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Sample records for activity results revealed

  1. A highly potent agonist to protease-activated receptor-2 reveals apical activation of the airway epithelium resulting in Ca2+-regulated ion conductance

    PubMed Central

    Sherwood, Cara L.; Daines, Michael O.; Price, Theodore J.; Vagner, Josef

    2014-01-01

    The airway epithelium provides a barrier that separates inhaled air and its various particulates from the underlying tissues. It provides key physiological functions in both sensing the environment and initiating appropriate innate immune defenses to protect the lung. Protease-activated receptor-2 (PAR2) is expressed both apically and basolaterally throughout the airway epithelium. One consequence of basolateral PAR2 activation is the rapid, Ca2+-dependent ion flux that favors secretion in the normally absorptive airway epithelium. However, roles for apically expressed PAR2 activation have not been demonstrated, in part due to the lack of specific, high-potency PAR2 ligands. In the present study, we used the newly developed PAR2 ligand 2at-LIGRLO(PEG3-Pam)-NH2 in combination with well-differentiated, primary cultured airway epithelial cells from wild-type and PAR2−/− mice to examine the physiological role of PAR2 in the conducting airway after apical activation. Using digital imaging microscopy of intracellular Ca2+ concentration changes, we verified ligand potency on PAR2 in primary cultured airway cells. Examination of airway epithelial tissue in an Ussing chamber showed that apical activation of PAR2 by 2at-LIGRLO(PEG3-Pam)-NH2 resulted in a transient decrease in transepithelial resistance that was due to increased apical ion efflux. We determined pharmacologically that this increase in ion conductance was through Ca2+-activated Cl− and large-conductance K+ channels that were blocked with a Ca2+-activated Cl− channel inhibitor and clotrimazole, respectively. Stimulation of Cl− efflux via PAR2 activation at the airway epithelial surface can increase airway surface liquid that would aid in clearing the airway of noxious inhaled agents. PMID:25143347

  2. Everett Weinreb, Photographer, April 1989 FOUNDATION DETAIL REVEALED AS RESULT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Everett Weinreb, Photographer, April 1989 FOUNDATION DETAIL REVEALED AS RESULT OF HOUSE DEMOLITION - Irvine Ranch Agricultural Headquarters, Boyd Tenant House, Southeast of Intersection of San Diego & Santa Ana Freeways, Irvine, Orange County, CA

  3. Deuterium reveals the dynamics of notch activation.

    PubMed

    Raphael, Kopan

    2011-04-13

    Notch activation requires unfolding of a juxtamembrane negative regulatory domain (NRR). Tiyanont et al. (2011) analyzed the dynamics of NRR unfolding in the presence of EGTA. As predicted from the crystal structure and deletion analyses, the lin-Notch repeats unfold first, facilitating access by ADAM proteases. Surprisingly, the heterodimerization domain remains stable.

  4. Small molecules reveal an alternative mechanism of Bax activation

    PubMed Central

    Brahmbhatt, Hetal; Uehling, David; Al-awar, Rima; Leber, Brian; Andrews, David

    2016-01-01

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. PMID:26916338

  5. Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

    PubMed Central

    Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; Hajighasemi, Mahbod; Nocek, Boguslaw; Khusnutdinova, Anna N.; Brown, Greg; Glinos, Julia; Flick, Robert; Skarina, Tatiana; Chernikova, Tatyana N.; Yim, Veronica; Brüls, Thomas; Paslier, Denis Le; Yakimov, Michail M.; Joachimiak, Andrzej; Ferrer, Manuel; Golyshina, Olga V.; Savchenko, Alexei; Golyshin, Peter N.; Yakunin, Alexander F.

    2017-01-01

    Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools. PMID:28272521

  6. Covert Waking Brain Activity Reveals Instantaneous Sleep Depth

    PubMed Central

    McKinney, Scott M.; Dang-Vu, Thien Thanh; Buxton, Orfeu M.; Solet, Jo M.; Ellenbogen, Jeffrey M.

    2011-01-01

    The neural correlates of the wake-sleep continuum remain incompletely understood, limiting the development of adaptive drug delivery systems for promoting sleep maintenance. The most useful measure for resolving early positions along this continuum is the alpha oscillation, an 8–13 Hz electroencephalographic rhythm prominent over posterior scalp locations. The brain activation signature of wakefulness, alpha expression discloses immediate levels of alertness and dissipates in concert with fading awareness as sleep begins. This brain activity pattern, however, is largely ignored once sleep begins. Here we show that the intensity of spectral power in the alpha band actually continues to disclose instantaneous responsiveness to noise—a measure of sleep depth—throughout a night of sleep. By systematically challenging sleep with realistic and varied acoustic disruption, we found that sleepers exhibited markedly greater sensitivity to sounds during moments of elevated alpha expression. This result demonstrates that alpha power is not a binary marker of the transition between sleep and wakefulness, but carries rich information about immediate sleep stability. Further, it shows that an empirical and ecologically relevant form of sleep depth is revealed in real-time by EEG spectral content in the alpha band, a measure that affords prediction on the order of minutes. This signal, which transcends the boundaries of classical sleep stages, could potentially be used for real-time feedback to novel, adaptive drug delivery systems for inducing sleep. PMID:21408616

  7. Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange Mass Spectrometry

    PubMed Central

    Landgraf, Rachelle R.; Goswami, Devrishi; Rajamohan, Francis; Harris, Melissa S.; Calabrese, Matthew; Hoth, Lise R.; Magyar, Rachelle; Pascal, Bruce D.; Chalmers, Michael J.; Busby, Scott A.; Kurumbail, Ravi; Griffin, Patrick R.

    2013-01-01

    Summary AMP-Activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme by hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit suggesting that the molecular binding site of latter resides between the α and β subunits. The distinct short and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands. PMID:24076403

  8. Active medulloblastoma enhancers reveal subgroup-specific cellular origins.

    PubMed

    Lin, Charles Y; Erkek, Serap; Tong, Yiai; Yin, Linlin; Federation, Alexander J; Zapatka, Marc; Haldipur, Parthiv; Kawauchi, Daisuke; Risch, Thomas; Warnatz, Hans-Jörg; Worst, Barbara C; Ju, Bensheng; Orr, Brent A; Zeid, Rhamy; Polaski, Donald R; Segura-Wang, Maia; Waszak, Sebastian M; Jones, David T W; Kool, Marcel; Hovestadt, Volker; Buchhalter, Ivo; Sieber, Laura; Johann, Pascal; Chavez, Lukas; Gröschel, Stefan; Ryzhova, Marina; Korshunov, Andrey; Chen, Wenbiao; Chizhikov, Victor V; Millen, Kathleen J; Amstislavskiy, Vyacheslav; Lehrach, Hans; Yaspo, Marie-Laure; Eils, Roland; Lichter, Peter; Korbel, Jan O; Pfister, Stefan M; Bradner, James E; Northcott, Paul A

    2016-02-04

    Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

  9. Active medulloblastoma enhancers reveal subgroup-specific cellular origins

    PubMed Central

    Lin, Charles Y.; Erkek, Serap; Tong, Yiai; Yin, Linlin; Federation, Alexander J.; Zapatka, Marc; Haldipur, Parthiv; Kawauchi, Daisuke; Risch, Thomas; Warnatz, Hans-Jörg; Worst, Barbara C.; Ju, Bensheng; Orr, Brent A.; Zeid, Rhamy; Polaski, Donald R.; Segura-Wang, Maia; Waszak, Sebastian M.; Jones, David T.W.; Kool, Marcel; Hovestadt, Volker; Buchhalter, Ivo; Sieber, Laura; Johann, Pascal; Chavez, Lukas; Gröschel, Stefan; Ryzhova, Marina; Korshunov, Andrey; Chen, Wenbiao; Chizhikov, Victor V.; Millen, Kathleen J.; Amstislavskiy, Vyacheslav; Lehrach, Hans; Yaspo, Marie-Laure; Eils, Roland; Lichter, Peter; Korbel, Jan O.; Pfister, Stefan M.; Bradner, James E.; Northcott, Paul A.

