of remnants is not dependent on syndecans but that the syndecans may modify the binding of remnants. These results establish that sequestration is a novel process, the clustering of remnants in the space of DISSE: The clustering involves remnants binding to the LRP, and this may be stabilized by binding with syndecans, eventually followed by endocytosis.

  1. Fragmentation scaling of percolation clusters in two and three dimensions: Large-cell Monte Carlo RG approach

    NASA Astrophysics Data System (ADS)

    Cheon, M.; Chang, I.

    1999-04-01

    The scaling behavior for a binary fragmentation of critical percolation clusters is investigated by a large-cell Monte Carlo real-space renormalization group method in two and three dimensions. We obtain accurate values of critical exponents λ and phi describing the scaling of fragmentation rate and the distribution of fragments' masses produced by a binary fragmentation. Our results for λ and phi show that the fragmentation rate is proportional to the size of mother cluster, and the scaling relation σ = 1 + λ - phi conjectured by Edwards et al. to be valid for all dimensions is satisfied in two and three dimensions, where σ is the crossover exponent of the average cluster number in percolation theory, which excludes the other scaling relations.

  2. Cerebellar contribution to higher and lower order rule learning and cognitive flexibility in mice.

    PubMed

    Dickson, P E; Cairns, J; Goldowitz, D; Mittleman, G

    2017-03-14

    Cognitive flexibility has traditionally been considered a frontal lobe function. However, converging evidence suggests involvement of a larger brain circuit which includes the cerebellum. Reciprocal pathways connecting the cerebellum to the prefrontal cortex provide a biological substrate through which the cerebellum may modulate higher cognitive functions, and it has been observed that cognitive inflexibility and cerebellar pathology co-occur in psychiatric disorders (e.g., autism, schizophrenia, addiction). However, the degree to which the cerebellum contributes to distinct forms of cognitive flexibility and rule learning is unknown. We tested lurcher↔wildtype aggregation chimeras which lose 0-100% of cerebellar Purkinje cells during development on a touchscreen-mediated attentional set-shifting task to assess the contribution of the cerebellum to higher and lower order rule learning and cognitive flexibility. Purkinje cells, the sole output of the cerebellar cortex, ranged from 0 to 108,390 in tested mice. Reversal learning and extradimensional set-shifting were impaired in mice with⩾95% Purkinje cell loss. Cognitive deficits were unrelated to motor deficits in ataxic mice. Acquisition of a simple visual discrimination and an attentional-set were unrelated to Purkinje cells. A positive relationship was observed between Purkinje cells and errors when exemplars from a novel, non-relevant dimension were introduced. Collectively, these data suggest that the cerebellum contributes to higher order cognitive flexibility, lower order cognitive flexibility, and attention to novel stimuli, but not the acquisition of higher and lower order rules. These data indicate that the cerebellar pathology observed in psychiatric disorders may underlie deficits involving cognitive flexibility and attention to novel stimuli. Copyright © 2016. Published by Elsevier Ltd.

  3. Multifunctional fluorescent iron quantum clusters for non-invasive radiofrequency ablationof cancer cells.

    PubMed

    Jose, Akhila; Surendran, Mrudula; Fazal, Sajid; Prasanth, Bindhu-Paul; Menon, Deepthy

    2018-05-01

    This work reports the potential of iron quantum clusters (FeQCs) as a hyperthermia agent for cancer, by testing its in-vitro response to shortwave (MHz range), radiofrequency (RF) waves non-invasively. Stable, fluorescent FeQCs of size ∼1 nm prepared by facile aqueous chemistry from endogenous protein haemoglobin were found to give a high thermal response, with a ΔT ∼50 °C at concentrationsas low as165 μg/mL. The as-prepared nanoclusters purified by lyophilization as well as dialysis showed a concentration, power and time-dependent RF response, with the lyophilized FeQCs exhibiting pronounced heating effects. FeQCs were found to be cytocompatible to NIH-3T3 fibroblast and 4T1 cancer cells treated at concentrations upto 1000 μg/mL for 24 h. Upon incubation with FeQCs and exposure to RF waves, significant cancer cell death was observed which proves its therapeutic ability. The fluorescent ability of the clusters could additionally be utilized for imaging cancer cells upon excitation at ∼450 nm. Further, to demonstrate the feasibility of imparting additional functionality such as drug/biomolecule/dye loading to FeQCs, they were self assembled with cationic polymers to form nanoparticles. Self assembly did not alter the RF heating potential of FeQCs and additionally enhanced its fluorescence. The multifunctional fluorescent FeQCs therefore show good promise as a novel therapeutic agent for RF hyperthermia and drug loading. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Overexpression of NGF ameliorates ethanol neurotoxicity in the developing cerebellum.

    PubMed

    Heaton, M B; Mitchell, J J; Paiva, M

    2000-11-05

    Transgenic mice overexpressing NGF in the central nervous system under the control of the glial fibrillary acidic protein (GFAP) promoter were exposed to ethanol via vapor inhalation on postnatal days 4 and 5 (P4-5), the period of maximal cerebellar Purkinje cell sensitivity to ethanol. Wild-type controls were exposed in a similar manner. There were no differences in body weight or size following these procedures, but the transgenic brain weights at this age were significantly greater than wild-type controls. In the wild-type animals, a significant 33.3% ethanol-mediated loss of Purkinje cells in lobule I was detected via unbiased three-dimensional stereological counting on P5. In the GFAP-NGF transgenic animals, however, the 17.6% difference in Purkinje cell number in control and ethanol-exposed animals was not significant. There was a similar difference in Purkinje cell density in both groups, which did reach statistical significance (-32.7% in wild-type ethanol-treated animals, -17% in transgenic ethanol-exposed animals). These results suggest that endogenous overexpression of neurotrophic factors, which have previously been shown to protect against ethanol neurotoxicity in culture, can serve a similar protective function in the intact animal. Copyright 2000 John Wiley & Sons, Inc.

  5. Organotin Dyes Bearing Anionic Boron Clusters as Cell-Staining Fluorescent Probes.

    PubMed

    Muñoz-Flores, Blanca M; Cabrera-González, Justo; Viñas, Clara; Chávez-Reyes, Arturo; Dias, H V Rasika; Jiménez-Pérez, Víctor M; Núñez, Rosario

    2018-04-11

    Within the cell nucleus, in the nucleoli, ribosomal RNAs are synthesized and participate in several biological processes. To better understand nucleoli-related processes, their visualization is often required, for which specific markers are needed. Herein, we report the design of novel fluorescent organotin compounds derived from 4-hydroxy-N'-((2-hydroxynaphthalen-1-yl)methylene)benzohydrazide and their cytoplasm and nucleoli staining of B16F10 cells in vitro. Tin compounds bearing an aliphatic carbon chain (-C 12 H 25 ) and an electron-donating group (-OH) were prepared, and the latter could be derivatized to bear the boron cluster anions [B 12 H 12 ] 2- and [3,3'-Co(1,2-C 2 B 9 H 11 ) 2 ] - (COSAN). All of the conjugates have been fully characterized and their luminescence properties have been assessed. In general, they show good quantum yields in solution (24-49 %), those for the COSAN derivatives being lower. Remarkably, the linking of [B 12 H 12 ] 2- and COSAN to the complexes made them more soluble, without being detrimental to their luminescence properties. Living B16F10 cells were treated with all of the compounds to determine their fluorescence staining properties; the compounds bearing the aliphatic chain showed a reduced staining capacity due to the formation of aggregates. Notably, the complexes bearing different boron clusters showed different staining effects; those bearing [B 12 H 12 ] 2- showed extraordinary staining of the nucleoli and cytoplasm, whereas those bearing COSAN were only detected in the cytoplasm. The remarkable fluorescence staining properties shown by these organotin compounds make them excellent candidates for fluorescence bioimaging in vitro. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines.

    PubMed

    Isbrucker, R; Daas, A; Wagner, L; Costanzo, A

    2016-01-01

    Current regulations for acellular pertussis (aP) vaccines require that they are tested for the presence of residual or reversion-derived pertussis toxin (PTx) activity using the mouse histamine sensitisation test (HIST). Although a CHO cell clustering assay can be used by manufacturers to verify if sufficient inactivation of the substance has occurred in-process, this assay cannot be used at present for the final product due to the presence of aluminium adjuvants which interfere with mammalian cell cultures. Recently, 2 modified CHO cell clustering assays which accommodate for the adjuvant effects have been proposed as alternatives to the HIST. These modified assays eliminate the adjuvant-induced cytotoxicity either through dilution of the vaccine (called the Direct Method) or by introducing a porous barrier between the adjuvant and the cells (the Indirect Method). Transferability and suitability of these methods for testing of products present on the European market were investigated during a collaborative study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). Thirteen laboratories participated in this study which included 4 aP-containing vaccines spiked by addition of PTx. This study also assessed the transferability of a standardised CHO cell clustering assay protocol for use with non-adjuvanted PTx preparations. Results showed that the majority of laboratories were able to detect the PTx spike in all 4 vaccines at concentrations of 4 IU/mL or lower using the Indirect Method. This sensitivity is in the range of the theoretical sensitivity of the HIST. The Direct Method however did not show the expected results and would need additional development work.

  7. Cerebellar abiotrophy in a family of Border Collie dogs.

    PubMed

    Sandy, J R; Slocombe, R E; Mitten, R W; Jedwab, D

    2002-11-01

    Cerebellar abiotrophies have a nonsex-linked, autosomal, recessively inherited basis in a number of species, and lesions typically reflect profound and progressive loss of Purkinje cells. In this report, an unusual form of abiotrophy is described for two sibling Border Collies. Extensive loss of the cerebellar granular cell layer was present with relative sparing of Purkinje cells of two female pups. The biochemical basis for this form of cerebellar abiotrophy is unknown, but the lack of disease in other siblings supports an autosomal recessive mode of inheritance.

  8. The Marr and Albus Theories of the Cerebellum: Two Eary Models of Associative Memory

    NASA Technical Reports Server (NTRS)

    Albus, James S.

    1989-01-01

    The Marr and Albus theories of the cerebellum are compared and contrasted. They are shown to be similar in their analysis of the function of the mossy fibers, granule cells, Golgi cells, and Purkinje cells. They both predict motor learning in the parallel fiber synapses on the Purkinje dendrites mediated by concurrent climbing fiber input. This prediction has been confirmed by experimental evidence. In contrast, Marr predicts these synapses would be facilitated by learning, while Albus predicts they would be weakened. Experimental evidence confirms synaptic weakening.

  9. Measuring Feedforward Inhibition and Its Impact on Local Circuit Function.

    PubMed

    Hull, Court

    2017-05-01

    This protocol describes a series of approaches to measure feedforward inhibition in acute brain slices from the cerebellar cortex. Using whole-cell voltage and current clamp recordings from Purkinje cells in conjunction with electrical stimulation of the parallel fibers, these methods demonstrate how to measure the relationship between excitation and inhibition in a feedforward circuit. This protocol also describes how to measure the impact of feedforward inhibition on Purkinje cell excitability, with an emphasis on spike timing. © 2017 Cold Spring Harbor Laboratory Press.

  10. Spiking neural networks on high performance computer clusters

    NASA Astrophysics Data System (ADS)

    Chen, Chong; Taha, Tarek M.

    2011-09-01

    In this paper we examine the acceleration of two spiking neural network models on three clusters of multicore processors representing three categories of processors: x86, STI Cell, and NVIDIA GPGPUs. The x86 cluster utilized consists of 352 dualcore AMD Opterons, the Cell cluster consists of 320 Sony Playstation 3s, while the GPGPU cluster contains 32 NVIDIA Tesla S1070 systems. The results indicate that the GPGPU platform can dominate in performance compared to the Cell and x86 platforms examined. From a cost perspective, the GPGPU is more expensive in terms of neuron/s throughput. If the cost of GPGPUs go down in the future, this platform will become very cost effective for these models.

  11. Expression of oncogenic miR-17-92 and tumor suppressive miR-143-145 clusters in basal cell carcinoma and cutaneous squamous cell carcinoma.

    PubMed

    Sand, Michael; Hessam, Schapoor; Amur, Susanne; Skrygan, Marina; Bromba, Michael; Stockfleth, Eggert; Gambichler, Thilo; Bechara, Falk G

    2017-05-01

    A variety of cancers are associated with the expression of the oncogenic miR-17-92 cluster (Oncomir-1) and tumor suppressor miR-143-5p/miR-145-5p. Epidermal skin cancer has not been investigated for the expression of miR-17-92 and miR-143-145 clusters, despite being extensively studied regarding global microRNA profiles. The goal of this study was to investigate the expression and possible correlation of expression of miR17-92 and miR-143-145 cluster members in epidermal skin cancer. We evaluated punch biopsies from patients with cutaneous squamous cell carcinoma (cSCC, n=15) and basal cell carcinoma (BCC, n=16), along with control specimens from non-lesional epidermal skin (n=16). Expression levels of the miR17-92 cluster (including miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-3p, miR-19b-1-5p, miR-20a-3p, miR-20a-5p, miR-92a-3p, and miR-92a-5p) and the tumor-suppressive cluster miR-143-145 (including miR-143-5p and miR-145-5p) were detected by quantitative real-time reverse transcriptase polymerase chain reaction. We noted a highly significant increased expression of the miR-17-92 members miR-17-5p, miR-18a-5p, miR19a-3p, and miR-19b-3p and tumor suppressor miR-143-5p (p<0.01) in cSCC. miR-145-5p had a significantly decreased expression (p<0.05) for in BCC. A correlation analysis revealed multiple correlating miRNA-pairs within and between the investigated clusters. This study marks the first evidence for the participation of members of the miR-17-92 cluster in cSCC and miR-143-145 cluster in BCC. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  12. MiR-17-92 cluster and immunity.

    PubMed

    Kuo, George; Wu, Chao-Yi; Yang, Huang-Yu

    2018-05-29

    MicroRNAs (MiR, MiRNA) are small single-stranded non-coding RNAs that play an important role in the regulation of gene expression. MircoRNAs exert their effect by binding to complementary nucleotide sequences of the targeted messenger RNA, thus forming an RNA-induced silencing complex. The mircoRNA-17-92 cluster encoded by the miR-17-92 host gene is first found in malignant B-cell lymphoma. Recent research identifies the miR-17-92 cluster as a crucial player in the development of the immune system, the heart, the lung, and oncogenic events. In light of the miR-17-92 cluster's increasing role in regulating the immune system, our review will discuss the latest knowledge regarding its involvement in cells of both innate and adaptive immunity, including B cells, subsets of T cells such as Th1, Th2, T follicular helper cells, regulatory T cells, monocytes/macrophages, NK cells, and dendritic cells, and the possible targets that are regulated by its members. Copyright © 2018. Published by Elsevier B.V.

  13. Density-based clustering analyses to identify heterogeneous cellular sub-populations

    NASA Astrophysics Data System (ADS)

    Heaster, Tiffany M.; Walsh, Alex J.; Landman, Bennett A.; Skala, Melissa C.

    2017-02-01

    Autofluorescence microscopy of NAD(P)H and FAD provides functional metabolic measurements at the single-cell level. Here, density-based clustering algorithms were applied to metabolic autofluorescence measurements to identify cell-level heterogeneity in tumor cell cultures. The performance of the density-based clustering algorithm, DENCLUE, was tested in samples with known heterogeneity (co-cultures of breast carcinoma lines). DENCLUE was found to better represent the distribution of cell clusters compared to Gaussian mixture modeling. Overall, DENCLUE is a promising approach to quantify cell-level heterogeneity, and could be used to understand single cell population dynamics in cancer progression and treatment.

  14. Stimulation of wound healing by positively charged dextran beads depends upon clustering of beads and cells in close proximity to the wound.

    PubMed

    Tawil, N J; Connors, D; Gies, D; Bennett, S; Gruskin, E; Mustoe, T

    1999-01-01

    We have previously shown that positively charged dextran (DEAE A25) increases wound breaking strength in linear incisions in rats and nonhuman primates at days 10-14 postwounding. In this article, we examined the cellular responses to different types of charged dextran beads (DEAE A50 and Cytodex-1) in culture studies and in rat incisional wounds. We show that Cytodex 1 and DEAE A50 beads also increased wound breaking strength in a rat linear incisional model. However, the increase was approximately 30-40% less than that observed in wounds treated with DEAE A25 beads. The main distinction between the three types of beads was the presence of bead clusters observed in tissue sections. Wounds treated with DEAE A25 beads formed distinct clusters while both Cytodex 1 and DEAE A50 beads clustered to a lesser extent or failed to cluster at all. We propose that the different types of charged dextran beads improve healing by promoting cell adhesion and encouraging proliferation in close proximity to the wound. We also hypothesize that the 30-40% improvement in wound breaking strength seen with DEAE A25 beads compared to other types of charged dextran beads (DEAE A50 and Cytodex-1) originates from the unique characteristic of DEAE A25 beads in forming cell-bead aggregates adjacent to the wounded area. This clustering, in turn, affects the distribution of cells infiltrating the wounded area (such as macrophages) during the healing process and, as a consequence, alters the distribution of matrix molecules and growth factors secreted by these cells.

  15. The cerebellum for jocks and nerds alike.

    PubMed

    Popa, Laurentiu S; Hewitt, Angela L; Ebner, Timothy J

    2014-01-01

    Historically the cerebellum has been implicated in the control of movement. However, the cerebellum's role in non-motor functions, including cognitive and emotional processes, has also received increasing attention. Starting from the premise that the uniform architecture of the cerebellum underlies a common mode of information processing, this review examines recent electrophysiological findings on the motor signals encoded in the cerebellar cortex and then relates these signals to observations in the non-motor domain. Simple spike firing of individual Purkinje cells encodes performance errors, both predicting upcoming errors as well as providing feedback about those errors. Further, this dual temporal encoding of prediction and feedback involves a change in the sign of the simple spike modulation. Therefore, Purkinje cell simple spike firing both predicts and responds to feedback about a specific parameter, consistent with computing sensory prediction errors in which the predictions about the consequences of a motor command are compared with the feedback resulting from the motor command execution. These new findings are in contrast with the historical view that complex spikes encode errors. Evaluation of the kinematic coding in the simple spike discharge shows the same dual temporal encoding, suggesting this is a common mode of signal processing in the cerebellar cortex. Decoding analyses show the considerable accuracy of the predictions provided by Purkinje cells across a range of times. Further, individual Purkinje cells encode linearly and independently a multitude of signals, both kinematic and performance errors. Therefore, the cerebellar cortex's capacity to make associations across different sensory, motor and non-motor signals is large. The results from studying how Purkinje cells encode movement signals suggest that the cerebellar cortex circuitry can support associative learning, sequencing, working memory, and forward internal models in non

  16. The cerebellum for jocks and nerds alike

    PubMed Central

    Popa, Laurentiu S.; Hewitt, Angela L.; Ebner, Timothy J.