    2016-01-01

    Summary Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Using H3K27ac and BRD4 ChIP-Seq, coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-Seq, that are responsible for subgroup divergence and implicate candidate cells-of-origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins. PMID:26814967

  10. Active Mars Revealed through HiRISE DTMs and Orthoimages

    NASA Astrophysics Data System (ADS)

    Mattson, Sarah; McEwen, Alfred S.; Bridges, Nathan; Byrne, Shane; Chojnacki, Matthew; Daubar, Ingrid; Dundas, Colin; Russell, Patrick

    2014-11-01

    Before the arrival of the Mars Reconnaissance Orbiter (MRO) with the High-Resolution Imaging Science Experiment (HiRISE), the amount of surface activity on Mars was not well known. HiRISE repeat imaging (often at ~30 cm/pixel), combined with the ability to take stereo images and generate high resolution Digital Terrain Models (DTMs) reveals the many types of surface processes that are currently active on Mars. Examples of active processes on Mars studied with HiRISE data include aeolian activity [Bridges et al., 2012, Nature 485; Chojnacki et al., 2014, Icarus 232], Recurring Slope Lineae (RSL) [McEwen et al., 2011, Science 333; 2014, Nature Geoscience 7], active gullies [Dundas et al., 2012, Icarus 220], polar processes [Hansen et al., 2011, Science 331; Portyankina et al. 2013, AGU], new impacts [Byrne et al., 2009, Science 325; Daubar et al., 2013, Icarus 225; Dundas et al., 2014, JGR 119], and north polar scarp avalanches [Russell et al., 2008, GRL 35, 2014, LPSC]. These studies utilize images from multiple Mars years and seasons. We generate animated gifs with sequences of orthorectified images to analyze temporal changes (see http://www.uahirise.org/sim/). HiRISE DTMs and orthoimages can be used to quantitatively map and record changes in geospatial software. More than 200 DTMs and 400 orthoimages are available through the Planetary Data System (see http://uahirise.org/dtm). Three-band color (blue-green, red, and near infrared) orthoimages are also available in many cases. The ability to monitor the surface of Mars at high spatial and temporal resolution provides insight into seasonal and annual changes, further increasing our understanding of Mars as an active planet.

  11. [Results of 2 years of activity].

    PubMed

    Panigazzi, M

    2010-01-01

    Work-related injuries and occupational diseases are a scourge of modern, western societies, which, although technologically advanced, have difficulty in preventing, treating and rehabilitating victims with speed and efficiency. The current hospital neuromotor rehabilitation centres, whether public or accredited private structures, have notable difficulty in meeting the demand, which despite annual fluctuations and variable needs, does not, overall, seem to be decreasing. We present the results of an organization model developed at the "Fondazione Maugeri" Scientific Institute (Pavia, Italy), the criteria used for the activity, the technological innovations employed to determine ability, and the prospects for further development. This model is effective from a health care-rehabilitative point of view, also in the light of the new legislative scenarios, and is sustainable from an economic points of view; overall it is, therefore, efficient.

  12. Tellurium in active volcanic environments: Preliminary results

    NASA Astrophysics Data System (ADS)

    Milazzo, Silvia; Calabrese, Sergio; D'Alessandro, Walter; Brusca, Lorenzo; Bellomo, Sergio; Parello, Francesco

    2014-05-01

    Tellurium is a toxic metalloid and, according to the Goldschmidt classification, a chalcophile element. In the last years its commercial importance has considerably increased because of its wide use in solar cells, thermoelectric and electronic devices of the last generation. Despite such large use, scientific knowledge about volcanogenic tellurium is very poor. Few previous authors report result of tellurium concentrations in volcanic plume, among with other trace metals. They recognize this element as volatile, concluding that volcanic gases and sulfur deposits are usually enriched with tellurium. Here, we present some results on tellurium concentrations in volcanic emissions (plume, fumaroles, ash leachates) and in environmental matrices (soils and plants) affected by volcanic emissions and/or deposition. Samples were collected at Etna and Vulcano (Italy), Turrialba (Costa Rica), Miyakejima, Aso, Asama (Japan), Mutnovsky (Kamchatka) at the crater rims by using common filtration techniques for aerosols (polytetrafluoroethylene filters). Filters were both eluted with Millipore water and acid microwave digested, and analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Volcanic ashes emitted during explosive events on Etna and Copahue (Argentina) were analyzed for tellurium bulk composition and after leaching experiments to evaluate the soluble fraction of tellurium. Soils and leaves of vegetation were also sampled close to active volcanic vents (Etna, Vulcano, Nisyros, Nyiragongo, Turrialba, Gorely and Masaya) and investigated for tellurium contents. Preliminary results showed very high enrichments of tellurium in volcanic emissions comparing with other volatile elements like mercury, arsenic, thallium and bismuth. This suggests a primary transport in the volatile phase, probably in gaseous form (as also suggested by recent studies) and/or as soluble salts (halides and/or sulfates) adsorbed on the surface of particulate particles and ashes. First

  13. Visualizing disaster attitudes resulting from terrorist activities.

    PubMed

    Khalid, Halimahtun M; Helander, Martin G; Hood, Nilwan A

    2013-09-01

    The purpose of this study was to analyze people's attitudes to disasters by investigating how people feel, behave and think during disasters. We focused on disasters induced by humans, such as terrorist attacks. Two types of textual information were collected - from Internet blogs and from research papers. The analysis enabled forecasting of attitudes for the design of proactive disaster advisory scheme. Text was analyzed using a text mining tool, Leximancer. The outcome of this analysis revealed core themes and concepts in the text concerning people's attitudes. The themes and concepts were sorted into three broad categories: Affect, Behaviour, and Cognition (ABC), and the data was visualized in semantic maps. The maps reveal several knowledge pathways of ABC for developing attitudinal ontologies, which describe the relations between affect, behaviour and cognition, and the sequence in which they develop. Clearly, terrorist attacks induced trauma and people became highly vulnerable.

  14. Cassava root membrane proteome reveals activities during storage root maturation.

    PubMed

    Naconsie, Maliwan; Lertpanyasampatha, Manassawe; Viboonjun, Unchera; Netrphan, Supatcharee; Kuwano, Masayoshi; Ogasawara, Naotake; Narangajavana, Jarunya

    2016-01-01

    Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.

  15. Standardization activities for harmonization of test results.

    PubMed

    Dati, F; Brand, B

    2000-07-01

    In the last years the search for sensitive and specific markers of renal damage and/or renal function has conducted to the development of laboratory assays for measurement of urinary proteins such as albumin, beta(2)-microglobulin, alpha(1)-microglobulin, cystatin C, etc. Furthermore, there have been new applications of already known markers based on different, reformulated methods which often rely on more advanced technologies. It is evident that such developments are connected with analytical and interpretative problems for laboratory managers and clinicians. In this situation, it is essential that international societies develop comprehensive measures for the quality management of these assays and issue uniform and carefully elaborated guidelines to ensure optimal test utilization. International activities are also directed to the development of optimized and standardized methods as well as to the production and evaluation of appropriate reference materials and, finally, to the establishment of appropriate reference ranges and cut-off values for specific analytes. The main use of reference materials is in the transfer of their accurately assigned values to the calibrators of diagnostic companies for calibration of commercially available test systems. These international standardization activities and strategies will allow a harmonized approach to disease management using a more reliable laboratory testing based on quality and value.

  16. Metatranscriptomics reveals overall active bacterial composition in caries lesions

    PubMed Central

    Simón-Soro, Aurea; Guillen-Navarro, Miriam; Mira, Alex

    2014-01-01

    Background Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms. Objective To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay. Design Non-cavitated enamel caries lesions (n=15) and dentin caries lesions samples (n=12) were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition. Results A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.02–0.73% of the total bacterial community. Conclusions The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci detected confirms that they

  17. Activities on Facebook Reveal the Depressive State of Users

    PubMed Central

    Kwak, Jinah

    2013-01-01

    Background As online social media have become prominent, much effort has been spent on identifying users with depressive symptoms in order to aim at early diagnosis, treatment, and even prevention by using various online social media. In this paper, we focused on Facebook to discern any correlations between the platform’s features and users’ depressive symptoms. This work may be helpful in trying to reach and detect large numbers of depressed individuals more easily. Objective Our goal was to develop a Web application and identify depressive symptom–related features from users of Facebook, a popular social networking platform. Methods 55 Facebook users (male=40, female=15, mean age 24.43, SD 3.90) were recruited through advertisement fliers distributed to students in a large university in Korea. Using EmotionDiary, the Facebook application we developed, we evaluated depressive symptoms using the Center for Epidemiological Studies-Depression (CES-D) scale. We also provided tips and facts about depression to participants and measured their responses using EmotionDiary. To identify the Facebook features related to depression, correlation analyses were performed between CES-D and participants’ responses to tips and facts or Facebook social features. Last, we interviewed depressed participants (CES-D≥25) to assess their depressive symptoms by a psychiatrist. Results Facebook activities had predictive power in distinguishing depressed and nondepressed individuals. Participants’ response to tips and facts, which can be explained by the number of app tips viewed and app points, had a positive correlation (P=.04 for both cases), whereas the number of friends and location tags had a negative correlation with the CES-D scale (P=.08 and P=.045 respectively). Furthermore, in finding group differences in Facebook social activities, app tips viewed and app points resulted in significant differences (P=.01 and P=.03 respectively) between probably depressed and

  18. Client satisfaction. Operations research activities and results.

    PubMed

    1998-06-01

    Operations research (OR) is a major component of the Quality Assurance Project's (QAP) strategy for improving the quality of health care delivery worldwide. QAP's Operations Research Program aims to improve the feasibility, utility, and cost-effectiveness of quality assurance strategies in developing countries. QAP and its field partners work to maximize the utility of each field study's findings. As such, the project hopes to disseminate information on all aspects of important OR projects, from the initial design to implementation and results. Over the course of the project, QAP's staff and their partners will develop studies in 16 technical areas. One key area of interest is the study of client satisfaction with health care delivery. The project currently has two major studies on client satisfaction underway in Niger and Peru. Phase one results from the Niger research and QAP and the Max Salud Institute in Peru are discussed.

  19. Revealing phosphoproteins playing role in tobacco pollen activated in vitro.

    PubMed

    Fíla, Jan; Matros, Andrea; Radau, Sonja; Zahedi, René Peiman; Capková, Věra; Mock, Hans-Peter; Honys, David

    2012-11-01

    The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.

  20. Single cell activity reveals direct electron transfer in methanotrophic consortia

    NASA Astrophysics Data System (ADS)

    McGlynn, Shawn E.; Chadwick, Grayson L.; Kempes, Christopher P.; Orphan, Victoria J.

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  1. Neural activity reveals perceptual grouping in working memory.

    PubMed

    Rabbitt, Laura R; Roberts, Daniel M; McDonald, Craig G; Peterson, Matthew S

    2017-03-01

    There is extensive evidence that the contralateral delay activity (CDA), a scalp recorded event-related brain potential, provides a reliable index of the number of objects held in visual working memory. Here we present evidence that the CDA not only indexes visual object working memory, but also the number of locations held in spatial working memory. In addition, we demonstrate that the CDA can be predictably modulated by the type of encoding strategy employed. When individual locations were held in working memory, the pattern of CDA modulation mimicked previous findings for visual object working memory. Specifically, CDA amplitude increased monotonically until working memory capacity was reached. However, when participants were instructed to group individual locations to form a constellation, the CDA was prolonged and reached an asymptote at two locations. This result provides neural evidence for the formation of a unitary representation of multiple spatial locations.

  2. Structure-activity relationships in carbohydrates revealed by their hydration.

    PubMed

    Maugeri, Laura; Busch, Sebastian; McLain, Sylvia E; Pardo, Luis Carlos; Bruni, Fabio; Ricci, Maria Antonietta

    2016-12-21

    One of the more intriguing aspects of carbohydrate chemistry is that despite having very similar molecular structures, sugars have very different properties. For instance, there is a sensible difference in sweet taste between glucose and trehalose, even though trehalose is a disaccharide that comprised two glucose units, suggesting a different ability of these two carbohydrates to bind to sweet receptors. Here we have looked at the hydration of specific sites and at the three-dimensional configuration of water molecules around three carbohydrates (glucose, cellobiose, and trehalose), combining neutron diffraction data with computer modelling. Results indicate that identical chemical groups can have radically different hydration patterns depending on their location on a given molecule. These differences can be linked with the specific activity of glucose, cellobiose, and trehalose as a sweet substance, as building block of cellulose fiber, and as a bioprotective agent, respectively. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

  3. Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants

    PubMed Central

    Llauro, Christel; Jobet, Edouard; Robakowska-Hyzorek, Dagmara; Lasserre, Eric; Ghesquière, Alain; Panaud, Olivier

    2017-01-01

    Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes. PMID:28212378

  4. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

    Bobrovsky, Pavel; Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-07-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection.

  5. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity

    PubMed Central

    Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-01-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis. Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection. PMID:27160295

  6. Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation.

    PubMed

    Cho, Joo-Youn; Kang, Dong Wook; Ma, Xiaochao; Ahn, Sung-Hoon; Krausz, Kristopher W; Luecke, Hans; Idle, Jeffrey R; Gonzalez, Frank J

    2009-05-01

    Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.

  7. Enceladus's activity as revealed by Cassini-Huygens

    NASA Astrophysics Data System (ADS)

    Schmidt, Juergen

    2015-08-01

    The activity of Enceladus has been monitored by Cassini for nearly one decade after its discovery (see Science, 2006, 311, special issue). Thus, crucial properties of the vapor and dust plumes, heat output, surface properties, and the gravity field of the satellite are constrained in a fairly detailed manner. In this paper I review key observational facts and discuss implications for the vent geometries as well as interior structure and composition. Special emphasize I will give to data recorded by the Cassini Cosmic Dust Analyzer, and the conclusions drawn from it, concerning the number, size, and composition of grains ejected by the plumes associated with the south polar activity.

  8. Revealing Student Blogging Activities Using RSS Feeds and LMS Logs

    ERIC Educational Resources Information Center

    Derntl, Michael

    2010-01-01

    Blogs are an easy-to-use, free alternative to classic means of computer-mediated communication. Moreover, they are authentically aligned with web activity patterns of today's students. The body of studies on integrating and implementing blogs in various educational settings has grown rapidly recently; however, it is often difficult to distill…

  9. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

    PubMed

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J; Adams, David J; Leung, Hing Y

    2016-07-19

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition.

  10. Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer

    PubMed Central

    Ahmad, Imran; Mui, Ernest; Galbraith, Laura; Patel, Rachana; Tan, Ee Hong; Salji, Mark; Rust, Alistair G.; Repiscak, Peter; Hedley, Ann; Markert, Elke; Loveridge, Carolyn; van der Weyden, Louise; Edwards, Joanne; Sansom, Owen J.; Adams, David J.; Leung, Hing Y.

    2016-01-01

    Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition. PMID:27357679

  11. Metaproteomic analysis reveals microbial metabolic activities in the deep ocean

    NASA Astrophysics Data System (ADS)

    Wang, Da-Zhi; Xie, Zhang-Xian; Zhang, Shu-Feng; Wang, Ming-Hua; Zhang, Hao; Kong, Ling-Fen; Lin, Lin

    2016-04-01

    The deep sea is the largest habitat on earth and holds many and varied microbial life forms. However, little is known about their metabolic activities in the deep ocean. Here, we characterized protein profiles of particulate (>0.22 μm) and dissolved (between 10 kDa and 0.22 μm) fractions collected from the deep South China Sea using a shotgun proteomic approach. SAR324, Alteromonadales and SAR11 were the most abundant groups, while Prasinophyte contributed most to eukaryotes and cyanophage to viruses. The dominant heterotrophic activity was evidenced by the abundant transporters (33%). Proteins participating in nitrification, methanogenesis, methyltrophy and CO2 fixation were detected. Notably, the predominance of unique cellular proteins in dissolved fraction suggested the presence of membrane structures. Moreover, the detection of translation proteins related to phytoplankton indicated that other process rather than sinking particles might be the downward export of living cells. Our study implied that novel extracellular activities and the interaction of deep water with its overlying water could be crucial to the microbial world of deep sea.

  12. Active Interaction Mapping Reveals the Hierarchical Organization of Autophagy.

    PubMed

    Kramer, Michael H; Farré, Jean-Claude; Mitra, Koyel; Yu, Michael Ku; Ono, Keiichiro; Demchak, Barry; Licon, Katherine; Flagg, Mitchell; Balakrishnan, Rama; Cherry, J Michael; Subramani, Suresh; Ideker, Trey

    2017-02-16

    We have developed a general progressive procedure, Active Interaction Mapping, to guide assembly of the hierarchy of functions encoding any biological system. Using this process, we assemble an ontology of functions comprising autophagy, a central recycling process implicated in numerous diseases. A first-generation model, built from existing gene networks in Saccharomyces, captures most known autophagy components in broad relation to vesicle transport, cell cycle, and stress response. Systematic analysis identifies synthetic-lethal interactions as most informative for further experiments; consequently, we saturate the model with 156,364 such measurements across autophagy-activating conditions. These targeted interactions provide more information about autophagy than all previous datasets, producing a second-generation ontology of 220 functions. Approximately half are previously unknown; we confirm roles for Gyp1 at the phagophore-assembly site, Atg24 in cargo engulfment, Atg26 in cytoplasm-to-vacuole targeting, and Ssd1, Did4, and others in selective and non-selective autophagy. The procedure and autophagy hierarchy are at http://atgo.ucsd.edu/.