    2014-01-01

    Historically the cerebellum has been implicated in the control of movement. However, the cerebellum's role in non-motor functions, including cognitive and emotional processes, has also received increasing attention. Starting from the premise that the uniform architecture of the cerebellum underlies a common mode of information processing, this review examines recent electrophysiological findings on the motor signals encoded in the cerebellar cortex and then relates these signals to observations in the non-motor domain. Simple spike firing of individual Purkinje cells encodes performance errors, both predicting upcoming errors as well as providing feedback about those errors. Further, this dual temporal encoding of prediction and feedback involves a change in the sign of the simple spike modulation. Therefore, Purkinje cell simple spike firing both predicts and responds to feedback about a specific parameter, consistent with computing sensory prediction errors in which the predictions about the consequences of a motor command are compared with the feedback resulting from the motor command execution. These new findings are in contrast with the historical view that complex spikes encode errors. Evaluation of the kinematic coding in the simple spike discharge shows the same dual temporal encoding, suggesting this is a common mode of signal processing in the cerebellar cortex. Decoding analyses show the considerable accuracy of the predictions provided by Purkinje cells across a range of times. Further, individual Purkinje cells encode linearly and independently a multitude of signals, both kinematic and performance errors. Therefore, the cerebellar cortex's capacity to make associations across different sensory, motor and non-motor signals is large. The results from studying how Purkinje cells encode movement signals suggest that the cerebellar cortex circuitry can support associative learning, sequencing, working memory, and forward internal models in non

  17. Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

    PubMed Central

    Maio, Nunziata; Rouault, Tracey. A.

    2014-01-01

    Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

  18. The Changeable Nervous System: Studies On Neuroplasticity In Cerebellar Cultures

    PubMed Central

    Seil, Fredrick J.

    2014-01-01

    Circuit reorganization after injury was studied in a cerebellar culture model. When cerebellar cultures derived from newborn mice were exposed at explantation to a preparation of cytosine arabinoside that destroyed granule cells and oligodendrocytes and compromised astrocytes, Purkinje cells surviving in greater than usual numbers were unensheathed by astrocytic processes and received twice the control number of inhibitory axosomatic synapses. Purkinje cell axon collaterals sprouted and many of their terminals formed heterotypical synapses with other Purkinje cell dendritic spines. The resulting circuit reorganization preserved inhibition in the cerebellar cortex. Following this reorganization, replacement of the missing granule cells and glia was followed by a restitution of the normal circuitry. Most of these developmental and reconstructive changes were not dependent on neuronal activity, the major exception being inhibitory synaptogenesis. The full complement of inhibitory synapses did not develop in the absence of neuronal activity, which could be mitigated by application of exogenous TrkB receptor ligands. Inhibitory synaptogenesis could also be promoted by activity-induced release of endogenous TrkB receptor ligands or by antibody activation of the TrkB receptor. PMID:24933693

  19. It's Lonely at the Top: Winning Climbing Fibers Ascend Dendrites Solo

    PubMed Central

    Draft, Ryan W.; Lichtman, Jeff W.

    2009-01-01

    In mammals, climbing fiber axons compete for sole innervation at each Purkinje cell. At the same time, synapses disappear from Purkinje somata and appear in great numbers on the dendrites. In this issue of Neuron, Hashimoto et al. show that, by the time climbing fibers ascend the dendrites, the winner and losers are already decided. PMID:19607787

  20. Integrative analysis of signaling pathways and diseases associated with the miR-106b/25 cluster and their function study in berberine-induced multiple myeloma cells.

    PubMed

    Gu, Chunming; Li, Tianfu; Yin, Zhao; Chen, Shengting; Fei, Jia; Shen, Jianping; Zhang, Yuan

    2017-05-01

    Berberine (BBR), a traditional Chinese herbal medicine compound, has emerged as a novel class of anti-tumor agent. Our previous microRNA (miRNA) microarray demonstrated that miR-106b/25 was significantly down-regulated in BBR-treated multiple myeloma (MM) cells. Here, systematic integration showed that miR-106b/25 cluster is involved in multiple cancer-related signaling pathways and tumorigenesis. MiREnvironment database revealed that multiple environmental factors (drug, ionizing radiation, hypoxia) affected the miR-106b/25 cluster expression. By targeting the seed region in the miRNA, tiny anti-mir106b/25 cluster (t-anti-mir106b/25 cluster) significantly induced suppression in cell viability and colony formation. Western blot validated that t-anti-miR-106b/25 cluster effectively inhibited the expression of P38 MAPK and phospho-P38 MAPK in MM cells. These findings indicated the miR-106b/25 cluster functioned as oncogene and might provide a novel molecular insight into MM.

  1. Mutant ataxin1 disrupts cerebellar development in spinocerebellar ataxia type 1.

    PubMed

    Edamakanti, Chandrakanth Reddy; Do, Jeehaeh; Didonna, Alessandro; Martina, Marco; Opal, Puneet

    2018-06-01

    Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the protein ATXN1, which is involved in transcriptional regulation. Although symptoms appear relatively late in life, primarily from cerebellar dysfunction, pathogenesis begins early, with transcriptional changes detectable as early as a week after birth in SCA1-knockin mice. Given the importance of this postnatal period for cerebellar development, we asked whether this region might be developmentally altered by mutant ATXN1. We found that expanded ATXN1 stimulates the proliferation of postnatal cerebellar stem cells in SCA1 mice. These hyperproliferating stem cells tended to differentiate into GABAergic inhibitory interneurons rather than astrocytes; this significantly increased the GABAergic inhibitory interneuron synaptic connections, disrupting cerebellar Purkinje cell function in a non-cell autonomous manner. We confirmed the increased basket cell-Purkinje cell connectivity in human SCA1 patients. Mutant ATXN1 thus alters the neural circuitry of the developing cerebellum, setting the stage for the later vulnerability of Purkinje cells to SCA1. We propose that other late-onset degenerative diseases may also be rooted in subtle developmental derailments.

  2. Zincergic innervation from the anterior olfactory nucleus to the olfactory bulb displays plastic responses after mitral cell loss.

    PubMed

    Airado, Carmen; Gómez, Carmela; Recio, Javier S; Baltanás, Fernando C; Weruaga, Eduardo; Alonso, José R

    2008-12-01

    Zinc ions are selectively accumulated in certain neurons (zinc-enriched neurons). The mouse olfactory bulb is richly innervated by zinc-enriched terminals. Here, the plasticity of the zincergic system was studied in the olfactory bulb of the Purkinje Cell Degeneration mutant mouse, an animal with specific postnatal neurodegeneration of the main projection neurons of the olfactory bulb. The analysis focused particularly on the anterior olfactory nucleus since most centrifugal afferents coming to the olfactory bulb arise from this structure. Zinc-enriched terminals in the olfactory bulb and zinc-enriched somata in the anterior olfactory nucleus were visualized after selenite injections. Immunohistochemistry against the vesicular zinc transporter was also carried out to confirm the distribution pattern of zinc-enriched terminals in the olfactory bulb. The mutant mice showed a clear reorganization of zincergic centrifugal projections from the anterior olfactory nucleus to the olfactory bulb. First, all zincergic contralateral neurons projecting to the olfactory bulb were absent in the mutant mice. Second, a significant increase in the number of stained somata was detected in the ipsilateral anterior olfactory nucleus. Since no noticeable changes were observed in the zinc-enriched terminals in the olfactory bulb, it is conceivable that mitral cell loss could induce a reorganization of zinc-enriched projections coming from the anterior olfactory nucleus, probably directed at balancing the global zincergic centrifugal modulation. These results show that zincergic anterior olfactory nucleus cells projecting to the olfactory bulb undergo plastic changes to adapt to the loss of mitral cells in the olfactory bulb of Purkinje Cell Degeneration mutant mice.

  3. Physiological oxygen prevents frequent silencing of the DLK1-DIO3 cluster during human embryonic stem cells culture.

    PubMed

    Xie, Pingyuan; Sun, Yi; Ouyang, Qi; Hu, Liang; Tan, Yueqiu; Zhou, Xiaoying; Xiong, Bo; Zhang, Qianjun; Yuan, Ding; Pan, Yi; Liu, Tiancheng; Liang, Ping; Lu, Guangxiu; Lin, Ge

    2014-02-01

    Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. © AlphaMed Press.

  4. Cluster-cluster clustering

    NASA Technical Reports Server (NTRS)

    Barnes, J.; Dekel, A.; Efstathiou, G.; Frenk, C. S.

    1985-01-01

    The cluster correlation function xi sub c(r) is compared with the particle correlation function, xi(r) in cosmological N-body simulations with a wide range of initial conditions. The experiments include scale-free initial conditions, pancake models with a coherence length in the initial density field, and hybrid models. Three N-body techniques and two cluster-finding algorithms are used. In scale-free models with white noise initial conditions, xi sub c and xi are essentially identical. In scale-free models with more power on large scales, it is found that the amplitude of xi sub c increases with cluster richness; in this case the clusters give a biased estimate of the particle correlations. In the pancake and hybrid models (with n = 0 or 1), xi sub c is steeper than xi, but the cluster correlation length exceeds that of the points by less than a factor of 2, independent of cluster richness. Thus the high amplitude of xi sub c found in studies of rich clusters of galaxies is inconsistent with white noise and pancake models and may indicate a primordial fluctuation spectrum with substantial power on large scales.

  5. Pili-taxis: Clustering of Neisseria gonorrhoeae bacteria

    NASA Astrophysics Data System (ADS)

    Taktikos, Johannes; Zaburdaev, Vasily; Biais, Nicolas; Stark, Holger; Weitz, David A.

    2012-02-01

    The first step of colonization of Neisseria gonorrhoeae bacteria, the etiological agent of gonorrhea, is the attachment to human epithelial cells. The attachment of N. gonorrhoeae bacteria to surfaces or other cells is primarily mediated by filamentous appendages, called type IV pili (Tfp). Cycles of elongation and retraction of Tfp are responsible for a common bacterial motility called twitching motility which allows the bacteria to crawl over surfaces. Experimentally, N. gonorrhoeae cells initially dispersed over a surface agglomerate into round microcolonies within hours. It is so far not known whether this clustering is driven entirely by the Tfp dynamics or if chemotactic interactions are needed. Thus, we investigate whether the agglomeration may stem solely from the pili-mediated attraction between cells. By developing a statistical model for pili-taxis, we try to explain the experimental measurements of the time evolution of the mean cluster size, number of clusters, and area fraction covered by the cells.

  6. Early Disruption of Extracellular Pleiotrophin Distribution Alters Cerebellar Neuronal Circuit Development and Function.

    PubMed

    Hamza, M M; Rey, S A; Hilber, P; Arabo, A; Collin, T; Vaudry, D; Burel, D

    2016-10-01

    The cerebellum is a structure of the central nervous system involved in balance, motor coordination, and voluntary movements. The elementary circuit implicated in the control of locomotion involves Purkinje cells, which receive excitatory inputs from parallel and climbing fibers, and are regulated by cerebellar interneurons. In mice as in human, the cerebellar cortex completes its development mainly after birth with the migration, differentiation, and synaptogenesis of granule cells. These cellular events are under the control of numerous extracellular matrix molecules including pleiotrophin (PTN). This cytokine has been shown to regulate the morphogenesis of Purkinje cells ex vivo and in vivo via its receptor PTPζ. Since Purkinje cells are the unique output of the cerebellar cortex, we explored the consequences of their PTN-induced atrophy on the function of the cerebellar neuronal circuit in mice. Behavioral experiments revealed that, despite a normal overall development, PTN-treated mice present a delay in the maturation of their flexion reflex. Moreover, patch clamp recording of Purkinje cells revealed a significant increase in the frequency of spontaneous excitatory postsynaptic currents in PTN-treated mice, associated with a decrease of climbing fiber innervations and an abnormal perisomatic localization of the parallel fiber contacts. At adulthood, PTN-treated mice exhibit coordination impairment on the rotarod test associated with an alteration of the synchronization gait. Altogether these histological, electrophysiological, and behavior data reveal that an early ECM disruption of PTN composition induces short- and long-term defaults in the establishment of proper functional cerebellar circuit.

  7. LTD, RP, and Motor Learning.

    PubMed

    Hirano, Tomoo; Yamazaki, Yoshito; Nakamura, Yoji

    2016-02-01

    Long-term depression (LTD) at excitatory synapses between parallel fibers and a Purkinje cell has been regarded as a critical cellular mechanism for motor learning. However, it was demonstrated that normal motor learning occurs under LTD suppression, suggesting that cerebellar plasticity mechanisms other than LTD also contribute to motor learning. One candidate for such plasticity is rebound potentiation (RP), which is long-term potentiation at inhibitory synapses between a stellate cell and a Purkinje cell. Both LTD and RP are induced by the increase in postsynaptic Ca(2+) concentration, and work to suppress the activity of a Purkinje cell. Thus, LTD and RP might work synergistically, and one might compensate defects of the other. RP induction is dependent on the interaction between GABAA receptor and GABAA receptor binding protein (GABARAP). Transgenic mice expressing a peptide which inhibits binding of GABARAP and GABAA receptor only in Purkinje cells show defects in both RP and adaptation of vestibulo-ocular reflex (VOR), a motor learning paradigm. However, another example of motor learning, adaptation of optokinetic response (OKR), is normal in the transgenic mice. Both VOR and OKR are reflex eye movements suppressing the slip of visual image on the retina during head movement. Previously, we reported that delphilin knockout mice show facilitated LTD induction and enhanced OKR adaptation, but we recently found that VOR adaptation was not enhanced in the knockout mice. These results together suggest that animals might use LTD and RP differently depending on motor learning tasks.

  8. A spatial hazard model for cluster detection on continuous indicators of disease: application to somatic cell score.

    PubMed

    Gay, Emilie; Senoussi, Rachid; Barnouin, Jacques

    2007-01-01

    Methods for spatial cluster detection dealing with diseases quantified by continuous variables are few, whereas several diseases are better approached by continuous indicators. For example, subclinical mastitis of the dairy cow is evaluated using a continuous marker of udder inflammation, the somatic cell score (SCS). Consequently, this study proposed to analyze spatialized risk and cluster components of herd SCS through a new method based on a spatial hazard model. The dataset included annual SCS for 34 142 French dairy herds for the year 2000, and important SCS risk factors: mean parity, percentage of winter and spring calvings, and herd size. The model allowed the simultaneous estimation of the effects of known risk factors and of potential spatial clusters on SCS, and the mapping of the estimated clusters and their range. Mean parity and winter and spring calvings were significantly associated with subclinical mastitis risk. The model with the presence of 3 clusters was highly significant, and the 3 clusters were attractive, i.e. closeness to cluster center increased the occurrence of high SCS. The three localizations were the following: close to the city of Troyes in the northeast of France; around the city of Limoges in the center-west; and in the southwest close to the city of Tarbes. The semi-parametric method based on spatial hazard modeling applies to continuous variables, and takes account of both risk factors and potential heterogeneity of the background population. This tool allows a quantitative detection but assumes a spatially specified form for clusters.

  9. Quantifying the pattern of microbial cell dispersion, density and clustering on surfaces of differing chemistries and topographies using multifractal analysis.

    PubMed

    Wickens, David; Lynch, Stephen; West, Glen; Kelly, Peter; Verran, Joanna; Whitehead, Kathryn A

    2014-09-01

    The effects of surface topography on bacterial distribution across a surface are of extreme importance when designing novel, hygienic or antimicrobial surface coatings. The majority of methods that are deployed to describe the pattern of cell dispersion, density and clustering across surfaces are currently qualitative. This paper presents a novel application of multifractal analysis to quantitatively measure these factors using medically relevant microorganisms (Staphylococcus aureus or Staphylococcus epidermidis). Surfaces (medical grade 316 stainless steel) and coatings (Ti-ZrN, Ti-ZrN/6.0%Ag, Ti-ZrN/15.6%Ag, TiZrN/24.7%Ag) were used in microbiological retention assays. Results demonstrated that S. aureus displayed a more heterogeneous cell dispersion (∆αAS<1) whilst the dispersion of S. epidermidis was more symmetric and homogeneous (∆αAS≥1). Further, although the surface topography and chemistry had an effect on cell dispersion, density and clustering, the type of bonding that occurred at the surface interface was also important. Both types of cells were influenced by both surface topographical and chemical effects; however, S. aureus was influenced marginally more by surface chemistry whilst S. epidermidis cells was influenced marginally more by surface topography. Thus, this effect was bacterially species specific. The results demonstrate that multifractal analysis is a method that can be used to quantitatively analyse the cell dispersion, density and clustering of retained microorganisms on surfaces. Using quantitative descriptors has the potential to aid the understanding the effect of surface properties on the production of hygienic and antimicrobial coatings. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

    PubMed Central

    Schaufler, Viktoria; Czichos-Medda, Helmi; Hirschfeld-Warnecken, Vera; Neubauer, Stefanie; Rechenmacher, Florian; Medda, Rebecca; Kessler, Horst; Geiger, Benjamin; Spatz, Joachim P.; Cavalcanti-Adam, E. Ada

    2016-01-01

    ABSTRACT Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions. PMID:27003228

  11. Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

    DOE PAGES

    Gao, Zhiwen; Gao, Yanfei

    2016-05-14

    We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

  12. Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gao, Zhiwen; Gao, Yanfei

    We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

  13. Helicobacter pylori with the Intact dupA Cluster is more Virulent than the Strains with the Incomplete dupA Cluster.

    PubMed

    Wang, Ming-yi; Shao, Chen; Li, Jie; Yang, Ya-Chao; Wang, Shao-bo; Hao, Jun-ling; Wu, Chun-mei; Gao, Xiao-zhong; Shao, Shi-he

    2015-07-01

    The duodenal ulcer promoting gene (dupA), located in the plasticity region of Helicobacter pylori (H. pylori), is predicted to form a type IV secretory system (T4SS) with vir genes around dupA. In the study, we investigated the association between the dupA cluster status and the virulence of H. pylori in a littoral region of Northeast China. Two hundred and sixty-two H. pylori strains isolated from the chronic gastritis were examined to evaluate the dupA cluster status, cag PAI genes and vacA genotype using PCR and Western blot. Histopathologic evaluations of biopsy specimens were performed to analysis the association between the dupA cluster and the inflammatory response. IL-8 productions in gastric mucosa and from GES-1 cells co-cultured with H. pylori were measured, respectively, to analysis the association between the dupA cluster status and IL-8 production. We found that gastric mucosal inflammatory cell infiltration was significantly higher in patients with dupA-positive H. pylori, including H. pylori with complete dupA cluster (2.71 ± 0.79) and incomplete dupA cluster (2.09 ± 0.61) than in patients with dupA-negative strain (1.73 ± 0.60, p < 0.01), whereas no significant difference in the gastric mucosal atrophy was found according to the status of dupA cluster. Gastric mucosal IL-8 levels were higher in the complete dupA cluster group than in other groups (p < 0.01), and IL-8 production from GES-1 cells was also significantly higher in strains with a complete dupA cluster (1527.9 ± 180.0 pg/ml) than in those with an incomplete dupA cluster (1229.4 ± 75.3 pg/ml, p < 0.01) or those with dupA negative (1201.9 ± 92.3 pg/ml, p < 0.01). In conclusion, the complete dupA cluster in H. pylori is associated with inflammatory cell infiltration and IL-8 secretion, and H. pylori strain with a complete dupA cluster seems to be more virulent than other strains with the incomplete dupA cluster or dupA negative.

  14. Model-based branching point detection in single-cell data by K-branches clustering

    PubMed Central

    Chlis, Nikolaos K.; Wolf, F. Alexander; Theis, Fabian J.

    2017-01-01

    Abstract Motivation The identification of heterogeneities in cell populations by utilizing single-cell technologies such as single-cell RNA-Seq, enables inference of cellular development and lineage trees. Several methods have been proposed for such inference from high-dimensional single-cell data. They typically assign each cell to a branch in a differentiation trajectory. However, they commonly assume specific geometries such as tree-like developmental hierarchies and lack statistically sound methods to decide on the number of branching events. Results We present K-Branches, a solution to the above problem by locally fitting half-lines to single-cell data, introducing a clustering algorithm similar to K-Means. These halflines are proxies for branches in the differentiation trajectory of cells. We propose a modified version of the GAP statistic for model selection, in order to decide on the number of lines that best describe the data locally. In this manner, we identify the location and number of subgroups of cells that are associated with branching events and full differentiation, respectively. We evaluate the performance of our method on single-cell RNA-Seq data describing the differentiation of myeloid progenitors during hematopoiesis, single-cell qPCR data of mouse blastocyst development, single-cell qPCR data of human myeloid monocytic leukemia and artificial data. Availability and implementation An R implementation of K-Branches is freely available at https://github.com/theislab/kbranches. Contact fabian.theis@helmholtz-muenchen.de Supplementary information Supplementary data are available at Bioinformatics online. PMID:28582478

  15. Self-organization and positioning of bacterial protein clusters

    NASA Astrophysics Data System (ADS)

    Murray, Seán M.; Sourjik, Victor

    2017-10-01

    Many cellular processes require proteins to be precisely positioned within the cell. In some cases this can be attributed to passive mechanisms such as recruitment by other proteins in the cell or by exploiting the curvature of the membrane. However, in bacteria, active self-positioning is likely to play a role in multiple processes, including the positioning of the future site of cell division and cytoplasmic protein clusters. How can such dynamic clusters be formed and positioned? Here, we present a model for the self-organization and positioning of dynamic protein clusters into regularly repeating patterns based on a phase-locked Turing pattern. A single peak in the concentration is always positioned at the midpoint of the model cell, and two peaks are positioned at the midpoint of each half. Furthermore, domain growth results in peak splitting and pattern doubling. We argue that the model may explain the regular positioning of the highly conserved structural maintenance of chromosomes complexes on the bacterial nucleoid and that it provides an attractive mechanism for the self-positioning of dynamic protein clusters in other systems.

  16. Collective Signal Processing in Cluster Chemotaxis: Roles of Adaptation, Amplification, and Co-attraction in Collective Guidance

    PubMed Central

    Camley, Brian A.; Zimmermann, Juliane; Levine, Herbert; Rappel, Wouter-Jan

    2016-01-01

    Single eukaryotic cells commonly sense and follow chemical gradients, performing chemotaxis. Recent experiments and theories, however, show that even when single cells do not chemotax, clusters of cells may, if their interactions are regulated by the chemoattractant. We study this general mechanism of “collective guidance” computationally with models that integrate stochastic dynamics for individual cells with biochemical reactions within the cells, and diffusion of chemical signals between the cells. We show that if clusters of cells use the well-known local excitation, global inhibition (LEGI) mechanism to sense chemoattractant gradients, the speed of the cell cluster becomes non-monotonic in the cluster’s size—clusters either larger or smaller than an optimal size will have lower speed. We argue that the cell cluster speed is a crucial readout of how the cluster processes chemotactic signals; both amplification and adaptation will alter the behavior of cluster speed as a function of size. We also show that, contrary to the assumptions of earlier theories, collective guidance does not require persistent cell-cell contacts and strong short range adhesion. If cell-cell adhesion is absent, and the cluster cohesion is instead provided by a co-attraction mechanism, e.g. chemotaxis toward a secreted molecule, collective guidance may still function. However, new behaviors, such as cluster rotation, may also appear in this case. Co-attraction and adaptation allow for collective guidance that is robust to varying chemoattractant concentrations while not requiring strong cell-cell adhesion. PMID:27367541

  17. Covalently Bound Clusters of Alpha-Substituted PDI—Rival Electron Acceptors to Fullerene for Organic Solar Cells

    DOE PAGES

    Wu, Qinghe; Zhao, Donglin; Schneider, Alexander M.; ...

    2016-05-24

    Here, a cluster type of electron acceptor, TPB, bearing four α-perylenediimides (PDIs), was developed, in which the four PDIs form a cross-like molecular conformation while still partially conjugated with the BDT-Th core. The blend TPB:PTB7-Th films shows favorable morphology and efficient charge dissociation. The inverted solar cells exhibited the highest PCE of 8.47 % with the extraordinary high J sc values (>18 mA/cm 2), comparable with those of the corresponding PC 71BM/PBT7-Th based solar cells.

  18. The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows

    NASA Astrophysics Data System (ADS)

    Brust, M.; Aouane, O.; Thiébaud, M.; Flormann, D.; Verdier, C.; Kaestner, L.; Laschke, M. W.; Selmi, H.; Benyoussef, A.; Podgorski, T.; Coupier, G.; Misbah, C.; Wagner, C.

    2014-03-01

    The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects.

  19. FLOCK cluster analysis of plasma cell flow cytometry data predicts bone marrow involvement by plasma cell neoplasia.

    PubMed

    Dorfman, David M; LaPlante, Charlotte D; Li, Betty

    2016-09-01

    We analyzed plasma cell populations in bone marrow samples from 353 patients with possible bone marrow involvement by a plasma cell neoplasm, using FLOCK (FLOw Clustering without K), an unbiased, automated, computational approach to identify cell subsets in multidimensional flow cytometry data. FLOCK identified discrete plasma cell populations in the majority of bone marrow specimens found by standard histologic and immunophenotypic criteria to be involved by a plasma cell neoplasm (202/208 cases; 97%), including 34 cases that were negative by standard flow cytometric analysis that included clonality assessment. FLOCK identified discrete plasma cell populations in only a minority of cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (38/145 cases; 26%). Interestingly, 55% of the cases negative by standard analysis, but containing a FLOCK-identified discrete plasma cell population, were positive for monoclonal gammopathy by serum protein electrophoresis and immunofixation. FLOCK-identified and quantitated plasma cell populations accounted for 3.05% of total cells on average in cases positive for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria, and 0.27% of total cells on average in cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (p<0.0001; area under the curve by ROC analysis=0.96). The presence of a FLOCK-identified discrete plasma cell population was predictive of the presence of plasma cell neoplasia with a sensitivity of 97%, compared with only 81% for standard flow cytometric analysis, and had specificity of 74%, PPV of 84% and NPV of 95%. FLOCK analysis, which has been shown to provide useful diagnostic information for evaluating patients with suspected systemic mastocytosis, is able to identify neoplastic plasma cell populations analyzed by flow cytometry, and may be helpful in the diagnostic

  20. Glycogen synthase kinase 3 beta inhibits microRNA-183-96-182 cluster via the β-Catenin/TCF/LEF-1 pathway in gastric cancer cells.

    PubMed

    Tang, Xiaoli; Zheng, Dong; Hu, Ping; Zeng, Zongyue; Li, Ming; Tucker, Lynne; Monahan, Renee; Resnick, Murray B; Liu, Manran; Ramratnam, Bharat

    2014-03-01

    Glycogen synthase kinase 3 beta (GSK3β) is a critical protein kinase that phosphorylates numerous proteins in cells and thereby impacts multiple pathways including the β-Catenin/TCF/LEF-1 pathway. MicroRNAs (miRs) are a class of noncoding small RNAs of ∼22 nucleotides in length. Both GSK3β and miR play myriad roles in cell functions including stem cell development, apoptosis, embryogenesis and tumorigenesis. Here we show that GSK3β inhibits the expression of miR-96, miR-182 and miR-183 through the β-Catenin/TCF/LEF-1 pathway. Knockout of GSK3β in mouse embryonic fibroblast cells increases expression of miR-96, miR-182 and miR-183, coinciding with increases in the protein level and nuclear translocation of β-Catenin. In addition, overexpression of β-Catenin enhances the expression of miR-96, miR-182 and miR-183 in human gastric cancer AGS cells. GSK3β protein levels are decreased in human gastric cancer tissue compared with surrounding normal gastric tissue, coinciding with increases of β-Catenin protein, miR-96, miR-182, miR-183 and primary miR-183-96-182 cluster (pri-miR-183). Furthermore, suppression of miR-183-96-182 cluster with miRCURY LNA miR inhibitors decreases the proliferation and migration of AGS cells. Knockdown of GSK3β with siRNA increases the proliferation of AGS cells. Mechanistically, we show that β-Catenin/TCF/LEF-1 binds to the promoter of miR-183-96-182 cluster gene and thereby activates the transcription of the cluster. In summary, our findings identify a novel role for GSK3β in the regulation of miR-183-96-182 biogenesis through β-Catenin/TCF/LEF-1 pathway in gastric cancer cells.

  1. Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

    PubMed Central

    Skogan, Gunnar

    2017-01-01

    ABSTRACT The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H2O, phosphate-buffered saline[PBS], spent culture medium[SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. IMPORTANCE The outcome of this work contributes to improved selection of simulants

  2. Progeny Clustering: A Method to Identify Biological Phenotypes

    PubMed Central

    Hu, Chenyue W.; Kornblau, Steven M.; Slater, John H.; Qutub, Amina A.

    2015-01-01

    Estimating the optimal number of clusters is a major challenge in applying cluster analysis to any type of dataset, especially to biomedical datasets, which are high-dimensional and complex. Here, we introduce an improved method, Progeny Clustering, which is stability-based and exceptionally efficient in computing, to find the ideal number of clusters. The algorithm employs a novel Progeny Sampling method to reconstruct cluster identity, a co-occurrence probability matrix to assess the clustering stability, and a set of reference datasets to overcome inherent biases in the algorithm and data space. Our method was shown successful and robust when applied to two synthetic datasets (datasets of two-dimensions and ten-dimensions containing eight dimensions of pure noise), two standard biological datasets (the Iris dataset and Rat CNS dataset) and two biological datasets (a cell phenotype dataset and an acute myeloid leukemia (AML) reverse phase protein array (RPPA) dataset). Progeny Clustering outperformed some popular clustering evaluation methods in the ten-dimensional synthetic dataset as well as in the cell phenotype dataset, and it was the only method that successfully discovered clinically meaningful patient groupings in the AML RPPA dataset. PMID:26267476

  3. Neuropathology of Cervical Dystonia

    PubMed Central

    Prudente, C.N.; Pardo, C.A.; Xiao, J.; Hanfelt, J.; Hess, E.J.; LeDoux, M.S.; Jinnah, H.A.

    2012-01-01

    The aim of this study was to search for neuropathological changes in postmortem brain tissue of individuals with cervical dystonia (CD). Multiple regions of formalin-preserved brains were collected from patients with CD and controls and examined with an extensive battery of histopathological stains in a two-stage study design. In stage one, 4 CD brains underwent a broad screening neuropathological examination. In stage two, these 4 CD brains were combined with 2 additional CD brains, and the subjective findings were quantified and compared to 16 age-matched controls. The initial subjective neuropathological assessment revealed only two regions with relatively consistent changes. The substantia nigra had frequent ubiquitin-positive intranuclear inclusions known as Marinesco bodies. Additionally, the cerebellum showed patchy loss of Purkinje cells, areas of focal gliosis and torpedo bodies. Other brain regions showed minor or inconsistent changes. In the second stage of the analysis, quantitative studies failed to reveal significant differences in the numbers of Marinesco bodies in CD versus controls, but confirmed a significantly lower Purkinje cell density in CD. Molecular investigations revealed 4 of the CD cases and 2 controls to harbor sequence variants in non-coding regions of THAP1, and these cases had lower Purkinje cell densities regardless of whether they had CD. The findings suggest that subtle neuropathological changes such as lower Purkinje cell density may be found in primary CD when relevant brain regions are investigated with appropriate methods. PMID:23195594

  4. Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation

    PubMed Central

    Leitão, Alexandre B; Sucena, Élio

    2015-01-01

    Virtually all species of coelomate animals contain blood cells that display a division of labor necessary for homeostasis. This functional partition depends upon the balance between proliferation and differentiation mostly accomplished in the hematopoietic organs. In Drosophila melanogaster, the lymph gland produces plasmatocytes and crystal cells that are not released until pupariation. Yet, throughout larval development, both hemocyte types increase in numbers. Mature plasmatocytes can proliferate but it is not known if crystal cell numbers increase by self-renewal or by de novo differentiation. We show that new crystal cells in third instar larvae originate through a Notch-dependent process of plasmatocyte transdifferentiation. This process occurs in the sessile clusters and is contingent upon the integrity of these structures. The existence of this hematopoietic tissue, relying on structure-dependent signaling events to promote blood homeostasis, creates a new paradigm for addressing outstanding questions in Drosophila hematopoiesis and establishing further parallels with vertebrate systems. DOI: http://dx.doi.org/10.7554/eLife.06166.001 PMID:25650737

  5. A Glutaredoxin·BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery*♦

    PubMed Central

    Frey, Avery G.; Palenchar, Daniel J.; Wildemann, Justin D.; Philpott, Caroline C.

    2016-01-01

    Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apoproteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live cell proximity-dependent biotinylation to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3·BolA2 as a [2Fe-2S] chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, whereas Glrx3 homodimer formation did not. Cellular Glrx3·BolA2 complexes increased 6–8-fold in response to increasing iron, forming a rapidly expandable pool of Fe-S clusters. Fe-S coordination by Glrx3·BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a [2Fe-2S] protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3·BolA is a [2Fe-2S] chaperone complex capable of transferring [2Fe-2S] clusters to apoproteins in human cells. PMID:27519415

  6. Nicotinic receptor abnormalities in the cerebellar cortex in autism.

    PubMed

    Lee, M; Martin-Ruiz, C; Graham, A; Court, J; Jaros, E; Perry, R; Iversen, P; Bauman, M; Perry, E

    2002-07-01

    Autism is a common developmental disorder associated with structural and inferred neurochemical abnormalities of the brain. Cerebellar abnormalities frequently have been identified, based on neuroimaging or neuropathology. Recently, the cholinergic neurotransmitter system has been implicated on the basis of nicotinic receptor loss in the cerebral cortex. Cerebellar cholinergic activities were therefore investigated in autopsy tissue from a series of autistic individuals. The presynaptic cholinergic enzyme, choline acetyltransferase, together with nicotinic and muscarinic receptor subtypes were compared in the cerebellum from age-matched mentally retarded autistic (eight), normal control (10) and non-autistic mentally retarded individuals (11). The nicotinic receptor binding the agonist epibatidine (the high affinity receptor subtype, consisting primarily of alpha3 and alpha4, together with beta2 receptor subunits) was significantly reduced by 40-50% in the granule cell, Purkinje and molecular layers in the autistic compared with the normal group (P < 0.05). There was an opposite increase (3-fold) in the nicotinic receptor binding alpha-bungarotoxin (to the alpha7 subunit) which reached significance in the granule cell layer (P < 0.05). These receptor changes were paralleled by a significant reduction (P < 0.05) and non-significant increase, respectively, of alpha4 and alpha7 receptor subunit immunoreactivity measured using western blotting. Immunohistochemically loss of alpha(4 )reactivity was apparent from Purkinje and the other cell layers, with increased alpha7 reactivity in the granule cell layer. There were no significant changes in choline acetyltransferase activity, or in muscarinic M1 and M2 receptor subtypes in autism. In the non-autistic mentally retarded group, the only significant abnormality was a reduction in epibatidine binding in the granule cell and Purkinje layers. In two autistic cases examined histologically, Purkinje cell loss was observed in

  7. Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

    PubMed Central

    Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

    2013-01-01

    Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and

  8. Quantum cluster algebras and quantum nilpotent algebras.

    PubMed

    Goodearl, Kenneth R; Yakimov, Milen T

    2014-07-08

    A major direction in the theory of cluster algebras is to construct (quantum) cluster algebra structures on the (quantized) coordinate rings of various families of varieties arising in Lie theory. We prove that all algebras in a very large axiomatically defined class of noncommutative algebras possess canonical quantum cluster algebra structures. Furthermore, they coincide with the corresponding upper quantum cluster algebras. We also establish analogs of these results for a large class of Poisson nilpotent algebras. Many important families of coordinate rings are subsumed in the class we are covering, which leads to a broad range of applications of the general results to the above-mentioned types of problems. As a consequence, we prove the Berenstein-Zelevinsky conjecture [Berenstein A, Zelevinsky A (2005) Adv Math 195:405-455] for the quantized coordinate rings of double Bruhat cells and construct quantum cluster algebra structures on all quantum unipotent groups, extending the theorem of Geiß et al. [Geiß C, et al. (2013) Selecta Math 19:337-397] for the case of symmetric Kac-Moody groups. Moreover, we prove that the upper cluster algebras of Berenstein et al. [Berenstein A, et al. (2005) Duke Math J 126:1-52] associated with double Bruhat cells coincide with the corresponding cluster algebras.

  9. Quantum cluster algebras and quantum nilpotent algebras

    PubMed Central

    Goodearl, Kenneth R.; Yakimov, Milen T.