  13. Oscillatory Brain Activity Reveals Linguistic Prints in the Quantity Code

    PubMed Central

    Salillas, Elena; Barraza, Paulo; Carreiras, Manuel

    2015-01-01

    Number representations change through education, although it is currently unclear whether and how language could impact the magnitude representation that we share with other species. The most prominent view is that language does not play any role in modulating the core numeric representation involved in the contrast of quantities. Nevertheless, possible cultural hints on the numerical magnitude representation are currently on discussion focus. In fact, the acquisition of number words provides linguistic input that the quantity system may not ignore. Bilingualism offers a window to the study of this question, especially in bilinguals where the two number wording systems imply also two different numerical systems, such as in Basque-Spanish bilinguals. The present study evidences linguistic prints in the core number representational system through the analysis of EEG oscillatory activity during a simple number comparison task. Gamma band synchronization appears when Basque-Spanish bilinguals compare pairs of Arabic numbers linked through the Basque base-20 wording system, but it does not if the pairs are related through the base-10 system. Crucially, this gamma activity, originated in a left fronto-parietal network, only appears in bilinguals who learned math in Basque and not in equivalent proficiency bilinguals who learned math in Spanish. Thus, this neural index reflected in gamma band synchrony appears to be triggered by early learning experience with the base-20 numerical associations in Basque number words. PMID:25875210

  14. Metatranscriptomic Analysis of Groundwater Reveals an Active Anammox Bacterial Population

    NASA Astrophysics Data System (ADS)

    Jewell, T. N. M.; Karaoz, U.; Thomas, B. C.; Banfield, J. F.; Brodie, E.; Williams, K. H.; Beller, H. R.

    2014-12-01

    Groundwater is a major natural resource, yet little is known about the contribution of microbial anaerobic ammonium oxidation (anammox) activity to subsurface nitrogen cycling. During anammox, energy is generated as ammonium is oxidized under anaerobic conditions to dinitrogen gas, using nitrite as the final electron acceptor. This process is a global sink for fixed nitrogen. Only a narrow range of monophyletic bacteria within the Planctomycetes carries out anammox, and the full extent of their metabolism, and subsequent impact on nitrogen cycling and microbial community structure, is still unknown. Here, we employ a metatranscriptomic analysis on enriched mRNA to identify the abundance and activity of a population of anammox bacteria within an aquifer at Rifle, CO. Planktonic biomass was collected over a two-month period after injection of up to 1.5 mM nitrate. Illumina-generated sequences were mapped to a phylogenetically binned Rifle metagenome database. We identified transcripts for genes with high protein sequence identities (81-98%) to those of anammox strain KSU-1 and to two of the five anammox bacteria genera, Brocadia and Kuenenia, suggesting an active, if not diverse, anammox population. Many of the most abundant anammox transcripts mapped to a single scaffold, indicative of a single dominant anammox species. Transcripts of the genes necessary for the anammox pathway were present, including an ammonium transporter (amtB), nitrite/formate transporter, nitrite reductase (nirK), and hydrazine oxidoreductase (hzoB). The form of nitrite reductase encoded by anammox is species-dependent, and we only identified nirK, with no evidence of anammox nirS. In addition to the anammox pathway we saw evidence of the anammox bacterial dissimilatory nitrate reduction to ammonium pathway (narH, putative nrfA, and nrfB), which provides an alternate means of generating substrates for anammox from nitrate, rather than relying on an external pool. Transcripts for hydroxylamine

  15. Long-term home cage activity scans reveal lowered exploratory behaviour in symptomatic female Rett mice.

    PubMed

    Robinson, Lianne; Plano, Andrea; Cobb, Stuart; Riedel, Gernot

    2013-08-01

    Numerous experimental models have been developed to reiterate endophenotypes of Rett syndrome, a neurodevelopmental disorder with a multitude of motor, cognitive and vegetative symptoms. Here, female Mecp2(Stop) mice [1] were characterised at mild symptomatic conditions in tests for anxiety (open field, elevated plus maze) and home cage observation systems for food intake, locomotor activity and circadian rhythms. Aged 8-9 months, Mecp2(Stop) mice presented with heightened body weight, lower overall activity in the open field, but no anxiety phenotype. Although home cage activity scans conducted in two different observation systems, PhenoMaster and PhenoTyper, confirmed normal circadian activity, they revealed severely compromised habituation to a novel environment in all parameters registered including those derived from a non-linear decay model such as initial exploration maximum, decay half-life of activity and span, as well as plateau. Furthermore, overall activity was significantly reduced in nocturnal periods due to reductions in both fast ambulatory movements, but also a slow lingering. In contrast, light-period activity profiles during which the amount of sleep was highest remained normal in Mecp2(Stop) mice. These data confirm the slow and progressive development of Rett-like symptoms in female Mecp2(Stop) mice resulting in a prominent reduction of overall locomotor activity, while circadian rhythms are maintained. Alterations in the time-course of habituation may indicate deficiencies in cognitive processing.

  16. Revealing the nature of the active site on the carbon catalyst for C-H bond activation.

    PubMed

    Sun, XiaoYing; Li, Bo; Su, Dangsheng

    2014-09-28

    A reactivity descriptor for the C-H bond activation on the nanostructured carbon catalyst is proposed. Furthermore the calculations reveal that the single ketone group can be an active site in ODH reaction.

  17. Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

    PubMed Central

    Mofford, David M.; Reddy, Gadarla Randheer; Miller, Stephen C.

    2014-01-01

    Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation. PMID:24616520

  18. Quantitative and Temporal Requirements Revealed for Zap-70 Catalytic Activity During T Cell Development

    PubMed Central

    Au-Yeung, Byron B.; Melichar, Heather J.; Ross, Jenny O.; Cheng, Debra A.; Zikherman, Julie; Shokat, Kevan M.; Robey, Ellen A.; Weiss, Arthur

    2014-01-01

    The catalytic activity of Zap-70 is crucial for T cell receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap-70 catalytic activity in a model of synchronized thymic selection, we showed that CD4+CD8+ thymocytes integrate multiple, transient, Zap-70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas one hour of signaling was sufficient for negative selection. Titration of Zap-70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, revealing heterogeneity, even among CD4+CD8+ thymocytes expressing identical TCRs undergoing positive selection. PMID:24908390

  19. Integrative analysis of breast cancer reveals prognostic haematopoietic activity and patient-specific immune response profiles

    PubMed Central

    Varn, Frederick S.; Andrews, Erik H.; Mullins, David W.; Cheng, Chao

    2016-01-01

    Transcriptional programmes active in haematopoietic cells enable a variety of functions including dedifferentiation, innate immunity and adaptive immunity. Understanding how these programmes function in the context of cancer can provide valuable insights into host immune response, cancer severity and potential therapy response. Here we present a method that uses the transcriptomes of over 200 murine haematopoietic cells, to infer the lineage-specific haematopoietic activity present in human breast tumours. Correlating this activity with patient survival and tumour purity reveals that the transcriptional programmes of many cell types influence patient prognosis and are found in environments of high lymphocytic infiltration. Collectively, these results allow for a detailed and personalized assessment of the patient immune response to a tumour. When combined with routinely collected patient biopsy genomic data, this method can enable a richer understanding of the complex interplay between the host immune system and cancer. PMID:26725977

  20. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    PubMed Central

    Mertsalov, Ilya B.; Novikov, Boris N.; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M.

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  1. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.

  2. Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism.

    PubMed

    Fan, Ying-Xin; Wong, Lily; Ding, Jinhui; Spiridonov, Nikolay A; Johnson, Richard C; Johnson, Gibbes R

    2008-01-18

    Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

  3. Crystal Structure of Escherichia coli Diaminopropionate Ammonia-lyase Reveals Mechanism of Enzyme Activation and Catalysis*

    PubMed Central

    Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R.; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N.; Savithri, Handanahal S.; Murthy, Mathur R. N.

    2012-01-01

    Pyridoxal 5′-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp120 and Lys77 is suggested. PMID:22505717

  4. Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

    PubMed

    Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N; Savithri, Handanahal S; Murthy, Mathur R N

    2012-06-08

    Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

  5. Revealing Time-Unlocked Brain Activity from MEG Measurements by Common Waveform Estimation

    PubMed Central

    Takeda, Yusuke; Yamanaka, Kentaro; Yamagishi, Noriko; Sato, Masa-aki

    2014-01-01

    Brain activities related to cognitive functions, such as attention, occur with unknown and variable delays after stimulus onsets. Recently, we proposed a method (Common Waveform Estimation, CWE) that could extract such brain activities from magnetoencephalography (MEG) or electroencephalography (EEG) measurements. CWE estimates spatiotemporal MEG/EEG patterns occurring with unknown and variable delays, referred to here as unlocked waveforms, without hypotheses about their shapes. The purpose of this study is to demonstrate the usefulness of CWE for cognitive neuroscience. For this purpose, we show procedures to estimate unlocked waveforms using CWE and to examine their role. We applied CWE to the MEG epochs during Go trials of a visual Go/NoGo task. This revealed unlocked waveforms with interesting properties, specifically large alpha oscillations around the temporal areas. To examine the role of the unlocked waveform, we attempted to estimate the strength of the brain activity of the unlocked waveform in various conditions. We made a spatial filter to extract the component reflecting the brain activity of the unlocked waveform, applied this spatial filter to MEG data under different conditions (a passive viewing, a simple reaction time, and Go/NoGo tasks), and calculated the powers of the extracted components. Comparing the powers across these conditions suggests that the unlocked waveforms may reflect the inhibition of the task-irrelevant activities in the temporal regions while the subject attends to the visual stimulus. Our results demonstrate that CWE is a potential tool for revealing new findings of cognitive brain functions without any hypothesis in advance. PMID:24879410

  6. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  7. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  8. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

  9. Plutonium recycle test reactor characterization activities and results

    SciTech Connect

    Cornwell, B.C.

    1997-05-01

    Report contains results of PRTR core and associated structures characterization performed in January and February of 1997. Radiation survey data are presented, along with recommendations for stabilization activities before transitioning to a decontamination and decommissioning function. Recommendations are also made about handling the waste generated by the stabilization activities, and actions suggested by the Decontamination and Decommissioning organization.

  10. Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

    PubMed Central

    Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

    2016-01-01

    Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

  11. Brain Network Activation (BNA) reveals scopolamine-induced impairment of visual working memory.

    PubMed

    Reches, Amit; Levy-Cooperman, Naama; Laufer, Ilan; Shani-Hershkovitch, Revital; Ziv, Keren; Kerem, Dani; Gal, Noga; Stern, Yaki; Cukierman, Guy; Romach, Myroslava K; Sellers, Edward M; Geva, Amir B

    2014-09-01

    The overarching goal of this event-related potential (ERP) study was to examine the effects of scopolamine on the dynamics of brain network activation using a novel ERP network analysis method known as Brain Network Activation (BNA). BNA was used for extracting group-common stimulus-activated network patterns elicited to matching probe stimuli in the context of a delayed matching-to-sample task following placebo and scopolamine treatments administered to healthy participants. The BNA extracted networks revealed the existence of two pathophysiological mechanisms following scopolamine, disconnection, and compensation. Specifically, weaker frontal theta and parietal alpha coupling was accompanied with enhanced fronto-centro-parietal theta activation relative to placebo. In addition, using the characteristic BNA network of each treatment as well as corresponding literature-guided selective subnetworks as combined biomarkers managed to differentiate between individual responses to each of the treatments. Behavioral effects associated with scopolamine included delayed response time and impaired response accuracy. These results indicate that the BNA method is sensitive to the effects of scopolamine on working memory and that it may potentially enable diagnosis and treatment assessment of dysfunctions associated with cholinergic deficiency.

  12. Pluto Revealed: First Results from the Historic 1st Fly-By Space Mission

    NASA Technical Reports Server (NTRS)

    Smith, Kimberly Ennico

    2015-01-01

    On July 14, 2015, after a 9.5 year trek across the solar system, NASAs New Horizons spacecraft successfully flew by the dwarf planet Pluto and its system of moons, taking imagery, spectra and in-situ particle data. Data obtained by New Horizons will address numerous outstanding questions on the geology and composition of Pluto and Charon, plus measurements of Plutos atmosphere, and provide revised understanding of the formation and evolution of Pluto and Charon and its smaller moons. This data set is an invaluable glimpse into the outer Third Zone of the Solar System. Data from the intense July 14th fly-by sequence will be downlinked to Earth over a period of 16 months, the duration set by the large data set (over 60 GBits), tempered by limited transmission bandwidth rates (1-2 kbps) and sharing the three 70m DSN assets. This presentation summarizes the New Horizons mission and early science results.

  13. Lithospheric geometries revealed through electromagnetic imaging: SAMTEX (Southern Africa MagnetoTelluric Experiment) observations and results

    NASA Astrophysics Data System (ADS)

    Jones, A. G.; Muller, M. R.; Evans, R. L.; Miensopust, M. P.; Khoza, D. T.; Samtex Team

    2011-12-01

    The Southern African Magnetotelluric Experiment (SAMTEX) is imaging the properties and geometries of the lithosphere below southern Africa to depths of 200+ km. Electrical conductivity is highly sensitive to ambient temperature, and to the presence of an interconnected conducting phase, such as a solid phase like graphite or sulphides, a fluid phase like partial melt, or bound water through hydrogen diffusion. Thus, primary geometrical information can be readily obtained from lithospheric-scale MT experiments about the three-dimensional variation in conductivity that can be related to formation and deformation processes. One important piece of information easily obtained from MT data is the depth to the lithosphere-asthenosphere boundary (LAB), due to the sensitivity of conductivity to small fractions (<1%) of partial melt and/or of some hundreds of ppm of bound water. SAMTEX measurements have been made at a total of more than 750 MT sites over an area in excess of a million square kilometres, making it by far the largest-ever regional MT project undertaken. One of the most significant results from SAMTEX is the mapping of the LAB beneath the Archean cratons and bounding mobile belts of Southern Africa, particularly of the previously unknown regions of Namibia and Botswana. The LAB is shallow (150 km) beneath the mobile belts, deep (250 km) in the centres of the cratons, and transitional at the edges. Diamondiferous kimberlites are located primarily where lithosphere is transitional in thickness, or where there is a change in its anisotropy properties, both of which are craton edge effects. The electrical properties of the continental mantle derived from SAMTEX data can be compared with seismic ones derived from data from the South African Seismic Experiment (SASE) of the Kaapvaal Project. Generally there is very good predictive linear agreement between seismic velocity and log(conductivity), indicative of both being influenced by the same bulk property factors

  14. Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity.

    PubMed

    Mangkalanan, Seksan; Sanguanrat, Piyachat; Utairangsri, Tanatchaporn; Sritunyalucksana, Kallaya; Krittanai, Chartchai

    2014-05-01

    This study focused on an isolation and characterization of the circulating hemocytes in mud crab, Scylla olivacea. Isolation of specific cell types of hemocytes from crab hemolymph was accomplished by using 60% Percoll density gradient centrifugation. Four separated bands of the hemocytes were successfully obtained. Characterization of these isolated hemocytes by light microscope using trypan blue-rose bengal staining, rose bengal-hematoxilin staining, and phase contrast revealed four distinct types of hemocyte cells. Using their specific morphology and granularity, they were identified as hyaline cell (HC), small granular cell (SGC), large granular cell (LGC) and mixed granular cell (MGC). Transmission electron microscopy (TEM) revealed more details on specific cell size, size of cytoplasmic granule, and nuclear to cytoplasmic ratio, and confirmed the classification. Relative abundance of these cells types in the hemolymph of an adult crab were 15.50±8.22% for HC, 55.50±7.15% for SGC, 13.50±5.28% for LGC, and 15.50±3.50% for MGC. Proteomic analysis of protein expression for each specific cell types by two-dimensional electrophoresis identified two highly abundant proteins, prophenoloxidase (ProPO) and peroxinectin in LGC. Determination of phenoloxidase (PO) activity in each isolated cell types using in vitro and in situ chemical assays confirmed the presence of PO activity only in LGC. Based on an increased PO activity of crab hemolymph during the course of White Spot Syndrome Virus (WSSV) infection, these results suggest that prophenoloxidase pathway was employed for host defense mechanism against WSSV and it may link to the role of large granular hemocyte.

  15. Structural Differences between Active Forms of Plasminogen Activator Inhibitor Type 1 Revealed by Conformationally Sensitive Ligands*

    PubMed Central

    Li, Shih-Hon; Gorlatova, Natalia V.; Lawrence, Daniel A.; Schwartz, Bradford S.