    2014-01-01

    A major direction in the theory of cluster algebras is to construct (quantum) cluster algebra structures on the (quantized) coordinate rings of various families of varieties arising in Lie theory. We prove that all algebras in a very large axiomatically defined class of noncommutative algebras possess canonical quantum cluster algebra structures. Furthermore, they coincide with the corresponding upper quantum cluster algebras. We also establish analogs of these results for a large class of Poisson nilpotent algebras. Many important families of coordinate rings are subsumed in the class we are covering, which leads to a broad range of applications of the general results to the above-mentioned types of problems. As a consequence, we prove the Berenstein–Zelevinsky conjecture [Berenstein A, Zelevinsky A (2005) Adv Math 195:405–455] for the quantized coordinate rings of double Bruhat cells and construct quantum cluster algebra structures on all quantum unipotent groups, extending the theorem of Geiß et al. [Geiß C, et al. (2013) Selecta Math 19:337–397] for the case of symmetric Kac–Moody groups. Moreover, we prove that the upper cluster algebras of Berenstein et al. [Berenstein A, et al. (2005) Duke Math J 126:1–52] associated with double Bruhat cells coincide with the corresponding cluster algebras. PMID:24982197

  10. White blood cell segmentation by color-space-based k-means clustering.

    PubMed

    Zhang, Congcong; Xiao, Xiaoyan; Li, Xiaomei; Chen, Ying-Jie; Zhen, Wu; Chang, Jun; Zheng, Chengyun; Liu, Zhi

    2014-09-01

    White blood cell (WBC) segmentation, which is important for cytometry, is a challenging issue because of the morphological diversity of WBCs and the complex and uncertain background of blood smear images. This paper proposes a novel method for the nucleus and cytoplasm segmentation of WBCs for cytometry. A color adjustment step was also introduced before segmentation. Color space decomposition and k-means clustering were combined for segmentation. A database including 300 microscopic blood smear images were used to evaluate the performance of our method. The proposed segmentation method achieves 95.7% and 91.3% overall accuracy for nucleus segmentation and cytoplasm segmentation, respectively. Experimental results demonstrate that the proposed method can segment WBCs effectively with high accuracy.

  11. Semiconductor hierarchically structured flower-like clusters for dye-sensitized solar cells with nearly 100% charge collection efficiency.

    PubMed

    Xin, Xukai; Liu, Hsiang-Yu; Ye, Meidan; Lin, Zhiqun

    2013-11-21

    By combining the ease of producing ZnO nanoflowers with the advantageous chemical stability of TiO2, hierarchically structured hollow TiO2 flower-like clusters were yielded via chemical bath deposition (CBD) of ZnO nanoflowers, followed by their conversion into TiO2 flower-like clusters in the presence of TiO2 precursors. The effects of ZnO precursor concentration, precursor amount, and reaction time on the formation of ZnO nanoflowers were systematically explored. Dye-sensitized solar cells fabricated by utilizing these hierarchically structured ZnO and TiO2 flower clusters exhibited a power conversion efficiency of 1.16% and 2.73%, respectively, under 100 mW cm(-2) illumination. The intensity modulated photocurrent/photovoltage spectroscopy (IMPS/IMVS) studies suggested that flower-like structures had a fast electron transit time and their charge collection efficiency was nearly 100%.

  12. Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

    PubMed

    Nagasawa, Hatsumi; Lin, Yu-Fen; Kato, Takamitsu A; Brogan, John R; Shih, Hung-Ying; Kurimasa, Akihiro; Bedford, Joel S; Chen, Benjamin P C; Little, John B

    2017-02-01

    The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G 0 /G 1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single

  13. Mitochondrial filaments and clusters as intracellular power-transmitting cables.

    PubMed

    Skulachev, V P

    2001-01-01

    Mitochondria exist in two interconverting forms; as small isolated particles, and as extended filaments, networks or clusters connected with intermitochondrial junctions. Extended mitochondria can represent electrically united systems, which can facilitate energy delivery from the cell periphery to the cell core and organize antioxidant defence of the cell interior when O2 is consumed by mitochondrial clusters near the the outer cell membrane, and protonic potential is transmitted to the cell core mitochondria to form ATP. As to small mitochondria, they might represent a transportable form of these organelles.

  14. Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

    PubMed

    Tsai, Ming-Yuan; Lu, Yu-Fen; Liu, Yu-Hsiu; Lien, Huang-Wei; Huang, Chang-Jen; Wu, Jen-Leih; Hwang, Sheng-Ping L

    2015-09-01

    Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos. © 2014 Wiley Periodicals, Inc.

  15. Cell-to-cell variation sets a tissue-rheology–dependent bound on collective gradient sensing

    PubMed Central

    Camley, Brian A.; Rappel, Wouter-Jan

    2017-01-01

    When a single cell senses a chemical gradient and chemotaxes, stochastic receptor–ligand binding can be a fundamental limit to the cell’s accuracy. For clusters of cells responding to gradients, however, there is a critical difference: Even genetically identical cells have differing responses to chemical signals. With theory and simulation, we show collective chemotaxis is limited by cell-to-cell variation in signaling. We find that when different cells cooperate, the resulting bias can be much larger than the effects of ligand–receptor binding. Specifically, when a strongly responding cell is at one end of a cell cluster, cluster motion is biased toward that cell. These errors are mitigated if clusters average measurements over times long enough for cells to rearrange. In consequence, fluid clusters are better able to sense gradients: We derive a link between cluster accuracy, cell-to-cell variation, and the cluster rheology. Because of this connection, increasing the noisiness of individual cell motion can actually increase the collective accuracy of a cluster by improving fluidity. PMID:29114053

  16. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.

    PubMed

    Cho, Won-Ki; Spille, Jan-Hendrik; Hecht, Micca; Lee, Choongman; Li, Charles; Grube, Valentin; Cisse, Ibrahim I

    2018-06-21

    Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. Here we used live cell super-resolution and light sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo. Copyright © 2018, American Association for the Advancement of Science.

  17. Dynamical clustering of red blood cells in capillary vessels.

    PubMed

    Boryczko, Krzysztof; Dzwinel, Witold; Yuen, David A

    2003-02-01

    We have modeled the dynamics of a 3-D system consisting of red blood cells (RBCs), plasma and capillary walls using a discrete-particle approach. The blood cells and capillary walls are composed of a mesh of particles interacting with harmonic forces between nearest neighbors. We employ classical mechanics to mimic the elastic properties of RBCs with a biconcave disk composed of a mesh of spring-like particles. The fluid particle method allows for modeling the plasma as a particle ensemble, where each particle represents a collective unit of fluid, which is defined by its mass, moment of inertia, translational and angular momenta. Realistic behavior of blood cells is modeled by considering RBCs and plasma flowing through capillaries of various shapes. Three types of vessels are employed: a pipe with a choking point, a curved vessel and bifurcating capillaries. There is a strong tendency to produce RBC clusters in capillaries. The choking points and other irregularities in geometry influence both the flow and RBC shapes, considerably increasing the clotting effect. We also discuss other clotting factors coming from the physical properties of blood, such as the viscosity of the plasma and the elasticity of the RBCs. Modeling has been carried out with adequate resolution by using 1 to 10 million particles. Discrete particle simulations open a new pathway for modeling the dynamics of complex, viscoelastic fluids at the microscale, where both liquid and solid phases are treated with discrete particles. Figure A snapshot from fluid particle simulation of RBCs flowing along a curved capillary. The red color corresponds to the highest velocity. We can observe aggregation of RBCs at places with the most stagnant plasma flow.

  18. Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

    PubMed Central

    Taleb, Omar; Kemmel, Véronique; Laux, Alexis; Miehe, Monique; Delalande, François; Roussel, Guy; Van Dorsselaer, Alain; Metz-Boutigue, Marie-Hélène; Aunis, Dominique; Goumon, Yannick

    2008-01-01

    Background Morphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system. Methodology and Principal Findings Microscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca2+-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10−10 M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies. Conclusions/Significance The presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a

  19. Clustering T cell GM1 Lipid Rafts Increases Cellular Resistance to Shear on Fibronectin through Changes in Integrin Affinity and Cytoskeletal Dynamics

    PubMed Central

    Mitchell, Jason S.; Brown, Wells S.; Woodside, Darren G.; Vanderslice, Peter; McIntyre, Bradley W.

    2008-01-01

    Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T cell functions. The aggregation of specific GM1 lipid rafts can control many T cell activation events, including their novel association with T cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F-actin, the resistance to shear induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin “inside-out” signaling mechanisms. This was seen through increased integrin-cytoskeleton associations and enhanced soluble binding of FN and VCAM-1 suggesting the induction of high affinity integrin conformations. The activation of these adhesion strengthening characteristics appear to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion strengthening processes. PMID:19139760

  20. c-MYC-regulated miR-23a/24-2/27a Cluster Promotes Mammary Carcinoma Cell Invasion and Hepatic Metastasis by Targeting Sprouty2*

    PubMed Central

    Li, Xiaoni; Liu, Xin; Xu, Weiyi; Zhou, Peng; Gao, Ping; Jiang, Songshan; Lobie, Peter E.; Zhu, Tao

    2013-01-01

    Emerging evidence indicates that the miR-23a/24-2/27a cluster may possess a causal role in mammary tumorigenesis and function as a novel class of oncogenes. However, the regulatory mechanism of the miR-23a/24-2/27a cluster in mammary carcinoma cell invasion and migration is still largely unknown. We observed that the expression levels of miR-23a, miR-24-2 and miR-27a were significantly higher in breast cancer with lymph node metastasis, compared with that from patients without lymph node metastasis or normal tissue. Forced expression of the miR-23a/24-2/27a cluster promoted mammary carcinoma cell migration, invasion, and hepatic metastasis, through targeting Sprouty2 (SPRY2) and consequent activation of p44/42 MAPK. Epidermal growth factor induced the expression of the transcription factor c-MYC, which promoted the expression of mature miR-23a, miR-24-2, and miR-27a and subsequently decreased expression of SPRY2 and activated p44/42 MAPK to promote mammary carcinoma cell migration and invasion. We therefore suggest a novel link between epidermal growth factor and the miR-23a/24-2/27a cluster via the regulation of c-MYC, providing the potential for the miR-23a/24-2/27a cluster to be used as biomarker in the diagnosis and/or treatment of breast cancer. PMID:23649631

  1. Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells

    PubMed Central

    Yi, Jason; Wu, Xufeng S.; Crites, Travis; Hammer, John A.

    2012-01-01

    Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin—a novel reporter for F-actin—we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function–associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse. PMID:22219382

  2. Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin-Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration.

    PubMed

    Karimi, Fatemeh; O'Connor, Andrea J; Qiao, Greg G; Heath, Daniel E

    2018-03-25

    Material systems that exhibit tailored interactions with cells are a cornerstone of biomaterial and tissue engineering technologies. One method of achieving these tailored interactions is to biofunctionalize materials with peptide ligands that bind integrin receptors present on the cell surface. However, cell biology research has illustrated that both integrin binding and integrin clustering are required to achieve a full adhesion response. This biophysical knowledge has motivated researchers to develop material systems biofunctionalized with nanoscale clusters of ligands that promote both integrin occupancy and clustering of the receptors. These materials have improved a wide variety of biological interactions in vitro including cell adhesion, proliferation, migration speed, gene expression, and stem cell differentiation; and improved in vivo outcomes including increased angiogenesis, tissue healing, and biomedical device integration. This review first introduces the techniques that enable the fabrication of these nanopatterned materials, describes the improved biological effects that have been achieved, and lastly discusses the current limitations of the technology and where future advances may occur. Although this technology is still in its nascency, it will undoubtedly play an important role in the future development of biomaterials and tissue engineering scaffolds for both in vitro and in vivo applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

    PubMed Central

    Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

    2014-01-01

    The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

  4. Fabrication of large size alginate beads for three-dimensional cell-cluster culture

    NASA Astrophysics Data System (ADS)

    Zhang, Zhengtao; Ruan, Meilin; Liu, Hongni; Cao, Yiping; He, Rongxiang

    2017-08-01

    We fabricated large size alginate beads using a simple microfluidic device under a co-axial injection regime. This device was made by PDMS casting with a mold formed by small diameter metal and polytetrafluorothylene tubes. Droplets of 2% sodium alginate were generated in soybean oil through the device and then cross-linked in a 2% CaCl2 solution, which was mixed tween80 with at a concentration of 0.4 to 40% (w/v). Our results showed that the morphology of the produced alginate beads strongly depends on the tween80 concentration. With the increase of concentration of tween80, the shape of the alginate beads varied from semi-spherical to tailed-spherical, due to the decrease of interface tension between oil and cross-link solution. To access the biocompatibility of the approach, MCF-7 cells were cultured with the alginate beads, showing the formation of cancer cells clusters which might be useful for future studies.

  5. Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli.

    PubMed

    Tan, Guoqiang; Yang, Jing; Li, Tang; Zhao, Jin; Sun, Shujuan; Li, Xiaokang; Lin, Chuxian; Li, Jianghui; Zhou, Huaibin; Lyu, Jianxin; Ding, Huangen

    2017-08-15

    While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells. IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they

  6. Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli

    PubMed Central

    Tan, Guoqiang; Yang, Jing; Li, Tang; Zhao, Jin; Sun, Shujuan; Li, Xiaokang; Lin, Chuxian; Li, Jianghui; Zhou, Huaibin

    2017-01-01

    ABSTRACT While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells. IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions

  7. The [Mo₆Cl14]2- Cluster is Biologically Secure and Has Anti-Rotavirus Activity In Vitro.

    PubMed

    Rojas-Mancilla, Edgardo; Oyarce, Alexis; Verdugo, Viviana; Morales-Verdejo, Cesar; Echeverria, Cesar; Velásquez, Felipe; Chnaiderman, Jonas; Valiente-Echeverría, Fernando; Ramirez-Tagle, Rodrigo

    2017-07-05

    The molybdenum cluster [Mo₆Cl 14 ] 2- is a fluorescent component with potential for use in cell labelling and pharmacology. Biological safety and antiviral properties of the cluster are as yet unknown. Here, we show the effect of acute exposition of human cells and red blood cells to the molybdenum cluster and its interaction with proteins and antiviral activity in vitro. We measured cell viability of HepG2 and EA.hy926 cell lines exposed to increasing concentrations of the cluster (0.1 to 250 µM), by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Hemolysis and morphological alterations of red blood cells, obtained from healthy donors, exposed to the cluster (10 to 200 µM) at 37 °C were analyzed. Furthermore, quenching of tryptophan residues of albumin was performed. Finally, plaque formation by rotavirus SA11 in MA104 cells treated with the cluster (100 to 300 µM) were analyzed. We found that all doses of the cluster showed similar cell viability, hemolysis, and morphology values, compared to control. Quenching of tryptophan residues of albumin suggests a protein-cluster complex formation. Finally, the cluster showed antiviral activity at 300 µM. These results indicate that the cluster [Mo₆Cl 14 ] 2- could be intravenously administered in animals at therapeutic doses for further in vivo studies and might be studied as an antiviral agent.

  8. Flexible method for monitoring fuel cell voltage

    DOEpatents

    Mowery, Kenneth D.; Ripley, Eugene V.

    2002-01-01

    A method for equalizing the measured voltage of each cluster in a fuel cell stack wherein at least one of the clusters has a different number of cells than the identical number of cells in the remaining clusters by creating a pseudo voltage for the different cell numbered cluster. The average cell voltage of the all of the cells in the fuel cell stack is calculated and multiplied by a constant equal to the difference in the number of cells in the identical cell clusters and the number of cells in the different numbered cell cluster. The resultant product is added to the actual voltage measured across the different numbered cell cluster to create a pseudo voltage which is equivalent in cell number to the number of cells in the other identical numbered cell clusters.

  9. SERA Scenarios of Early Market Fuel Cell Electric Vehicle Introductions: Modeling Framework, Regional Markets, and Station Clustering

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bush, B.; Melaina, M.; Penev, M.

    This report describes the development and analysis of detailed temporal and spatial scenarios for early market hydrogen fueling infrastructure clustering and fuel cell electric vehicle rollout using the Scenario Evaluation, Regionalization and Analysis (SERA) model. The report provides an overview of the SERA scenario development framework and discusses the approach used to develop the nationwidescenario.

  10. Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of prickle.

    PubMed

    Cho, Bomsoo; Pierre-Louis, Gandhy; Sagner, Andreas; Eaton, Suzanne; Axelrod, Jeffrey D

    2015-05-01

    The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

  11. densityCut: an efficient and versatile topological approach for automatic clustering of biological data

    PubMed Central

    Ding, Jiarui; Shah, Sohrab; Condon, Anne

    2016-01-01

    Motivation: Many biological data processing problems can be formalized as clustering problems to partition data points into sensible and biologically interpretable groups. Results: This article introduces densityCut, a novel density-based clustering algorithm, which is both time- and space-efficient and proceeds as follows: densityCut first roughly estimates the densities of data points from a K-nearest neighbour graph and then refines the densities via a random walk. A cluster consists of points falling into the basin of attraction of an estimated mode of the underlining density function. A post-processing step merges clusters and generates a hierarchical cluster tree. The number of clusters is selected from the most stable clustering in the hierarchical cluster tree. Experimental results on ten synthetic benchmark datasets and two microarray gene expression datasets demonstrate that densityCut performs better than state-of-the-art algorithms for clustering biological datasets. For applications, we focus on the recent cancer mutation clustering and single cell data analyses, namely to cluster variant allele frequencies of somatic mutations to reveal clonal architectures of individual tumours, to cluster single-cell gene expression data to uncover cell population compositions, and to cluster single-cell mass cytometry data to detect communities of cells of the same functional states or types. densityCut performs better than competing algorithms and is scalable to large datasets. Availability and Implementation: Data and the densityCut R package is available from https://bitbucket.org/jerry00/densitycut_dev. Contact: condon@cs.ubc.ca or sshah@bccrc.ca or jiaruid@cs.ubc.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153661

  12. Intra-cluster Globular Clusters in a Simulated Galaxy Cluster

    NASA Astrophysics Data System (ADS)

    Ramos-Almendares, Felipe; Abadi, Mario; Muriel, Hernán; Coenda, Valeria

    2018-01-01

    Using a cosmological dark matter simulation of a galaxy-cluster halo, we follow the temporal evolution of its globular cluster population. To mimic the red and blue globular cluster populations, we select at high redshift (z∼ 1) two sets of particles from individual galactic halos constrained by the fact that, at redshift z = 0, they have density profiles similar to observed ones. At redshift z = 0, approximately 60% of our selected globular clusters were removed from their original halos building up the intra-cluster globular cluster population, while the remaining 40% are still gravitationally bound to their original galactic halos. As the blue population is more extended than the red one, the intra-cluster globular cluster population is dominated by blue globular clusters, with a relative fraction that grows from 60% at redshift z = 0 up to 83% for redshift z∼ 2. In agreement with observational results for the Virgo galaxy cluster, the blue intra-cluster globular cluster population is more spatially extended than the red one, pointing to a tidally disrupted origin.