    2008-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) in which the reactive center loop (RCL) spontaneously inserts into a central β-sheet, β-sheet A, resulting in inactive inhibitor. Available x-ray crystallographic studies of PAI-1 in an active conformation relied on the use of stabilizing mutations. Recently it has become evident that these structural models do not adequately explain the behavior of wild-type PAI-1 (wtPAI-1) in solution. To probe the structure of native wtPAI-1, we used three conformationally sensitive ligands: the physiologic cofactor, vitronectin; a monoclonal antibody, 33B8, that binds preferentially to RCL-inserted forms of PAI-1; and RCL-mimicking peptides that insert into β-sheet A. From patterns of interaction with wtPAI-1 and the stable mutant, 14-1B, we propose a model of the native conformation of wtPAI-1 in which the bottom of the central sheet is closed, whereas the top of the β-sheet A is open to allow partial insertion of the RCL. Because the incorporation of RCL-mimicking peptides into wtPAI-1 is accelerated by vitronectin, we further propose that vitronectin alters the conformation of the RCL to allow increased accessibility to β-sheet A, yielding a structural hypothesis that is contradictory to the current structural model of PAI-1 in solution and its interaction with vitronectin. PMID:18436534

  16. Abnormal Spontaneous Brain Activity in Women with Premenstrual Syndrome Revealed by Regional Homogeneity

    PubMed Central

    Liao, Hai; Pang, Yong; Liu, Peng; Liu, Huimei; Duan, Gaoxiong; Liu, Yanfei; Tang, Lijun; Tao, Jien; Wen, Danhong; Li, Shasha; Liang, Lingyan; Deng, Demao

    2017-01-01

    Background: Previous studies have revealed that the etiologies of premenstrual syndrome (PMS) refer to menstrual cycle related brain changes. However, its intrinsic neural mechanism is still unclear. The aim of the present study was to assess abnormal spontaneous brain activity and to explicate the intricate neural mechanism of PMS using resting state functional magnetic resonance imaging (RS-fMRI). Materials and Methods: The data of 20 PMS patients (PMS group) and 21 healthy controls (HC group) were analyzed by regional homogeneity (ReHo) method during the late luteal phase of menstrual cycle. In addition, all the participants were asked to complete a daily record of severity of problems (DRSP) questionnaire. Results: Compared with HC group, the results showed that PMS group had increased ReHo mainly in the bilateral precuneus, left inferior temporal cortex (ITC), right inferior frontal cortex (IFC) and left middle frontal cortex (MFC) and decreased ReHo in the right anterior cingulate cortex (ACC) at the luteal phase. Moreover, the PMS group had higher DRSP scores, and the DRSP scores positively correlated with ReHo in left MFC and negatively correlated with ReHo in the right ACC. Conclusion: Our results suggest that abnormal spontaneous brain activity is found in PMS patients and the severity of symptom is specifically related to the left MFC and right ACC. The present findings may be beneficial to explicate the intricate neural mechanism of PMS. PMID:28243196

  17. Analysis of miRNA market trends reveals hotspots of research activity.

    PubMed

    Oosta, Gary; Razvi, Enal

    2012-04-01

    We have conducted an analysis of the miRNA research marketplace by evaluating the publication trends in the field. In this article, we present the results of our analysis which reveals that hotspots exist in terms of research activities in the miRNA space--these hotspots illustrate the areas in the miRNA research space where specific miRNAs have been extensively studied, and other areas that represent new territory. We frame these data into the context of areas of opportunity for miRNA content harvest versus segments of opportunity for the development of research tools. Also presented in this article are the primary market data from online surveys we have performed with researchers involved in miRNA research around the world. Taken together, these data frame the current state of the miRNA marketplace and provide niches of opportunity for new entrants into this space.

  18. Nanoscale electrochemical patterning reveals the active sites for catechol oxidation at graphite surfaces.

    PubMed

    Patel, Anisha N; McKelvey, Kim; Unwin, Patrick R

    2012-12-19

    Graphite-based electrodes (graphite, graphene, and nanotubes) are used widely in electrochemistry, and there is a long-standing view that graphite step edges are needed to catalyze many reactions, with the basal surface considered to be inert. In the present work, this model was tested directly for the first time using scanning electrochemical cell microscopy reactive patterning and shown to be incorrect. For the electro-oxidation of dopamine as a model process, the reaction rate was measured at high spatial resolution across a surface of highly oriented pyrolytic graphite. Oxidation products left behind in a pattern defined by the scanned electrochemical cell served as surface-site markers, allowing the electrochemical activity to be correlated directly with the graphite structure on the nanoscale. This process produced tens of thousands of electrochemical measurements at different locations across the basal surface, unambiguously revealing it to be highly electrochemically active, with step edges providing no enhanced activity. This new model of graphite electrodes has significant implications for the design of carbon-based biosensors, and the results are additionally important for understanding electrochemical processes on related sp(2)-hybridized materials such as pristine graphene and nanotubes.

  19. Stepwise multiphoton activation fluorescence reveals a new method of melanin detection.

    PubMed

    Lai, Zhenhua; Kerimo, Josef; Mega, Yair; Dimarzio, Charles A

    2013-06-01

    The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

  20. Community Lenses Revealing the Role of Sociocultural Environment on Physical Activity

    PubMed Central

    Belon, Ana Paula; Nieuwendyk, Laura M.; Vallianatos, Helen; Nykiforuk, Candace I. J.

    2016-01-01

    Purpose To identify perceptions of how sociocultural environment enabled and hindered physical activity (PA) participation. Design Community-based participatory research. Setting Two semirural and two urban communities located in Alberta, Canada. Participants Thirty-five people (74.3% females, 71.4% aged 25–64 years) across the four communities. Method PhotoVoice activities occurred over 3 months during the spring of 2009. Participants were asked to document perceived environmental attributes that might foster or inhibit PA in their community. Photographs and narratives were shared in one-on-one interviews. Line-by-line coding of the transcripts was independently conducted by two researchers using an inductive approach. Codes were arranged into themes and subthemes, which were then organized into the Analysis Grid for Environments Linked to Obesity (ANGELO) framework. Results Six main themes (accompanied by subthemes) emerged: sociocultural aesthetics, safety, social involvement, PA motivation, cultural ideas of recreation, and car culture. Representative quotes and photographs illustrate enablers and obstacles identified by participants. Conclusion This PhotoVoice study revealed how aspects of participants’ sociocultural environments shaped their decisions to be physically active. Providing more PA resources is only one step in the promotion of supportive environments. Strategies should also account for the beautification and maintenance of communities, increasing feelings of safety, enhancement of social support among community members, popularization of PA, and mitigating car culture, among others. PMID:25973966

  1. What Do the California Standards Test Results Reveal about the Movement toward Eighth-Grade Algebra for All?

    ERIC Educational Resources Information Center

    Liang, Jian-Hua; Heckman, Paul E.; Abedi, Jamal

    2012-01-01

    In California, an increasing number of 8th graders have taken algebra courses since 2003. This study examines students' California Standards Test (CST) results in grades 7 through 11, aiming to reveal who took the CST for Algebra I in 8th grade and whether the increase has led to a rise in students' taking higher-level mathematics CSTs and an…

  2. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

    PubMed

    Tan, Haiyan; Yang, Kai; Li, Yuxin; Shaw, Timothy I; Wang, Yanyan; Blanco, Daniel Bastardo; Wang, Xusheng; Cho, Ji-Hoon; Wang, Hong; Rankin, Sherri; Guy, Cliff; Peng, Junmin; Chi, Hongbo

    2017-03-21

    The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

  3. Active Aging Promotion: Results from the Vital Aging Program

    PubMed Central

    Caprara, Mariagiovanna; Molina, María Ángeles; Schettini, Rocío; Santacreu, Marta; Orosa, Teresa; Mendoza-Núñez, Víctor Manuel; Rojas, Macarena; Fernández-Ballesteros, Rocío

    2013-01-01

    Active aging is one of the terms in the semantic network of aging well, together with others such as successful, productive, competent aging. All allude to the new paradigm in gerontology, whereby aging is considered from a positive perspective. Most authors in the field agree active aging is a multidimensional concept, embracing health, physical and cognitive fitness, positive affect and control, social relationships and engagement. This paper describes Vital Aging, an individual active aging promotion program implemented through three modalities: Life, Multimedia, and e-Learning. The program was developed on the basis of extensive evidence about individual determinants of active aging. The different versions of Vital Aging are described, and four evaluation studies (both formative and summative) are reported. Formative evaluation reflected participants' satisfaction and expected changes; summative evaluations yielded some quite encouraging results using quasi-experimental designs: those who took part in the programs increased their physical exercise, significantly improved their diet, reported better memory, had better emotional balance, and enjoyed more cultural, intellectual, affective, and social activities than they did before the course, thus increasing their social relationships. These results are discussed in the context of the common literature within the field and, also, taking into account the limitations of the evaluations accomplished. PMID:23476644

  4. A Global Genomic Screening Strategy Reveals Diverse Activators of Constitutive Activated Receptor (CAR)

    EPA Science Inventory

    A comprehensive survey of conditions that activate CAR in the mouse liver has not been carried out but would be useful in understanding their impact on CAR-dependent liver tumor induction. A gene signature dependent on CAR activation was identified by comparing the transcript pr...