  13. Rapid Onset of Motor Deficits in a Mouse Model of Spinocerebellar Ataxia Type 6 Precedes Late Cerebellar Degeneration1,2,3

    PubMed Central

    Ljungberg, Lovisa; Cormier, Alexander; Quilez, Sabrina

    2015-01-01

    Abstract Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant cerebellar ataxia that has been associated with loss of cerebellar Purkinje cells. Disease onset is typically at midlife, although it can vary widely from late teens to old age in SCA6 patients. Our study focused on an SCA6 knock-in mouse model with a hyper-expanded (84X) CAG repeat expansion that displays midlife-onset motor deficits at ∼7 months old, reminiscent of midlife-onset symptoms in SCA6 patients, although a detailed phenotypic analysis of these mice has not yet been reported. Here, we characterize the onset of motor deficits in SCA684Q mice using a battery of behavioral assays to test for impairments in motor coordination, balance, and gait. We found that these mice performed normally on these assays up to and including at 6 months, but motor impairment was detected at 7 months with all motor coordination assays used, suggesting that motor deficits emerge rapidly during a narrow age window in SCA684Q mice. In contrast to what is seen in SCA6 patients, the decrease in motor coordination was observed without alterations in gait. No loss of cerebellar Purkinje cells or striatal neurons were observed at 7 months, the age at which motor deficits were first detected, but significant Purkinje cell loss was observed in 2-year-old SCA684Q mice, arguing that Purkinje cell death does not significantly contribute to the early stages of SCA6. PMID:26730403

  14. Cellular and genetic regulation of the development of the cerebellar system.

    PubMed

    Sotelo, Constantino

    2004-04-01

    Recent advances in molecular biology have drastically changed our vision on the development of the nervous system, the cerebellum in particular. After a classical descriptive period, we are now in a modern mechanistic epoch as we begin to answer crucial questions in our quest to understand the mechanisms underlying the emergence of brain complexity. This review begins with an analysis of the role of the "isthmic organizer" in the induction and specification of the cerebellar territory and progresses through cerebellar development to the formation of cerebellar maps. It gathers information about the control of the proliferation of granule cell precursors by Purkinje cells and the role of Shh/Gli-patched signaling. The migratory routes for cerebellar and precerebellar neurons, together with the long-range and short-range cues guiding gliophilic and, particularly, neurophilic migrations, are also discussed. Because these cues are similar to those involved in axon guidance, both processes are under the same molecular constraints. Finally, using primarily the olivocerebellar projection as a model, the cellular and molecular mechanisms involved in the formation of cerebellar maps are discussed. During embryonic development, Purkinje cells in the cerebellum and neurons in the inferior olive follow a simultaneous, but independent, process of intrinsic parcellation, giving rise to subsets of biochemically different cortical compartments. The occurrence of positional information shared between olivary axons and their postsynaptic targets, the Purkinje cells, provides a molecular code for the formation of coarse-grained maps. Activity-dependent mechanisms are required for the transition from crude to fine-grained maps. This important refinement, which confers ultimate specificity to the maps, is under the regulation of parallel fiber-Purkinje cell synaptic activity.

  15. Delayed repair of radiation induced clustered DNA damage: Friend or foe?

    PubMed Central

    Eccles, Laura J.; O’Neill, Peter; Lomax, Martine E.

    2011-01-01

    A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue. PMID:21130102

  16. Real-time dynamics of neutrophil clustering in response to phototoxicity-induced cell death and tissue damage in mouse ear dermis.

    PubMed

    Park, Sang A; Choe, Young Ho; Park, Eunji; Hyun, Young-Min

    2018-05-22

    Neutrophils are highly motile innate immune cells; they actively migrate in response to inflammatory signals. Using two-photon intravital microscopy, we discovered that neutrophils form stable clusters upon phototoxicity at a certain threshold. Without significant damage to the collagen structure of mouse dermis, neutrophils aggregated together with nearby neutrophils. Surprisingly, this in situ neutrophil clustering resulted in rigorous changes of migratory direction. The density of residing neutrophils was also a critical factor affecting clustering. Additionally, we found that the triggering point of neutrophil aggregation was correlated with the structure of the extracellular matrix in the ear dermis, where autofluorescence was strongly observed. This swarming behavior of neutrophils may reflect an unknown communication mechanism of neutrophils during migration under sterile injury.

  17. Enhanced Cell Capture on Functionalized Graphene Oxide Nanosheets through Oxygen Clustering.

    PubMed

    Bardhan, Neelkanth M; Kumar, Priyank V; Li, Zeyang; Ploegh, Hidde L; Grossman, Jeffrey C; Belcher, Angela M; Chen, Guan-Yu

    2017-02-28

    With the global rise in incidence of cancer and infectious diseases, there is a need for the development of techniques to diagnose, treat, and monitor these conditions. The ability to efficiently capture and isolate cells and other biomolecules from peripheral whole blood for downstream analyses is a necessary requirement. Graphene oxide (GO) is an attractive template nanomaterial for such biosensing applications. Favorable properties include its two-dimensional architecture and wide range of functionalization chemistries, offering significant potential to tailor affinity toward aromatic functional groups expressed in biomolecules of interest. However, a limitation of current techniques is that as-synthesized GO nanosheets are used directly in sensing applications, and the benefits of their structural modification on the device performance have remained unexplored. Here, we report a microfluidic-free, sensitive, planar device on treated GO substrates to enable quick and efficient capture of Class-II MHC-positive cells from murine whole blood. We achieve this by using a mild thermal annealing treatment on the GO substrates, which drives a phase transformation through oxygen clustering. Using a combination of experimental observations and MD simulations, we demonstrate that this process leads to improved reactivity and density of functionalization of cell capture agents, resulting in an enhanced cell capture efficiency of 92 ± 7% at room temperature, almost double the efficiency afforded by devices made using as-synthesized GO (54 ± 3%). Our work highlights a scalable, cost-effective, general approach to improve the functionalization of GO, which creates diverse opportunities for various next-generation device applications.

  18. Inflammation-induced formation of fat-associated lymphoid clusters

    PubMed Central

    Bénézech, Cécile; Kruglov, Andrei A.; Loo, Yunhua; Nakamura, Kyoko; Zhang, Yang; Nayar, Saba; Jones, Lucy H.; Flores-Langarica, Adriana; McIntosh, Alistair; Marshall, Jennifer; Barone, Francesca; Besra, Gurdyal; Miles, Katherine; Allen, Judith E.; Gray, Mohini; Kollias, George; Cunningham, Adam F.; Withers, David R.; Toellner, Kai Michael; Jones, Nick D.; Veldhoen, Marc; Nedospasov, Sergei A.; McKenzie, Andrew N.J.; Caamaño, Jorge H.

    2015-01-01

    Fat-associated lymphoid clusters (FALCs) are a recently discovered type of lymphoid tissue associated with visceral fat. Here we show that distribution of FALCs was heterogeneous with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 B cells in the peritoneal cavity through high expression of the chemokine CXCL13 and supported B cell proliferation and germinal center differentiation during peritoneal immune challenges. FALC formation was induced by inflammation, which triggered recruitment of myeloid cells that express tumor necrosis factor (TNF) necessary for TNF receptor-signaling in stromal cells. CD1d-restricted Natural killer T (NKT) cells were likewise required for inducible formation of FALCs. Thus, FALCs support and coordinate innate B and T cell activation during serosal immune responses. PMID:26147686

  19. Receptor Type Protein Tyrosine Phosphatase ζ-Pleiotrophin Signaling Controls Endocytic Trafficking of DNER That Regulates Neuritogenesis▿ †

    PubMed Central

    Fukazawa, Nobuna; Yokoyama, Seisuke; Eiraku, Mototsugu; Kengaku, Mineko; Maeda, Nobuaki

    2008-01-01

    Protein tyrosine phosphatase ζ (PTPζ) is a receptor type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTPζ, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTPζ and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTPζ and DNER displayed patchy colocalization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTPζ, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic-acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased the tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTPζ signaling controls subcellular localization of DNER and thereby regulates neuritogenesis. PMID:18474614

  20. Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

    PubMed

    Sun, Ying-Jie; Nishikawa, Kaori; Yuda, Hideki; Wang, Yu-Lai; Osaka, Hitoshi; Fukazawa, Nobuna; Naito, Akira; Kudo, Yoshihisa; Wada, Keiji; Aoki, Shunsuke

    2006-09-01

    With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

  1. Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster.

    PubMed

    Wüst, Stas; Dröse, Stefan; Heidler, Juliana; Wittig, Ilka; Klockner, Ina; Franko, Andras; Bonke, Erik; Günther, Stefan; Gärtner, Ulrich; Boettger, Thomas; Braun, Thomas

    2018-05-01

    Muscle stem cells undergo a dramatic metabolic switch to oxidative phosphorylation during differentiation, which is achieved by massively increased mitochondrial activity. Since expression of the muscle-specific miR-1/133a gene cluster correlates with increased mitochondrial activity during muscle stem cell (MuSC) differentiation, we examined the potential role of miR-1/133a in metabolic maturation of skeletal muscles in mice. We found that miR-1/133a downregulate Mef2A in differentiated myocytes, thereby suppressing the Dlk1-Dio3 gene cluster, which encodes multiple microRNAs inhibiting expression of mitochondrial genes. Loss of miR-1/133a in skeletal muscles or increased Mef2A expression causes continuous high-level expression of the Dlk1-Dio3 gene cluster, compromising mitochondrial function. Failure to terminate the stem cell-like metabolic program characterized by high-level Dlk1-Dio3 gene cluster expression initiates profound changes in muscle physiology, essentially abrogating endurance running. Our results suggest a major role of miR-1/133a in metabolic maturation of skeletal muscles but exclude major functions in muscle development and MuSC maintenance. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering*

    PubMed Central

    Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; Stanley, Christopher B.; Do, Changwoo; Heller, William T.; Aggarwal, Aneel K.; Callaway, David J. E.; Bu, Zimei

    2015-01-01

    The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. PMID:25572402

  3. Scaffold Architecture Controls Insulinoma Clustering, Viability, and Insulin Production

    PubMed Central

    Blackstone, Britani N.; Palmer, Andre F.; Rilo, Horacio R.

    2014-01-01

    Recently, in vitro diagnostic tools have shifted focus toward personalized medicine by incorporating patient cells into traditional test beds. These cell-based platforms commonly utilize two-dimensional substrates that lack the ability to support three-dimensional cell structures seen in vivo. As monolayer cell cultures have previously been shown to function differently than cells in vivo, the results of such in vitro tests may not accurately reflect cell response in vivo. It is therefore of interest to determine the relationships between substrate architecture, cell structure, and cell function in 3D cell-based platforms. To investigate the effect of substrate architecture on insulinoma organization and function, insulinomas were seeded onto 2D gelatin substrates and 3D fibrous gelatin scaffolds with three distinct fiber diameters and fiber densities. Cell viability and clustering was assessed at culture days 3, 5, and 7 with baseline insulin secretion and glucose-stimulated insulin production measured at day 7. Small, closely spaced gelatin fibers promoted the formation of large, rounded insulinoma clusters, whereas monolayer organization and large fibers prevented cell clustering and reduced glucose-stimulated insulin production. Taken together, these data show that scaffold properties can be used to control the organization and function of insulin-producing cells and may be useful as a 3D test bed for diabetes drug development. PMID:24410263

  4. Time-lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes.

    PubMed

    Wang, M D; Axelrod, D

    1994-09-01

    To study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time-lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine alpha-bungarotoxin (R-BT) continuously present in the medium. Since TIRF illuminates only cell-substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the culture during imaging procedures is much enhanced relative to standard epi- (or trans-) illumination. During the experiment, cells were kept alive on the microscope stage at 37 degrees C in an atmosphere of 10% CO2. Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week-long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest-lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell-cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour.

  5. Development and Structure of Winter Monsoon Cloud Clusters On 10 December 1978.

    NASA Astrophysics Data System (ADS)

    Churchill, Dean D.; Houze, Robert A., Jr.

    1984-03-01

    Data from the Winter Monsoon Experiment (WMONEX) are used to study cloud clusters that occurred over the South China Sea on 10 December 1978. These clusters underwent life cycles in which they began as groups of intense convective cells and then developed into mesoscale systems consisting partly of convective cells and partly of stratiform precipitation. In the cellular regions of clusters, ice particle concentrations (at the 8 km, or 17°C, level) were found to be of the order of hundreds per liter, local convective updrafts of 4-17 m s1 were observed, and the dominant ice-particle growth mechanism appeared to be riming. In the stratiform regions of clusters, the ice particles appeared to grow by vapor deposition and aggregation and weaker but more widespread mesoscale updraft motion was indicated at mid to upper levels by the observed ice-crystal structures, while unsaturated mesoscale downdraft motion was indicated at mid to lower levels by sounding data. The ice-particle concentrations at 8 km in stratiform regions were one to two orders of magnitude lower than in the convective regions; in areas of weaker stratiform precipitation the concentrations were 1-10 per liter, while in areas of stronger stratiform precipitation they were 20-70 per liter. Substructure within the stratiform regions was indicated by variations in observed ice-crystal habits over horizontal distances of 10-100 km.The dominant cluster observed on this day was initiated when a group of convective cells formed just off the Borneo coast, apparently in response to land-breeze convergence. During the mature stage of this cluster, the stratiform rain area became nearly surrounded by a broken line of convective cells of various intensity. The greatest concentration of intense cores was maintained in a generally stationary region on the southeast side of the cluster where the land-breeze convergence was located, while convection on the northwest side of the cluster propagated out to sea. The

  6. Basolateral Sorting of Furin in MDCK Cells Requires a Phenylalanine-Isoleucine Motif Together with an Acidic Amino Acid Cluster

    PubMed Central

    Simmen, Thomas; Nobile, Massimo; Bonifacino, Juan S.; Hunziker, Walter

    1999-01-01

    Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin. PMID:10082580

  7. How Escherichia coli lands and forms cell clusters on a surface: a new role of surface topography

    PubMed Central

    Gu, Huan; Chen, Aaron; Song, Xinran; Brasch, Megan E.; Henderson, James H.; Ren, Dacheng

    2016-01-01

    Bacterial response to surface topography during biofilm formation was studied using 5 μm tall line patterns of poly (dimethylsiloxane) (PDMS). Escherichia coli cells attached on top of protruding line patterns were found to align more perpendicularly to the orientation of line patterns when the pattern narrowed. Consistently, cell cluster formation per unit area on 5 μm wide line patterns was reduced by 14-fold compared to flat PDMS. Contrasting the reduced colony formation, cells attached on narrow patterns were longer and had higher transcriptional activities, suggesting that such unfavorable topography may present a stress to attached cells. Results of mutant studies indicate that flagellar motility is involved in the observed preference in cell orientation on narrow patterns, which was corroborated by the changes in cell rotation pattern before settling on different surface topographies. These findings led to a set of new design principles for creating antifouling topographies, which was validated using 10 μm tall hexagonal patterns. PMID:27412365

  8. Localization of a bacterial cytoplasmic receptor is dynamic and changes with cell-cell contacts

    PubMed Central

    Mauriello, Emilia M. F.; Astling, David P.; Sliusarenko, Oleksii; Zusman, David R.

    2009-01-01

    Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor, does not form membrane-bound polar clusters typical for most bacteria, but rather cytoplasmic clusters that appear helically arranged and span the cell length. The distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters that continuously changed their size, number, and position. The number of FrzCD clusters was correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing mutants and additional clusters were observed in hyper-reversing mutants. When moving cells made side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These events were frequently followed by one of the interacting cells reversing. These observations suggest that FrzCD detects signals from a cell contact-sensitive signaling system and then re-localizes as it directs reversals to distributed motility engines. PMID:19273862

  9. Advanced analysis of forest fire clustering

    NASA Astrophysics Data System (ADS)

    Kanevski, Mikhail; Pereira, Mario; Golay, Jean

    2017-04-01

    Analysis of point pattern clustering is an important topic in spatial statistics and for many applications: biodiversity, epidemiology, natural hazards, geomarketing, etc. There are several fundamental approaches used to quantify spatial data clustering using topological, statistical and fractal measures. In the present research, the recently introduced multi-point Morisita index (mMI) is applied to study the spatial clustering of forest fires in Portugal. The data set consists of more than 30000 fire events covering the time period from 1975 to 2013. The distribution of forest fires is very complex and highly variable in space. mMI is a multi-point extension of the classical two-point Morisita index. In essence, mMI is estimated by covering the region under study by a grid and by computing how many times more likely it is that m points selected at random will be from the same grid cell than it would be in the case of a complete random Poisson process. By changing the number of grid cells (size of the grid cells), mMI characterizes the scaling properties of spatial clustering. From mMI, the data intrinsic dimension (fractal dimension) of the point distribution can be estimated as well. In this study, the mMI of forest fires is compared with the mMI of random patterns (RPs) generated within the validity domain defined as the forest area of Portugal. It turns out that the forest fires are highly clustered inside the validity domain in comparison with the RPs. Moreover, they demonstrate different scaling properties at different spatial scales. The results obtained from the mMI analysis are also compared with those of fractal measures of clustering - box counting and sand box counting approaches. REFERENCES Golay J., Kanevski M., Vega Orozco C., Leuenberger M., 2014: The multipoint Morisita index for the analysis of spatial patterns. Physica A, 406, 191-202. Golay J., Kanevski M. 2015: A new estimator of intrinsic dimension based on the multipoint Morisita index

  10. Host Cell Contact-Induced Transcription of the Type IV Fimbria Gene Cluster of Actinobacillus pleuropneumoniae

    PubMed Central

    Boekema, Bouke K. H. L.; Van Putten, Jos P. M.; Stockhofe-Zurwieden, Norbert; Smith, Hilde E.