  5. Angiogenesis Interactome and Time Course Microarray Data Reveal the Distinct Activation Patterns in Endothelial Cells

    PubMed Central

    Chu, Liang-Hui; Lee, Esak; Bader, Joel S.; Popel, Aleksander S.

    2014-01-01

    Angiogenesis involves stimulation of endothelial cells (EC) by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the “angiome”) could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A). We used the Short Time-series Expression Miner (STEM) to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME) show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC) and human microvascular EC (MEC). The results show that VEGFR1–VEGFR2 levels are more closely coupled than VEGFR1–VEGFR3 or VEGFR2–VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle. PMID:25329517

  6. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    PubMed

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

  7. Activation of nanoscale allosteric protein domain motion revealed by neutron spin echo spectroscopy

    NASA Astrophysics Data System (ADS)

    Bu, Zimei; Farago, Bela; Callaway, David

    2012-02-01

    NHERF1 is a multi-domain scaffolding protein that assembles the signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by a membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 angstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length scales and on sub-microsecond time scales upon forming a complex with ezrin. We show that a much simplified coarse-grained model is sufficient to describe inter-domain motion of a multi-domain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. The results demonstrate that propagation of allosteric signals to distal sites involves the activation of long-range coupled domain motions on submicrosecond time scales, and that these coupled motions can be distinguished and characterized by NSE.

  8. Multitaxon activity profiling reveals differential microbial response to reduced seawater pH and oil pollution.

    PubMed

    Coelho, Francisco J R C; Cleary, Daniel F R; Costa, Rodrigo; Ferreira, Marina; Polónia, Ana R M; Silva, Artur M S; Simões, Mário M Q; Oliveira, Vanessa; Gomes, Newton C M

    2016-09-01

    There is growing concern that predicted changes to global ocean chemistry will interact with anthropogenic pollution to significantly alter marine microbial composition and function. However, knowledge of the compounding effects of climate change stressors and anthropogenic pollution is limited. Here, we used 16S and 18S rRNA (cDNA)-based activity profiling to investigate the differential responses of selected microbial taxa to ocean acidification and oil hydrocarbon contamination under controlled laboratory conditions. Our results revealed that a lower relative abundance of sulphate-reducing bacteria (Desulfosarcina/Desulfococcus clade) due to an adverse effect of seawater acidification and oil hydrocarbon contamination (reduced pH-oil treatment) may be coupled to changes in sediment archaeal communities. In particular, we observed a pronounced compositional shift and marked reduction in the prevalence of otherwise abundant operational taxonomic units (OTUs) belonging to the archaeal Marine Benthic Group B and Marine Hydrothermal Vent Group (MHVG) in the reduced pH-oil treatment. Conversely, the abundance of several putative hydrocarbonoclastic fungal OTUs was higher in the reduced pH-oil treatment. Sediment hydrocarbon profiling, furthermore, revealed higher concentrations of several alkanes in the reduced pH-oil treatment, corroborating the functional implications of the structural changes to microbial community composition. Collectively, our results advance the understanding of the response of a complex microbial community to the interaction between reduced pH and anthropogenic pollution. In future acidified marine environments, oil hydrocarbon contamination may alter the typical mixotrophic and k-/r-strategist composition of surface sediment microbiomes towards a more heterotrophic state with lower doubling rates, thereby impairing the ability of the ecosystem to recover from acute oil contamination events.

  9. First Results of the TIGRE Chromospheric Activity Survey

    NASA Astrophysics Data System (ADS)

    Mittag, M.; Hempelmann, A.; Gonzalez-Perez, J. N.; Schmitt, J. H. M. M.

    2015-01-01

    We present the first results of the stellar activity survey with TIGRE (Telescopio Internacional de Guanajuato, Robótico-Espectroscópico). This long term program was started in August 2013 with the monitoring of a larger number of stars. We aim at measuring the short- and long-term variability of stellar activity for stars of different spectral types and luminosity classes, using indicators of different spectral lines (mainly Ca II S-Index, Ca II IR triplet, H_α and sodium D). A transformation equation of the TIGRE S-Index into the Mount Wilson S-index was derived in order to compare our results to the vast body of existing S-index measurements. Furthermore, the correlation between the S-index and the lines of the Ca II IR triplet has been studied, based on strictly simultaneous observations.

  10. Human Ebola virus infection results in substantial immune activation.

    PubMed

    McElroy, Anita K; Akondy, Rama S; Davis, Carl W; Ellebedy, Ali H; Mehta, Aneesh K; Kraft, Colleen S; Lyon, G Marshall; Ribner, Bruce S; Varkey, Jay; Sidney, John; Sette, Alessandro; Campbell, Shelley; Ströher, Ute; Damon, Inger; Nichol, Stuart T; Spiropoulou, Christina F; Ahmed, Rafi

    2015-04-14

    Four Ebola patients received care at Emory University Hospital, presenting a unique opportunity to examine the cellular immune responses during acute Ebola virus infection. We found striking activation of both B and T cells in all four patients. Plasmablast frequencies were 10-50% of B cells, compared with less than 1% in healthy individuals. Many of these proliferating plasmablasts were IgG-positive, and this finding coincided with the presence of Ebola virus-specific IgG in the serum. Activated CD4 T cells ranged from 5 to 30%, compared with 1-2% in healthy controls. The most pronounced responses were seen in CD8 T cells, with over 50% of the CD8 T cells expressing markers of activation and proliferation. Taken together, these results suggest that all four patients developed robust immune responses during the acute phase of Ebola virus infection, a finding that would not have been predicted based on our current assumptions about the highly immunosuppressive nature of Ebola virus. Also, quite surprisingly, we found sustained immune activation after the virus was cleared from the plasma, observed most strikingly in the persistence of activated CD8 T cells, even 1 mo after the patients' discharge from the hospital. These results suggest continued antigen stimulation after resolution of the disease. From these convalescent time points, we identified CD4 and CD8 T-cell responses to several Ebola virus proteins, most notably the viral nucleoprotein. Knowledge of the viral proteins targeted by T cells during natural infection should be useful in designing vaccines against Ebola virus.

  11. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes.

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-06-14

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the "hot-spot" within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  12. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    NASA Astrophysics Data System (ADS)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  13. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    PubMed Central

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  14. Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

    PubMed

    Kremmydas, Gerasimos F; Tampakaki, Anastasia P; Georgakopoulos, Dimitrios G

    2013-01-01

    Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ), and two genes (sup5 and sup6) which seem to be organized in a putative operon. This operon (named supX) consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon.

  15. An insight into synthetic Schiff bases revealing antiproliferative activities in vitro.

    PubMed

    Sztanke, Krzysztof; Maziarka, Agata; Osinka, Anna; Sztanke, Małgorzata

    2013-07-01

    Schiff bases or azomethines are among the most important groups of biomolecules. These compounds have been found to reveal both remarkable biological activities and a variety of valuable practical applications. An interest in the exploration of novel series of synthetic Schiff bases has undoubtedly been growing due to their proven utility as attractive lead structures for the design of novel cytotoxic and cytostatic agents with a mechanism of action that sometimes differs from that of clinically authorized anticancer agents. Therefore, in the present paper we have focussed our attention on the collected synthetic simple Schiff bases of aldimine- and ketimine-types revealing anticancer activities in vitro, that have been described in the scientific literature during the last decade, and on structural variations whose affect the antiproliferative activity in sets of the designed molecules.

  16. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    SciTech Connect

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  17. Nuclear RNA-seq of single neurons reveals molecular signatures of activation.

    PubMed

    Lacar, Benjamin; Linker, Sara B; Jaeger, Baptiste N; Krishnaswami, Suguna; Barron, Jerika; Kelder, Martijn; Parylak, Sarah; Paquola, Apuã; Venepally, Pratap; Novotny, Mark; O'Connor, Carolyn; Fitzpatrick, Conor; Erwin, Jennifer; Hsu, Jonathan Y; Husband, David; McConnell, Michael J; Lasken, Roger; Gage, Fred H

    2016-04-19

    Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.