    2004-01-01

    Type IV pili (Tfp) of gram-negative species share many characteristics, including a common architecture and conserved biogenesis pathway. Much less is known about the regulation of Tfp expression in response to changing environmental conditions. We investigated the diversity of Tfp regulatory systems by searching for the molecular basis of the reported variable expression of the Tfp gene cluster of the pathogen Actinobacillus pleuropneumoniae. Despite the presence of an intact Tfp gene cluster consisting of four genes, apfABCD, no Tfp were formed under standard growth conditions. Sequence analysis of the predicted major subunit protein ApfA showed an atypical alanine residue at position −1 from the prepilin peptidase cleavage site in 42 strains. This alanine deviates from the consensus glycine at this position in Tfp from other species. Yet, cloning of the apfABCD genes under a constitutive promoter in A. pleuropneumoniae resulted in pilin and Tfp assembly. Tfp promoter-luxAB reporter gene fusions demonstrated that the Tfp promoter was intact but tightly regulated. Promoter activity varied with bacterial growth phase and was detected only when bacteria were grown in chemically defined medium. Infection experiments with cultured epithelial cells demonstrated that Tfp promoter activity was upregulated upon adherence of the pathogen to primary cultures of lung epithelial cells. Nonadherent bacteria in the culture supernatant exhibited virtually no promoter activity. A similar upregulation of Tfp promoter activity was observed in vivo during experimental infection of pigs. The host cell contact-induced and in vivo-upregulated Tfp promoter activity in A. pleuropneumoniae adds a new dimension to the diversity of Tfp regulation. PMID:14742510

  11. In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2.

    PubMed

    Hoff, Kevin G; Culler, Stephanie J; Nguyen, Peter Q; McGuire, Ryan M; Silberg, Jonathan J; Smolke, Christina D

    2009-12-24

    A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins. Copyright 2009 Elsevier Ltd. All rights reserved.

  12. Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells

    PubMed Central

    Huang, Cheng-Chiu; Sugino, Ken; Shima, Yasuyuki; Guo, Caiying; Bai, Suxia; Mensh, Brett D; Nelson, Sacha B; Hantman, Adam W

    2013-01-01

    Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI: http://dx.doi.org/10.7554/eLife.00400.001 PMID:23467508

  13. Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum.

    PubMed

    Obana, Edwin A; Lundell, Travis G; Yi, Kevin J; Radomski, Kryslaine L; Zhou, Qiong; Doughty, Martin L

    2015-06-01

    Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.

  14. miR-15a/miR-16 cluster inhibits invasion of prostate cancer cells by suppressing TGF-β signaling pathway.

    PubMed

    Jin, Wei; Chen, Fangjie; Wang, Kefeng; Song, Yan; Fei, Xiang; Wu, Bin

    2018-05-23

    To determine whether and how miR15a/16 regulate TGF-β signaling pathways during the progression of prostate cancer. We used bioinformatics prediction, reporter gene assay, real-time PCR, Matrigel invasion assay and Western blot to dissect the molecular mechanism of how miR-15a/miR-16 may cause metastasis in prostate tumor. MiR-15a/16 targeted and inhibited the expression of endogenous Smad3 and ACVR2A proteins. The overexpression of miR15a/16 down-regulated p-smad3 expression, affected the expression of both MMP2 and E-cadherin, and down-regulated the expression of the EMT-mediated factors Snail and Twist in LNCaP prostate cancer cells. The overexpression of miR15a/16 decreased the invasion of LNCaP cells. MiR-15a/miR-16 cluster could reverse the invasion of activin A-mediated prostate cancer cells. After the inhibition of the activin/smad signaling pathway, the inhibitory effect of invasion in prostate cancer cells by miR-15a/miR-16 cluster disappeared. Our data indicated that miR15a/16 inhibited the components of TGF-β signaling pathways in LNCaP cell line, which might relate to the progression and metastasis of prostate cancer. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  15. Recent Developments of the Local Effect Model (LEM) - Implications of clustered damage on cell transformation

    NASA Astrophysics Data System (ADS)

    Elsässer, Thilo

    Exposure to radiation of high-energy and highly charged ions (HZE) causes a major risk to human beings, since in long term space explorations about 10 protons per month and about one HZE particle per month hit each cell nucleus (1). Despite the larger number of light ions, the high ionisation power of HZE particles and its corresponding more complex damage represents a major hazard for astronauts. Therefore, in order to get a reasonable risk estimate, it is necessary to take into account the entire mixed radiation field. Frequently, neoplastic cell transformation serves as an indicator for the oncogenic potential of radiation exposure. It can be measured for a small number of ion and energy combinations. However, due to the complexity of the radiation field it is necessary to know the contribution to the radiation damage of each ion species for the entire range of energies. Therefore, a model is required which transfers the few experimental data to other particles with different LETs. We use the Local Effect Model (LEM) (2) with its cluster extension (3) to calculate the relative biological effectiveness (RBE) of neoplastic transformation. It was originally developed in the framework of hadrontherapy and is applicable for a large range of ions and energies. The input parameters for the model include the linear-quadratic parameters for the induction of lethal events as well as for the induction of transformation events per surviving cell. Both processes of cell inactivation and neoplastic transformation per viable cell are combined to eventually yield the RBE for cell transformation. We show that the Local Effect Model is capable of predicting the RBE of neoplastic cell transformation for a broad range of ions and energies. The comparison of experimental data (4) with model calculations shows a reasonable agreement. We find that the cluster extension results in a better representation of the measured RBE values. With this model it should be possible to better

  16. Size Effects in Dye-Sensitized TiO2 Clusters

    NASA Astrophysics Data System (ADS)

    Marom, Noa; Körzdörfer, Thomas; Ren, Xinguo; Tkatchenko, Alexandre; Chelikowsky, James

    2014-03-01

    The development of solar cells is driven by the need for clean and sustainable energy. Organic and dye sensitized cells are considered as promising technologies, particularly for large area, low cost applications. However, the efficiency of such cells is still far from the theoretical limit. Ab initio simulations may be used for computer-aided design of new materials and nano-structures for more efficient solar cells. It is essential to obtain an accurate description of the electronic structure, including the fundamental gaps and energy level alignment at the interfaces in the device active region. This requires going beyond ground-state DFT to the GW approximation. A recently developed GW method [PRB 86, 041110R (2012)] is applied to dye-sensitized TiO2 clusters [PRB 84, 245115 (2011)]. The effect of cluster size on the energy level alignment at the dye-TiO2 interface is discussed. With the increase in the TiO2 cluster size its gap narrows. The gap of the molecule attached to the cluster subsequently narrows due to screening. As a result, the energy level alignment at the interface changes in an unexpected way [Marom, Körzdörfer, Ren, Tkatchenko, Chelikowsky, to be published].

  17. Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering.

    PubMed

    Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K; Stanley, Christopher B; Do, Changwoo; Heller, William T; Aggarwal, Aneel K; Callaway, David J E; Bu, Zimei

    2015-03-06

    The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Functional roles of Cot/Tpl2 in mast cell responses to lipopolysaccharide and FcεRI-clustering.

    PubMed

    Chiba, Norika; Kakimoto, Kyoko; Masuda, Akio; Matsuguchi, Tetsuya

    2010-11-05

    Cot/Tpl2, a member of MAP kinase kinase kinase (MAPKKK), is indispensable for the ERK activation, as well as the production of TNF-α, IL-1β, IL-23, and PGE(2) in lipopolysaccharide (LPS)-stimulated macrophages. However, the expression and the functional roles of Cot/Tpl2 in mast cells have not been elucidated. The administration of LPS impairs allergic airway inflammation in a mast cell-dependent manner, and LPS stimulates mast cells to produce not only pro-inflammatory cytokines, such as IL-6 and TNF-α, but also Th2-type cytokines, such as IL-5, IL-10 and IL-13. Here, we examine the role of Cot/Tpl2 by using bone marrow-derived mast cells (BMMCs) from cot/tpl2 gene-deficient mice. Phosphorylation of ERKs was significantly decreased, whereas that of JNKs and p38 kinase was normal in LPS-stimulated cot/tpl2(-/-) BMMCs compared with wild-type counterparts. LPS-induced mRNA increase was significantly impaired for IL-5, IL-10, IL-13, and TNF-α, but was normal for IL-6, in cot/tpl2(-/-) BMMCs. On the other hand, degranulation by FcεRI-clustering from cot/tpl2(-/-) BMMCs was significantly enhanced compared with the WT control. Although the phosphorylation of ERKs and p38 kinase by FcεRI-clustering was similar in WT and cot/tpl2(-/-) BMMCs, the phosphorylation of Syk was significantly enhanced in cot/tpl2(-/-) BMMCs, which seemed to be due to the increased protein concentration of Syk. These results imply the functional importance of Cot/Tpl2 in mast cells during the course of allergic diseases such as asthma. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Why not make a PC cluster of your own? 5. AppleSeed: A Parallel Macintosh Cluster for Scientific Computing

    NASA Astrophysics Data System (ADS)

    Decyk, Viktor K.; Dauger, Dean E.

    We have constructed a parallel cluster consisting of Apple Macintosh G4 computers running both Classic Mac OS as well as the Unix-based Mac OS X, and have achieved very good performance on numerically intensive, parallel plasma particle-in-cell simulations. Unlike other Unix-based clusters, no special expertise in operating systems is required to build and run the cluster. This enables us to move parallel computing from the realm of experts to the mainstream of computing.

  20. Acoustical sensing of cardiomyocyte cluster beating

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tymchenko, Nina; Kunze, Angelika; Dahlenborg, Kerstin

    2013-06-14

    Highlights: •An example of the application of QCM-D to live cell studies. •Detection of human pluripotent stem cell-derived cardiomyocyte cluster beating. •Clusters were studied in a thin liquid film and in a large liquid volume. •The QCM-D beating profile provides an individual fingerprint of the hPS-CMCs. -- Abstract: Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cellmore » quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to μm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66–168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.« less

  1. Anatomical and Electrophysiological Clustering of Superficial Medial Entorhinal Cortex Interneurons

    PubMed Central

    2017-01-01

    Abstract Local GABAergic interneurons regulate the activity of spatially-modulated principal cells in the medial entorhinal cortex (MEC), mediating stellate-to-stellate connectivity and possibly enabling grid formation via recurrent inhibitory circuitry. Despite the important role interneurons seem to play in the MEC cortical circuit, the combination of low cell counts and functional diversity has made systematic electrophysiological studies of these neurons difficult. For these reasons, there remains a paucity of knowledge on the electrophysiological profiles of superficial MEC interneuron populations. Taking advantage of glutamic acid decarboxylase 2 (GAD2)-IRES-tdTomato and PV-tdTomato transgenic mice, we targeted GABAergic interneurons for whole-cell patch-clamp recordings and characterized their passive membrane features, basic input/output properties and action potential (AP) shape. These electrophysiologically characterized cells were then anatomically reconstructed, with emphasis on axonal projections and pial depth. K-means clustering of interneuron anatomical and electrophysiological data optimally classified a population of 106 interneurons into four distinct clusters. The first cluster is comprised of layer 2- and 3-projecting, slow-firing interneurons. The second cluster is comprised largely of PV+ fast-firing interneurons that project mainly to layers 2 and 3. The third cluster contains layer 1- and 2-projecting interneurons, and the fourth cluster is made up of layer 1-projecting horizontal interneurons. These results, among others, will provide greater understanding of the electrophysiological characteristics of MEC interneurons, help guide future in vivo studies, and may aid in uncovering the mechanism of grid field formation. PMID:29085901

  2. Opsoclonus: clinical and immunological features.

    PubMed

    Jen, Joanna C; Lopez, Ivan; Baloh, Robert W

    2012-09-15

    Opsoclonus is felt to be a saccadic oscillation disorder but the neuroanatomical substrate for generating the abnormal eye movements is poorly understood. We recorded eye movements and studied serum samples from 7 patients who presented with opsoclonus and with either myoclonus or generalized tremor. Anti neuronal antibodies were detected by immunohistochemestry using rat and human cerebellar sections. In all patients but one the opsoclonus resolved within 2weeks (after immunosuppression in 4, resection of the underlying neoplasm in 1 and spontaneously in 1). Opsoclonus was arrhythmic and multidirectional with a wide frequency range (4-10Hz). No known paraneoplastic antibodies were found in the initial commercial screen. Three patients had antiPurkinje cell antibodies with a characteristic punctate staining in the molecular layer. The clinical and immunological findings are consistent with the hypothesis, that in some patients, opsoclonus results from antibodies directed at the parallel fiber-Purkinje cell synapse. The antibodies block parallel fiber input to Purkinje cells allowing spontaneous oscillating activity generated in the inferior olives to be passed on to the oculomotor nuclei via the flocculus. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Delta-like 1 regulates Bergmann glial monolayer formation during cerebellar development.

    PubMed

    Hiraoka, Yuichi; Komine, Okiru; Nagaoka, Mai; Bai, Ning; Hozumi, Katsuto; Tanaka, Kohichi

    2013-05-21

    Bergmann glia (BG) are unipolar cerebellar astrocytes. The somata of mature BG reside in the Purkinje cell layer and extend radially arranged processes to the pial surface. BG have multiple branched processes, which enwrap the synapses of Purkinje cell dendrites. They migrate from the ventricular zone and align next to the Purkinje cell layer during development. Previously, we reported that Notch1, Notch2, and RBPj genes in the BG play crucial roles in the monolayer formation and morphogenesis of BG. However, it remains to be determined which ligand activates Nocth1 and Notch 2 on BG. Delta-like 1 (Dll1) is a major ligand of Notch receptors that is expressed in the developing cerebellum. In this study, we used human glial fibrillary acidic protein (hGFAP) promoter-driven Cre-mediated recombination to delete Dll1 in BG. Dll1-conditional mutant mice showed disorganization of Bergmann fibers, ectopic localization of BG in the molecular layer and a reduction in the number of BG. These results suggest that Dll1 is required for the formation of the BG layer and its morphological maturation, apparently through a Notch1/2-RBPj dependent signaling pathway.

  4. Melatonin Protects Human Cells from Clustered DNA Damages, Killing and Acquisition of Soft Agar Growth Induced by X-rays or 970 MeV/n Fe ions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Das, B.; Sutherland, B.; Bennett, P. V.

    We tested the ability of melatonin (N-acetyl-5 methoxytryptamine), a highly effective radical scavenger and human hormone, to protect DNA in solution and in human cells against induction of complex DNA clusters and biological damage induced by low or high linear energy transfer radiation (100 kVp X-rays, 970 MeV/nucleon Fe ions). Plasmid DNA in solution was treated with increasing concentrations of melatonin (0.0-3.5 mM) and were irradiated with X-rays. Human cells (28SC monocytes) were also irradiated with X-rays and Fe ions with and without 2 mM melatonin. Agarose plugs containing genomic DNA were subjected to Contour Clamped Homogeneous Electrophoretic Field (CHEF)more » followed by imaging and clustered DNA damages were measured by using Number Average length analysis. Transformation experiments on human primary fibroblast cells using soft agar colony assay were carried out which were irradiated with Fe ions with or without 2 mM melatonin. In plasmid DNA in solution, melatonin reduced the induction of single- and double-strand breaks. Pretreatment of human 28SC cells for 24 h before irradiation with 2 mM melatonin reduced the level of X-ray induced double-strand breaks by {approx}50%, of abasic clustered damages about 40%, and of Fe ion-induced double-strand breaks (41% reduction) and abasic clusters (34% reduction). It decreased transformation to soft agar growth of human primary cells by a factor of 10, but reduced killing by Fe ions only by 20-40%. Melatonin's effective reduction of radiation-induced critical DNA damages, cell killing, and striking decrease of transformation suggest that it is an excellent candidate as a countermeasure against radiation exposure, including radiation exposure to astronaut crews in space travel.« less

  5. Hierarchical Dirichlet process model for gene expression clustering

    PubMed Central

    2013-01-01

    Clustering is an important data processing tool for interpreting microarray data and genomic network inference. In this article, we propose a clustering algorithm based on the hierarchical Dirichlet processes (HDP). The HDP clustering introduces a hierarchical structure in the statistical model which captures the hierarchical features prevalent in biological data such as the gene express data. We develop a Gibbs sampling algorithm based on the Chinese restaurant metaphor for the HDP clustering. We apply the proposed HDP algorithm to both regulatory network segmentation and gene expression clustering. The HDP algorithm is shown to outperform several popular clustering algorithms by revealing the underlying hierarchical structure of the data. For the yeast cell cycle data, we compare the HDP result to the standard result and show that the HDP algorithm provides more information and reduces the unnecessary clustering fragments. PMID:23587447

  6. Preclinical Evaluation of An Anti-HCV miRNA Cluster for Treatment of HCV Infection

    PubMed Central

    Yang, Xiao; Marcucci, Katherine; Anguela, Xavier; Couto, Linda B.

    2013-01-01

    We developed a strategy to treat hepatitis C virus (HCV) infection by replacing five endogenous microRNA (miRNA) sequences of a natural miRNA cluster (miR-17–92) with sequences that are complementary to the HCV genome. This miRNA cluster (HCV-miR-Cluster 5) is delivered to cells using adeno-associated virus (AAV) vectors and the miRNAs are expressed in the liver, the site of HCV replication and assembly. AAV-HCV-miR-Cluster 5 inhibited bona fide HCV replication in vitro by up to 95% within 2 days, and the spread of HCV to uninfected cells was prevented by continuous expression of the anti-HCV miRNAs. Furthermore, the number of cells harboring HCV RNA replicons decreased dramatically by sustained expression of the anti-HCV miRNAs, suggesting that the vector is capable of curing cells of HCV. Delivery of AAV-HCV-miR-Cluster 5 to mice resulted in efficient transfer of the miRNA gene cluster and expression of all five miRNAs in liver tissue, at levels up to 1,300 copies/cell. These levels achieved up to 98% gene silencing of cognate HCV sequences, and no liver toxicity was observed, supporting the safety of this approach. Therefore, AAV-HCV-miR-Cluster 5 represents a different paradigm for the treatment of HCV infection. PMID:23295950

  7. Flow over gravel beds with clusters

    NASA Astrophysics Data System (ADS)

    Little, M.; Venditti, J. G.

    2014-12-01

    The structure of a gravel bed has been shown to alter the entrainment threshold. Structures such as clusters, reticulate stone cells and other discrete structures lock grains together, making it more difficult for them to be mobilized. These structures also generate form drag, reducing the shear stress available for mobilization. Form drag over gravel beds is often assumed to be negligible, but this assumption is not well supported. Here, we explore how cluster density and arrangement affect flow resistance and the flow structure over a fixed gravel bed in a flume experiment. Cluster density was varied from 6 to 68.3 clusters per square meter which corresponds to areal bed coverages of 2 to 17%. We used regular, irregular and random arrangements of the clusters. Our results show that flow resistance over a planar gravel bed initially declines, then increases with flow depth. The addition of clusters increases flow resistance, but the effect is dependent on cluster density, flow depth and arrangement. At the highest density, clusters can increase flow resistance as by as much as 8 times when compared to flat planar bed with no grain-related form drag. Spatially resolved observations of flow over the clusters indicate that a well-defined wake forms in the lee of each cluster. At low cluster density, the wakes are isolated and weak. As cluster density increases, the wakes become stronger. At the highest density, the wakes interact and the within cluster flow field detaches from the overlying flow. This generates a distinct shear layer at the height of the clusters. In spite of this change in the flow field at high density, our results suggest that flow resistance simply increases with cluster density. Our results suggest that the form drag associated with a gravel bed can be substantial and that it depends on the arrangement of the grains on the bed.

  8. A phosphorescent silver(I)-gold (I) cluster complex that specifically lights up the nucleolus of living cells with FLIM imaging.

    PubMed

    Chen, Min; Lei, Zhen; Feng, Wei; Li, Chunyan; Wang, Quan-Ming; Li, Fuyou

    2013-06-01

    The phosphorescent silver(I)-gold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1) selectively stains the nucleolus, with a much lower uptake in the nucleus and cytoplasm, and exhibits excellent photostability. This Ag-Au cluster, which has a photoluminescent lifetime of microseconds, is particularly attractive as a probe in applications of time-gated microscopy. Investigation of the pathway of cellular entry indicated that N1 permeates the outer membrane and nuclear membrane of living cells through an energy-dependent and non-endocytic route within 10 min. High concentrations of N1 in the nucleolus have been quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA), which also helped to elucidate the mechanism of the specific staining. Intracellular selective staining may be correlated with the microenvironment of the nucleolus, which is consistent with experiments conducted at different phases of the cell cycle. These results prove that N1 is a very attractive phosphorescent staining reagent for visualizing the nucleolus of living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

    PubMed Central

    Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

    2018-01-01

    Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

  10. Ionic mechanisms of transient inward current in the absence of Na(+)-Ca2+ exchange in rabbit cardiac Purkinje fibres.

    PubMed Central

    Han, X; Ferrier, G R

    1992-01-01

    1. Membrane currents were measured with a two-microelectrode technique in voltage clamped rabbit cardiac Purkinje fibres under conditions known to cause intracellular calcium overload and to eliminate or minimize Na(+)-Ca2+ exchange. 2. Increasing [Ca2+]o from 2.5 to 5 mM or above and substituting external sodium with either sucrose, choline or Li+ induced an oscillatory transient inward current (TI) which peaked 200-300 ms after repolarization from a previous depolarizing pulse. The TI quickly disappeared upon return to normal Tyrode solution. Both the rate and configuration of action potentials of Purkinje fibres also returned to control upon return to Tyrode solution after 30 min of high Ca2+ exposure, if the Ca2+ concentration was 30 mM or less. 3. The TI in Na(+)-free solution was Ca2+ dependent. Either zero or low (2.5 mM) [Ca2+]o, or replacement of [Ca2+]o by BaCl prevented induction of the TI current upon repolarization from a previous depolarizing pulse. 4. In the presence of 30 mM-CaCl2 and with choline chloride as the substitute for NaCl, TI had a distinct reversal potential (Erev) of -25 mV. The time-to-peak TI, either inward or outward, did not shift significantly with change in voltage. Both inward and outward TI were simultaneously abolished by exposure to 1 microM-ryanodine, suggesting they were both activated by transient release of Ca2+ from the sarcoplasmic reticulum. The occurrence of TI in the absence of [Na+]o is not compatible with an electrogenic Na(+)-Ca2+ exchange mechanism. The existence of a clear-cut reversal potential suggests that an ionic channel may be responsible for the TI under these conditions. 5. Both the magnitude of peak TI and the Erev were affected by changes of CaCl2 concentration. (i) Under steady-state conditions, peak inward TI was significantly increased when the [Ca2+]o was elevated from 5 to 15 mM. The peak TI in the outward direction was significantly increased when [Ca2+]o was elevated from 15 to 30 mM; however

  11. Subcellular TSC22D4 localization in cerebellum granule neurons of the mouse depends on development and differentiation.

    PubMed

    Canterini, Sonia; Bosco, Adriana; Carletti, Valentina; Fuso, Andrea; Curci, Armando; Mangia, Franco; Fiorenza, Maria Teresa

    2012-03-01

    We previously demonstrated that TSC22D4, a protein encoded by the TGF-β1-activated gene Tsc22d4 (Thg-1pit) and highly expressed in postnatal and adult mouse cerebellum with multiple post-translationally modified protein forms, moves to nucleus when in vitro differentiated cerebellum granule neurons (CGNs) are committed to apoptosis by hyperpolarizing KCl concentrations in the culture medium. We have now studied TSC22D4 cytoplasmic/nuclear localization in CGNs and Purkinje cells: (1) during CGN differentiation/maturation in vivo, (2) during CGN differentiation in vitro, and (3) by in vitro culturing ex vivo cerebellum slices under conditions favoring/inhibiting CGN/Purkinje cell differentiation. We show that TSC22D4 displays both nuclear and cytoplasmic localizations in undifferentiated, early postnatal cerebellum CGNs, irrespectively of CGN proliferation/migration from external to internal granule cell layer, and that it specifically accumulates in the somatodendritic and synaptic compartments when CGNs mature, as indicated by TSC22D4 abundance at the level of adult cerebellum glomeruli and apparent lack in CGN nuclei. These features were also observed in cerebellum slices cultured in vitro under conditions favoring/inhibiting CGN/Purkinje cell differentiation. In vitro TSC22D4 silencing with siRNAs blocked CGN differentiation and inhibited neurite elongation in N1E-115 neuroblastoma cells, pinpointing the relevance of this protein to CGN differentiation.

  12. Analysis of temporal gene expression profiles: clustering by simulated annealing and determining the optimal number of clusters.

    PubMed

    Lukashin, A V; Fuchs, R

    2001-05-01

    Cluster analysis of genome-wide expression data from DNA microarray hybridization studies has proved to be a useful tool for identifying biologically relevant groupings of genes and samples. In the present paper, we focus on several important issues related to clustering algorithms that have not yet been fully studied. We describe a simple and robust algorithm for the clustering of temporal gene expression profiles that is based on the simulated annealing procedure. In general, this algorithm guarantees to eventually find the globally optimal distribution of genes over clusters. We introduce an iterative scheme that serves to evaluate quantitatively the optimal number of clusters for each specific data set. The scheme is based on standard approaches used in regular statistical tests. The basic idea is to organize the search of the optimal number of clusters simultaneously with the optimization of the distribution of genes over clusters. The efficiency of the proposed algorithm has been evaluated by means of a reverse engineering experiment, that is, a situation in which the correct distribution of genes over clusters is known a priori. The employment of this statistically rigorous test has shown that our algorithm places greater than 90% genes into correct clusters. Finally, the algorithm has been tested on real gene expression data (expression changes during yeast cell cycle) for which the fundamental patterns of gene expression and the assignment of genes to clusters are well understood from numerous previous studies.

  13. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    PubMed

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  14. Investigation of dislocation cluster evolution during directional solidification of multicrystalline silicon

    NASA Astrophysics Data System (ADS)

    Oriwol, Daniel; Trempa, Matthias; Sylla, Lamine; Leipner, Hartmut S.

    2017-04-01

    Dislocation clusters are the main crystal defects in multicrystalline silicon and are detrimental for solar cell efficiency. They were formed during the silicon ingot casting due to the relaxation of strain energy. The evolution of the dislocation clusters was studied by means of automated analysing tools of the standard wafer and cell production giving information about the cluster development as a function of the ingot height. Due to the observation of the whole wafer surface the point of view is of macroscopic nature. It was found that the dislocations tend to build clusters of high density which usually expand in diameter as a function of ingot height. According to their structure the dislocation clusters can be divided into light and dense clusters. The appearance of both types shows a clear dependence on the orientation of the grain growth direction. Additionally, a process of annihilation of dislocation clusters during the crystallization has been observed. To complement the macroscopic description, the dislocation clusters were also investigates by TEM. It is shown that the dislocations within the subgrain boundaries are closely arranged. Distances of 40-30 nm were found. These results lead to the conclusion that the dislocation density within the cluster structure is impossible to quantify by means of etch pit counting.

  15. Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters.

    PubMed

    Beamish, Christine A; Strutt, Brenda J; Arany, Edith J; Hill, David J

    2016-04-18

    Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future.

  16. Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters

    PubMed Central

    Beamish, Christine A.; Strutt, Brenda J.; Arany, Edith J.; Hill, David J.

    2016-01-01

    ABSTRACT Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP+/+ mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP+ cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP+Ck19+ cells had derived from insulin-positive, glucose-transporter-2-low (Ins+Glut2LO) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin+Glut2LO cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin+Glut2+ cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins+Glut2LO cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future. PMID:27010375

  17. Interaction of plasticity and circuit organization during the acquisition of cerebellum-dependent motor learning

    PubMed Central

    Yang, Yan; Lisberger, Stephen G

    2013-01-01

    Motor learning occurs through interactions between the cerebellar circuit and cellular plasticity at different sites. Previous work has established plasticity in brain slices and suggested plausible sites of behavioral learning. We now reveal what actually happens in the cerebellum during short-term learning. We monitor the expression of plasticity in the simple-spike firing of cerebellar Purkinje cells during trial-over-trial learning in smooth pursuit eye movements of monkeys. Our findings imply that: 1) a single complex-spike response driven by one instruction for learning causes short-term plasticity in a Purkinje cell’s mossy fiber/parallel-fiber input pathways; 2) complex-spike responses and simple-spike firing rate are correlated across the Purkinje cell population; and 3) simple-spike firing rate at the time of an instruction for learning modulates the probability of a complex-spike response, possibly through a disynaptic feedback pathway to the inferior olive. These mechanisms may participate in long-term motor learning. DOI: http://dx.doi.org/10.7554/eLife.01574.001 PMID:24381248

  18. T cell receptor repertoires of mice and humans are clustered in similarity networks around conserved public CDR3 sequences

    PubMed Central

    Madi, Asaf; Poran, Asaf; Shifrut, Eric; Reich-Zeliger, Shlomit; Greenstein, Erez; Zaretsky, Irena; Arnon, Tomer; Laethem, Francois Van; Singer, Alfred; Lu, Jinghua; Sun, Peter D; Cohen, Irun R; Friedman, Nir

    2017-01-01

    Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity. DOI: http://dx.doi.org/10.7554/eLife.22057.001 PMID:28731407

  19. Expression of Gas1 in Mouse Brain: Release and Role in Neuronal Differentiation.

    PubMed

    Bautista, Elizabeth; Zarco, Natanael; Aguirre-Pineda, Nicolás; Lara-Lozano, Manuel; Vergara, Paula; González-Barrios, Juan Antonio; Aguilar-Roblero, Raúl; Segovia, José

    2018-05-01

    Growth arrest-specific 1 (Gas1) is a pleiotropic protein that induces apoptosis of tumor cells and has important roles during development. Recently, the presence of two forms of Gas1 was reported: one attached to the cell membrane by a GPI anchor; and a soluble extracellular form shed by cells. Previously, we showed that Gas1 is expressed in different areas of the adult mouse CNS. Here, we report the levels of Gas1 mRNA protein in different regions and analyzed its expressions in glutamatergic, GABAergic, and dopaminergic neurons. We found that Gas1 is expressed in GABAergic and glutamatergic neurons in the Purkinje-molecular layer of the cerebellum, hippocampus, thalamus, and fastigial nucleus, as well as in dopaminergic neurons of the substantia nigra. In all cases, Gas1 was found in the cell bodies, but not in the neuropil. The Purkinje and the molecular layers show the highest levels of Gas1, whereas the granule cell layer has low levels. Moreover, we detected the expression and release of Gas1 from primary cultures of Purkinje cells and from hippocampal neurons as well as from neuronal cell lines, but not from cerebellar granular cells. In addition, using SH-SY5Y cells differentiated with retinoic acid as a neuronal model, we found that extracellular Gas1 promotes neurite outgrowth, increases the levels of tyrosine hydroxylase, and stimulates the inhibition of GSK3β. These findings demonstrate that Gas1 is expressed and released by neurons and promotes differentiation, suggesting an important role for Gas1 in cellular signaling in the CNS.

  20. The miRNA-17∼92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K/AKT pathway activation.

    PubMed

    Rao, E; Jiang, C; Ji, M; Huang, X; Iqbal, J; Lenz, G; Wright, G; Staudt, L M; Zhao, Y; McKeithan, T W; Chan, W C; Fu, K

    2012-05-01

    The median survival of patients with mantle cell lymphoma (MCL) ranges from 3 to 5 years with current chemotherapeutic regimens. A common secondary genomic alteration detected in MCL is chromosome 13q31-q32 gain/amplification, which targets a microRNA (miRNA) cluster, miR-17∼92. On the basis of gene expression profiling, we found that high level expression of C13orf25, the primary transcript from which these miRNAs are processed, was associated with poorer survival in patients with MCL (P=0.021). We demonstrated that the protein phosphatase PHLPP2, an important negative regulator of the PI3K/AKT pathway, was a direct target of miR-17∼92 miRNAs, in addition to PTEN and BIM. These proteins were down-modulated in MCL cells with overexpression of the miR-17∼92 cluster. Overexpression of miR-17∼92 activated the PI3K/AKT pathway and inhibited chemotherapy-induced apoptosis in MCL cell lines. Conversely, inhibition of miR-17∼92 expression suppressed the PI3K/AKT pathway and inhibited tumor growth in a xenograft MCL mouse model. Targeting the miR-17∼92 cluster may therefore provide a novel therapeutic approach for patients with MCL.

  1. Mesenchymal Stem Cells and Their Conditioned Medium Improve Integration of Purified Induced Pluripotent Stem Cell–Derived Cardiomyocyte Clusters into Myocardial Tissue

    PubMed Central

    Rubach, Martin; Adelmann, Roland; Haustein, Moritz; Drey, Florian; Pfannkuche, Kurt; Xiao, Bing; Koester, Annette; Udink ten Cate, Floris E.A.; Choi, Yeong-Hoon; Neef, Klaus; Fatima, Azra; Hannes, Tobias; Pillekamp, Frank; Hescheler, Juergen; Šarić, Tomo; Brockmeier, Konrad

    2014-01-01

    Induced pluripotent stem cell–derived cardiomyocytes (iPS-CMs) might become therapeutically relevant to regenerate myocardial damage. Purified iPS-CMs exhibit poor functional integration into myocardial tissue. The aim of this study was to investigate whether murine mesenchymal stem cells (MSCs) or their conditioned medium (MScond) improves the integration of murine iPS-CMs into myocardial tissue. Vital or nonvital embryonic murine ventricular tissue slices were cocultured with purified clusters of iPS-CMs in combination with murine embryonic fibroblasts (MEFs), MSCs, or MScond. Morphological integration was assessed by visual scoring and functional integration by isometric force and field potential measurements. We observed a moderate morphological integration of iPS-CM clusters into vital, but a poor integration into nonvital, slices. MEFs and MSCs but not MScond improved morphological integration of CMs into nonvital slices and enabled purified iPS-CMs to confer force. Coculture of vital slices with iPS-CMs and MEFs or MSCs resulted in an improved electrical integration. A comparable improvement of electrical coupling was achieved with the cell-free MScond, indicating that soluble factors secreted by MSCs were involved in electrical coupling. We conclude that cells such as MSCs support the engraftment and adhesion of CMs, and confer force to noncontractile tissue. Furthermore, soluble factors secreted by MSCs mediate electrical coupling of purified iPS-CM clusters to myocardial tissue. These data suggest that MSCs may increase the functional engraftment and therapeutic efficacy of transplanted iPS-CMs into infarcted myocardium. PMID:24219308

  2. Constrained clusters of gene expression profiles with pathological features.

    PubMed

    Sese, Jun; Kurokawa, Yukinori; Monden, Morito; Kato, Kikuya; Morishita, Shinichi

    2004-11-22

    Gene expression profiles should be useful in distinguishing variations in disease, since they reflect accurately the status of cells. The primary clustering of gene expression reveals the genotypes that are responsible for the proximity of members within each cluster, while further clustering elucidates the pathological features of the individual members of each cluster. However, since the first clustering process and the second classification step, in which the features are associated with clusters, are performed independently, the initial set of clusters may omit genes that are associated with pathologically meaningful features. Therefore, it is important to devise a way of identifying gene expression clusters that are associated with pathological features. We present the novel technique of 'itemset constrained clustering' (IC-Clustering), which computes the optimal cluster that maximizes the interclass variance of gene expression between groups, which are divided according to the restriction that only divisions that can be expressed using common features are allowed. This constraint automatically labels each cluster with a set of pathological features which characterize that cluster. When applied to liver cancer datasets, IC-Clustering revealed informative gene expression clusters, which could be annotated with various pathological features, such as 'tumor' and 'man', or 'except tumor' and 'normal liver function'. In contrast, the k-means method overlooked these clusters.

  3. Receptor clustering drives polarized assembly of ankyrin.

    PubMed

    Jefford, G; Dubreuil, R R

    2000-09-08

    Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.

  4. Scalable multi-sample single-cell data analysis by Partition-Assisted Clustering and Multiple Alignments of Networks

    PubMed Central

    Samusik, Nikolay; Wang, Xiaowei; Guan, Leying; Nolan, Garry P.

    2017-01-01

    Mass cytometry (CyTOF) has greatly expanded the capability of cytometry. It is now easy to generate multiple CyTOF samples in a single study, with each sample containing single-cell measurement on 50 markers for more than hundreds of thousands of cells. Current methods do not adequately address the issues concerning combining multiple samples for subpopulation discovery, and these issues can be quickly and dramatically amplified with increasing number of samples. To overcome this limitation, we developed Partition-Assisted Clustering and Multiple Alignments of Networks (PAC-MAN) for the fast automatic identification of cell populations in CyTOF data closely matching that of expert manual-discovery, and for alignments between subpopulations across samples to define dataset-level cellular states. PAC-MAN is computationally efficient, allowing the management of very large CyTOF datasets, which are increasingly common in clinical studies and cancer studies that monitor various tissue samples for each subject. PMID:29281633

  5. Intrauterine Growth Restriction Affects Cerebellar Granule Cells in the Developing Guinea Pig Brain.

    PubMed

    Tolcos, Mary; McDougall, Annie; Shields, Amy; Chung, Yoonyoung; O'Dowd, Rachael; Turnley, Ann; Wallace, Megan; Rees, Sandra

    2018-01-01

    Intrauterine growth restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. However, the effects of IUGR on the cerebellum are still to be fully elucidated. A major determinant of growth and development of the cerebellum is proliferation and subsequent migration of cerebellar granule cells. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in guinea pigs, results in abnormal cerebellar development due to deficits suggestive of impaired granule cell proliferation and/or migration. CPI was induced by unilateral ligation of the uterine artery at mid-gestation, producing growth-restricted (GR) foetuses at 52 and 60 days of gestation (dg), and neonates at 1 week postnatal age (term approx. 67 dg). Controls were from sham-operated animals. In GR foetuses compared with controls at 52 dg, the external granular layer (EGL) width and internal granular layer (IGL) area were similar. In GR foetuses compared with controls at 60 dg: (a) the EGL width was greater (p < 0.005); (b) the IGL area was smaller (p < 0.005); (c) the density of Ki67-negative (postmitotic) granule cells in the EGL was greater (p < 0.01); (d) the somal area of Purkinje cells was reduced (p < 0.005), and (e) the linear density of Bergmann glia was similar. The EGL width in GR foetuses at 60 dg was comparable to that of 52 dg control and GR foetuses. The pattern of p27-immunoreactivity in the EGL was the inverse of Ki67-immunoreactivity at both foetal ages; there was no difference between control and GR foetuses at either age in the width of p27-immunoreactivity, or in the percentage of the EGL width that it occupied. In the molecular layer of GR neonates compared with controls there was an increase in the areal density of granule cells (p < 0.05) and in the percentage of migrating to total number of granule cells (p < 0.01) at 1 week but not at 60 dg (p > 0.05). Thus, we found no specific evidence that IUGR affects granule cell

  6. Live Dynamics of 53BP1 Foci Following Simultaneous Induction of Clustered and Dispersed DNA Damage in U2OS Cells

    PubMed Central

    Sollazzo, Alice; Brzozowska, Beata; Cheng, Lei; Lundholm, Lovisa; Scherthan, Harry

    2018-01-01

    Cells react differently to clustered and dispersed DNA double strand breaks (DSB). Little is known about the initial reaction to simultaneous induction of DSBs with different complexities. Here, we used live cell microscopy to analyse the behaviour of 53BP1-GFP (green fluorescence protein) foci formation at DSBs induced in U2OS cells by alpha particles, X-rays or mixed beams over a 75 min period post irradiation. X-ray-induced foci rapidly increased and declined over the observation interval. After an initial increase, mixed beam-induced foci remained at a constant level over the observation interval, similarly as alpha-induced foci. The average areas of radiation-induced foci were similar for mixed beams and X-rays, being significantly smaller than those induced by alpha particles. Pixel intensities were highest for mixed beam-induced foci and showed the lowest level of variability over time as compared to foci induced by alphas and X-rays alone. Finally, mixed beam-exposed foci showed the lowest level of mobility as compared to alpha and X-ray exposure. The results suggest paralysation of chromatin around foci containing clustered DNA damage. PMID:29419809

  7. *K-means and cluster models for cancer signatures.

    PubMed

    Kakushadze, Zura; Yu, Willie

    2017-09-01

    We present *K-means clustering algorithm and source code by expanding statistical clustering methods applied in https://ssrn.com/abstract=2802753 to quantitative finance. *K-means is statistically deterministic without specifying initial centers, etc. We apply *K-means to extracting cancer signatures from genome data without using nonnegative matrix factorization (NMF). *K-means' computational cost is a fraction of NMF's. Using 1389 published samples for 14 cancer types, we find that 3 cancers (liver cancer, lung cancer and renal cell carcinoma) stand out and do not have cluster-like structures. Two clusters have especially high within-cluster correlations with 11 other cancers indicating common underlying structures. Our approach opens a novel avenue for studying such structures. *K-means is universal and can be applied in other fields. We discuss some potential applications in quantitative finance.

  8. Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression.

    PubMed

    Pascual, Marta; Abasolo, Ibane; Mingorance-Le Meur, Ana; Martínez, Albert; Del Rio, José A; Wright, Christopher V E; Real, Francisco X; Soriano, Eduardo

    2007-03-20

    We report in this study that, in the cerebellum, the pancreatic transcription factor Ptf1a is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell progenitors in the external GCL (EGL) appear to be unaffected by deletion of Ptf1a. Cell lineage analysis in Ptf1a(Cre/Cre) mice was used to establish that, in the absence of Ptf1a expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that Ptf1a is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that Ptf1a is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.

  9. Voltage-gated calcium channel autoimmune cerebellar degeneration

    PubMed Central

    McKasson, Marilyn; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Carlson, Noel; Bromberg, Mark; Greenlee, John E.

    2016-01-01

    Objectives: To describe response to treatment in a patient with autoantibodies against voltage-gated calcium channels (VGCCs) who presented with autoimmune cerebellar degeneration and subsequently developed Lambert-Eaton myasthenic syndrome (LEMS), and to study the effect of the patient's autoantibodies on Purkinje cells in rat cerebellar slice cultures. Methods: Case report and study of rat cerebellar slice cultures incubated with patient VGCC autoantibodies. Results: A 53-year-old man developed progressive incoordination with ataxic speech. Laboratory evaluation revealed VGCC autoantibodies without other antineuronal autoantibodies. Whole-body PET scans 6 and 12 months after presentation detected no malignancy. The patient improved significantly with IV immunoglobulin G (IgG), prednisone, and mycophenolate mofetil, but worsened after IV IgG was halted secondary to aseptic meningitis. He subsequently developed weakness with electrodiagnostic evidence of LEMS. The patient's IgG bound to Purkinje cells in rat cerebellar slice cultures, followed by neuronal death. Reactivity of the patient's autoantibodies with VGCCs was confirmed by blocking studies with defined VGCC antibodies. Conclusions: Autoimmune cerebellar degeneration associated with VGCC autoantibodies may precede onset of LEMS and may improve with immunosuppressive treatment. Binding of anti-VGCC antibodies to Purkinje cells in cerebellar slice cultures may be followed by cell death. Patients with anti-VGCC autoantibodies may be at risk of irreversible neurologic injury over time, and treatment should be initiated early. PMID:27088118

  10. Autophagy in lurcher mice: indicted but yet to be acquitted for the death of Purkinje cells.

    PubMed

    Yue, Zhenyu

    2010-05-01

    A recent study published in the Journal of Neuroscience by Nishiyama et al., has revisited an autophagy-neurodegeneration model of lurcher (Lc) mice and promoted further discussion regarding the "autophagic cell death" hypothesis. While the study confirmed the previous report by Yue et al., that GluRD2Lc induces autophagy both in vitro and in vivo, it also suggests that GluRD2 (Lc)-mediated autophagy and cell death occur via pathways outside the nPIST-Beclin 1 pathway. For example, the study makes an interesting observation that GluRD2 (Lc)-induced degeneration is associated with energy crisis and an aberrant AMPK activity. The result provides insight into the downstream events induced by GluRD2 (Lc); however, it is not surprising considering that constitutive ion influx caused by the Lc mutation is expected to cause activation of multiple cellular pathways or responses. In conclusion, the authors state that "constitutive ion flux causes cell death with, but not by, autophagy." The conclusion appears consistent with the primary function of autophagy, from an evolutionary point of view, as a survival mechanism.

  11. Self-confinement of finite dust clusters in isotropic plasmas.

    PubMed

    Miloshevsky, G V; Hassanein, A

    2012-05-01

    Finite two-dimensional dust clusters are systems of a small number of charged grains. The self-confinement of dust clusters in isotropic plasmas is studied using the particle-in-cell method. The energetically favorable configurations of grains in plasma are found that are due to the kinetic effects of plasma ions and electrons. The self-confinement phenomenon is attributed to the change in the plasma composition within a dust cluster resulting in grain attraction mediated by plasma ions. This is a self-consistent state of a dust cluster in which grain's repulsion is compensated by the reduced charge and floating potential on grains, overlapped ion clouds, and depleted electrons within a cluster. The common potential well is formed trapping dust clusters in the confined state. These results provide both valuable insights and a different perspective to the classical view on the formation of boundary-free dust clusters in isotropic plasmas.

  12. Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

    PubMed Central

    Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K.; Dancis, Andrew; Pain, Debkumar

    2015-01-01

    Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. PMID:25398879

  13. Cell Therapy From Bench to Bedside Translation in CNS Neurorestoratology Era

    PubMed Central

    Huang, Hongyun; Chen, Lin; Sanberg, Paul

    2010-01-01

    Recent advances in cell biology, neural injury and repair, and the progress towards development of neurorestorative interventions are the basis for increased optimism. Based on the complexity of the processes of demyelination and remyelination, degeneration and regeneration, damage and repair, functional loss and recovery, it would be expected that effective therapeutic approaches will require a combination of strategies encompassing neuroplasticity, immunomodulation, neuroprotection, neurorepair, neuroreplacement, and neuromodulation. Cell-based restorative treatment has become a new trend, and increasing data worldwide have strongly proven that it has a pivotal therapeutic value in CNS disease. Moreover, functional neurorestoration has been achieved to a certain extent in the CNS clinically. Up to now, the cells successfully used in preclinical experiments and/or clinical trial/treatment include fetal/embryonic brain and spinal cord tissue, stem cells (embryonic stem cells, neural stem/progenitor cells, hematopoietic stem cells, adipose-derived adult stem/precursor cells, skin-derived precursor, induced pluripotent stem cells), glial cells (Schwann cells, oligodendrocyte, olfactory ensheathing cells, astrocytes, microglia, tanycytes), neuronal cells (various phenotypic neurons and Purkinje cells), mesenchymal stromal cells originating from bone marrow, umbilical cord, and umbilical cord blood, epithelial cells derived from the layer of retina and amnion, menstrual blood-derived stem cells, Sertoli cells, and active macrophages, etc. Proof-of-concept indicates that we have now entered a new era in neurorestoratology. PMID:21359168

  14. Scoring clustering solutions by their biological relevance.

    PubMed

    Gat-Viks, I; Sharan, R; Shamir, R

    2003-12-12

    A central step in the analysis of gene expression data is the identification of groups of genes that exhibit similar expression patterns. Clustering gene expression data into homogeneous groups was shown to be instrumental in functional annotation, tissue classification, regulatory motif identification, and other applications. Although there is a rich literature on clustering algorithms for gene expression analysis, very few works addressed the systematic comparison and evaluation of clustering results. Typically, different clustering algorithms yield different clustering solutions on the same data, and there is no agreed upon guideline for choosing among them. We developed a novel statistically based method for assessing a clustering solution according to prior biological knowledge. Our method can be used to compare different clustering solutions or to optimize the parameters of a clustering algorithm. The method is based on projecting vectors of biological attributes of the clustered elements onto the real line, such that the ratio of between-groups and within-group variance estimators is maximized. The projected data are then scored using a non-parametric analysis of variance test, and the score's confidence is evaluated. We validate our approach using simulated data and show that our scoring method outperforms several extant methods, including the separation to homogeneity ratio and the silhouette measure. We apply our method to evaluate results of several clustering methods on yeast cell-cycle gene expression data. The software is available from the authors upon request.

  15. The organization of plasticity in the cerebellar cortex: from synapses to control.

    PubMed

    D'Angelo, Egidio

    2014-01-01

    The cerebellum is thought to play a critical role in procedural learning, but the relationship between this function and the underlying cellular and synaptic mechanisms remains largely speculative. At present, at least nine forms of long-term synaptic and nonsynaptic plasticity (some of which are bidirectional) have been reported in the cerebellar cortex and deep cerebellar nuclei. These include long-term potentiation (LTP) and long-term depression at the mossy fiber-granule cell synapse, at the synapses formed by parallel fibers, climbing fibers, and molecular layer interneurons on Purkinje cells, and at the synapses formed by mossy fibers and Purkinje cells on deep cerebellar nuclear cells, as well as LTP of intrinsic excitability in granule cells, Purkinje cells, and deep cerebellar nuclear cells. It is suggested that the complex properties of cerebellar learning would emerge from the distribution of plasticity in the network and from its dynamic remodeling during the different phases of learning. Intrinsic and extrinsic factors may hold the key to explain how the different forms of plasticity cooperate to select specific transmission channels and to regulate the signal-to-noise ratio through the cerebellar cortex. These factors include regulation of neuronal excitation by local inhibitory networks, engagement of specific molecular mechanisms by spike bursts and theta-frequency oscillations, and gating by external neuromodulators. Therefore, a new and more complex view of cerebellar plasticity is emerging with respect to that predicted by the original "Motor Learning Theory," opening issues that will require experimental and computational testing. © 2014 Elsevier B.V. All rights reserved.

  16. Molecular-receptor-specific, non-toxic, near-infrared-emitting Au cluster-protein nanoconjugates for targeted cancer imaging

    NASA Astrophysics Data System (ADS)

    Retnakumari, Archana; Setua, Sonali; Menon, Deepthy; Ravindran, Prasanth; Muhammed, Habeeb; Pradeep, Thalappil; Nair, Shantikumar; Koyakutty, Manzoor

    2010-02-01

    Molecular-receptor-targeted imaging of folate receptor positive oral carcinoma cells using folic-acid-conjugated fluorescent Au25 nanoclusters (Au NCs) is reported. Highly fluorescent Au25 clusters were synthesized by controlled reduction of Au+ ions, stabilized in bovine serum albumin (BSA), using a green-chemical reducing agent, ascorbic acid (vitamin-C). For targeted-imaging-based detection of cancer cells, the clusters were conjugated with folic acid (FA) through amide linkage with the BSA shell. The bioconjugated clusters show excellent stability over a wide range of pH from 4 to 14 and fluorescence efficiency of ~5.7% at pH 7.4 in phosphate buffer saline (PBS), indicating effective protection of nanoclusters by serum albumin during the bioconjugation reaction and cell-cluster interaction. The nanoclusters were characterized for their physico-chemical properties, toxicity and cancer targeting efficacy in vitro. X-ray photoelectron spectroscopy (XPS) suggests binding energies correlating to metal Au 4f7/2~83.97 eV and Au 4f5/2~87.768 eV. Transmission electron microscopy and atomic force microscopy revealed the formation of individual nanoclusters of size ~1 nm and protein cluster aggregates of size ~8 nm. Photoluminescence studies show bright fluorescence with peak maximum at ~674 nm with the spectral profile covering the near-infrared (NIR) region, making it possible to image clusters at the 700-800 nm emission window where the tissue absorption of light is minimum. The cell viability and reactive oxygen toxicity studies indicate the non-toxic nature of the Au clusters up to relatively higher concentrations of 500 µg ml-1. Receptor-targeted cancer detection using Au clusters is demonstrated on FR+ve oral squamous cell carcinoma (KB) and breast adenocarcinoma cell MCF-7, where the FA-conjugated Au25 clusters were found internalized in significantly higher concentrations compared to the negative control cell lines. This study demonstrates the potential of using

  17. Conformational B-cell epitopes prediction from sequences using cost-sensitive ensemble classifiers and spatial clustering.

    PubMed

    Zhang, Jian; Zhao, Xiaowei; Sun, Pingping; Gao, Bo; Ma, Zhiqiang

    2014-01-01

    B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use.

  18. The role of p38 in mitochondrial respiration in male and female mice.

    PubMed

    Ju, Xiaohua; Wen, Yi; Metzger, Daniel; Jung, Marianna

    2013-06-07

    p38 is a mitogen-activated protein kinase and mediates cell growth, cell differentiation, and synaptic plasticity. The aim of this study is to determine the extent to which p38 plays a role in maintaining mitochondrial respiration in male and female mice under a normal condition. To achieve this aim, we have generated transgenic mice that lack p38 in cerebellar Purkinje neurons by crossing Pcp2 (Purkinje cell protein 2)-Cre mice with p38(loxP/loxP) mice. Mitochondria from cerebellum were then isolated from the transgenic and wild-type mice to measure mitochondrial respiration using XF24 respirometer. The mRNA and protein expression of cytochrome c oxidase (COX) in cerebellum were also measured using RT-PCR and immunoblot methods. Separately, HT22 cells were used to determine the involvement of 17β-estradiol (E2) and COX in mitochondrial respiration. The genetic knockout of p38 in Purkinje neurons suppressed the mitochondrial respiration only in male mice and increased COX expression only in female mice. The inhibition of COX by sodium azide (SA) sharply suppressed mitochondrial respiration of HT22 cells in a manner that was protected by E2. These data suggest that p38 is required for the mitochondrial respiration of male mice. When p38 is below a normal level, females may maintain mitochondrial respiration through COX up-regulation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Neuronal and Astrocytic Monoacylglycerol Lipase Limit the Spread of Endocannabinoid Signaling in the Cerebellum.

    PubMed

    Chen, Yao; Liu, Xiaojie; Vickstrom, Casey R; Liu, Michelle J; Zhao, Li; Viader, Andreu; Cravatt, Benjamin F; Liu, Qing-Song

    2016-01-01

    Endocannabinoids are diffusible lipophilic molecules that may spread to neighboring synapses. Monoacylglycerol lipase (MAGL) is the principal enzyme that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG). Using knock-out mice in which MAGL is deleted globally or selectively in neurons and astrocytes, we investigated the extent to which neuronal and astrocytic MAGL limit the spread of 2-AG-mediated retrograde synaptic depression in cerebellar slices. A brief tetanic stimulation of parallel fibers in the molecular layer induced synaptically evoked suppression of excitation (SSE) in Purkinje cells, and both neuronal and astrocytic MAGL contribute to the termination of this form of endocannabinoid-mediated synaptic depression. The spread of SSE among Purkinje cells occurred only after global knock-out of MAGL or pharmacological blockade of either MAGL or glutamate uptake, but no spread was detected following neuron- or astrocyte-specific deletion of MAGL. The spread of endocannabinoid signaling was also influenced by the spatial pattern of synaptic stimulation, because it did not occur at spatially dispersed parallel fiber synapses induced by stimulating the granular layer. The tetanic stimulation of parallel fibers did not induce endocannabinoid-mediated synaptic suppression in Golgi cells even after disruption of MAGL and glutamate uptake, suggesting that heightened release of 2-AG by Purkinje cells does not spread the retrograde signal to parallel fibers that innervate Golgi cells. These results suggest that both neuronal and astrocytic MAGL limit the spatial diffusion of 2-AG and confer synapse-specificity of endocannabinoid signaling.

  20. A Parametric Computational Model of the Action Potential of Pacemaker Cells.

    PubMed

    Ai, Weiwei; Patel, Nitish D; Roop, Partha S; Malik, Avinash; Andalam, Sidharta; Yip, Eugene; Allen, Nathan; Trew, Mark L

    2018-01-01

    A flexible, efficient, and verifiable pacemaker cell model is essential to the design of real-time virtual hearts that can be used for closed-loop validation of cardiac devices. A new parametric model of pacemaker action potential is developed to address this need. The action potential phases are modeled using hybrid automaton with one piecewise-linear continuous variable. The model can capture rate-dependent dynamics, such as action potential duration restitution, conduction velocity restitution, and overdrive suppression by incorporating nonlinear update functions. Simulated dynamics of the model compared well with previous models and clinical data. The results show that the parametric model can reproduce the electrophysiological dynamics of a variety of pacemaker cells, such as sinoatrial node, atrioventricular node, and the His-Purkinje system, under varying cardiac conditions. This is an important contribution toward closed-loop validation of cardiac devices using real-time heart models.