  18. Nuclear RNA-seq of single neurons reveals molecular signatures of activation

    PubMed Central

    Lacar, Benjamin; Linker, Sara B.; Jaeger, Baptiste N.; Krishnaswami, Suguna; Barron, Jerika; Kelder, Martijn; Parylak, Sarah; Paquola, Apuã; Venepally, Pratap; Novotny, Mark; O'Connor, Carolyn; Fitzpatrick, Conor; Erwin, Jennifer; Hsu, Jonathan Y.; Husband, David; McConnell, Michael J.; Lasken, Roger; Gage, Fred H.

    2016-01-01

    Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo. PMID:27090946

  19. Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon

    PubMed Central

    Leis, Benedikt; Heinze, Simon; Angelov, Angel; Pham, Vu Thuy Trang; Thürmer, Andrea; Jebbar, Mohamed; Golyshin, Peter N.; Streit, Wolfgang R.; Daniel, Rolf; Liebl, Wolfgang

    2015-01-01

    Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8–70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70°C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12) endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45% sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92°C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential. PMID:26191525

  20. Detailed investigation of the microbial community in foaming activated sludge reveals novel foam formers

    NASA Astrophysics Data System (ADS)

    Guo, Feng; Wang, Zhi-Ping; Yu, Ke; Zhang, T.

    2015-01-01

    Foaming of activated sludge (AS) causes adverse impacts on wastewater treatment operation and hygiene. In this study, we investigated the microbial communities of foam, foaming AS and non-foaming AS in a sewage treatment plant via deep-sequencing of the taxonomic marker genes 16S rRNA and mycobacterial rpoB and a metagenomic approach. In addition to Actinobacteria, many genera (e.g., Clostridium XI, Arcobacter, Flavobacterium) were more abundant in the foam than in the AS. On the other hand, deep-sequencing of rpoB did not detect any obligate pathogenic mycobacteria in the foam. We found that unknown factors other than the abundance of Gordonia sp. could determine the foaming process, because abundance of the same species was stable before and after a foaming event over six months. More interestingly, although the dominant Gordonia foam former was the closest with G. amarae, it was identified as an undescribed Gordonia species by referring to the 16S rRNA gene, gyrB and, most convincingly, the reconstructed draft genome from metagenomic reads. Our results, based on metagenomics and deep sequencing, reveal that foams are derived from diverse taxa, which expands previous understanding and provides new insight into the underlying complications of the foaming phenomenon in AS.

  1. Genome-wide analysis of LXRα activation reveals new transcriptional networks in human atherosclerotic foam cells.

    PubMed

    Feldmann, Radmila; Fischer, Cornelius; Kodelja, Vitam; Behrens, Sarah; Haas, Stefan; Vingron, Martin; Timmermann, Bernd; Geikowski, Anne; Sauer, Sascha

    2013-04-01

    Increased physiological levels of oxysterols are major risk factors for developing atherosclerosis and cardiovascular disease. Lipid-loaded macrophages, termed foam cells, are important during the early development of atherosclerotic plaques. To pursue the hypothesis that ligand-based modulation of the nuclear receptor LXRα is crucial for cell homeostasis during atherosclerotic processes, we analysed genome-wide the action of LXRα in foam cells and macrophages. By integrating chromatin immunoprecipitation-sequencing (ChIP-seq) and gene expression profile analyses, we generated a highly stringent set of 186 LXRα target genes. Treatment with the nanomolar-binding ligand T0901317 and subsequent auto-regulatory LXRα activation resulted in sequence-dependent sharpening of the genome-binding patterns of LXRα. LXRα-binding loci that correlated with differential gene expression revealed 32 novel target genes with potential beneficial effects, which in part explained the implications of disease-associated genetic variation data. These observations identified highly integrated LXRα ligand-dependent transcriptional networks, including the APOE/C1/C4/C2-gene cluster, which contribute to the reversal of cholesterol efflux and the dampening of inflammation processes in foam cells to prevent atherogenesis.

  2. Chlorophyll a fluorescence lifetime reveals reversible UV-induced photosynthetic activity in the green algae Tetraselmis.

    PubMed

    Kristoffersen, Arne S; Hamre, Børge; Frette, Øyvind; Erga, Svein R

    2016-04-01

    The fluorescence lifetime is a very useful parameter for investigating biological materials on the molecular level as it is mostly independent of the fluorophore concentration. The green alga Tetraselmis blooms in summer, and therefore its response to UV irradiation is of particular interest. In vivo fluorescence lifetimes of chlorophyll a were measured under both normal and UV-stressed conditions of Tetraselmis. Fluorescence was induced by two-photon excitation using a femtosecond laser and laser scanning microscope. The lifetimes were measured in the time domain by time-correlated single-photon counting. Under normal conditions, the fluorescence lifetime was 262 ps, while after 2 h of exposure to UV radiation the lifetime increased to 389 ps, indicating decreased photochemical quenching, likely caused by a damaged and down-regulated photosynthetic apparatus. This was supported by a similar increase in the lifetime to 425 ps when inhibiting photosynthesis chemically using DCMU. Furthermore, the UV-stressed sample was dark-adapted overnight, resulting in a return of the lifetime to 280 ps, revealing that the damage caused by UV radiation is repairable on a relatively short time scale. This reversal of photosynthetic activity was also confirmed by [Formula: see text] measurements.

  3. Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice

    PubMed Central

    Zhang, Limin; Hatzakis, Emmanuel; Nichols, Robert G.; Hao, Ruixin; Correll, Jared; Smith, Philip B.; Chiaro, Christopher R.; Perdew, Gary H.; Patterson, Andrew D.

    2016-01-01

    Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) were assessed using global 1H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolic profiling of extracts obtained from serum and liver. 1H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an Ahr-dependent manner. Targeted analyses using gas chromatography mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of stearoyl coenzyme A desaturase 1 was also observed. In addition, TCDF exposure resulted in inhibition of de novo fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from Ahr-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure. PMID:26023891

  4. The anti-obesity drug orlistat reveals anti-viral activity.

    PubMed

    Ammer, Elisabeth; Nietzsche, Sandor; Rien, Christian; Kühnl, Alexander; Mader, Theresa; Heller, Regine; Sauerbrei, Andreas; Henke, Andreas

    2015-12-01

    The administration of drugs to inhibit metabolic pathways not only reduces the risk of obesity-induced diseases in humans but may also hamper the replication of different viral pathogens. In order to investigate the value of the US Food and Drug Administration-approved anti-obesity drug orlistat in view of its anti-viral activity against different human-pathogenic viruses, several anti-viral studies, electron microscopy analyses as well as fatty acid uptake experiments were performed. The results indicate that administrations of non-cytotoxic concentrations of orlistat reduced the replication of coxsackievirus B3 (CVB3) in different cell types significantly. Moreover, orlistat revealed cell protective effects and modified the formation of multi-layered structures in CVB3-infected cells, which are necessary for viral replication. Lowering fatty acid uptake from the extracellular environment by phloretin administrations had only marginal impact on CVB3 replication. Finally, orlistat reduced also the replication of varicella-zoster virus moderately but had no significant influence on the replication of influenza A viruses. The data support further experiments into the value of orlistat as an inhibitor of the fatty acid synthase to develop new anti-viral compounds, which are based on the modulation of cellular metabolic pathways.

  5. Detailed investigation of the microbial community in foaming activated sludge reveals novel foam formers

    PubMed Central

    Guo, Feng; Wang, Zhi-Ping; Yu, Ke; Zhang, T.

    2015-01-01

    Foaming of activated sludge (AS) causes adverse impacts on wastewater treatment operation and hygiene. In this study, we investigated the microbial communities of foam, foaming AS and non-foaming AS in a sewage treatment plant via deep-sequencing of the taxonomic marker genes 16S rRNA and mycobacterial rpoB and a metagenomic approach. In addition to Actinobacteria, many genera (e.g., Clostridium XI, Arcobacter, Flavobacterium) were more abundant in the foam than in the AS. On the other hand, deep-sequencing of rpoB did not detect any obligate pathogenic mycobacteria in the foam. We found that unknown factors other than the abundance of Gordonia sp. could determine the foaming process, because abundance of the same species was stable before and after a foaming event over six months. More interestingly, although the dominant Gordonia foam former was the closest with G. amarae, it was identified as an undescribed Gordonia species by referring to the 16S rRNA gene, gyrB and, most convincingly, the reconstructed draft genome from metagenomic reads. Our results, based on metagenomics and deep sequencing, reveal that foams are derived from diverse taxa, which expands previous understanding and provides new insight into the underlying complications of the foaming phenomenon in AS. PMID:25560234

  6. Revealing a new activity of the human Dicer DUF283 domain in vitro

    PubMed Central

    Kurzynska-Kokorniak, Anna; Pokornowska, Maria; Koralewska, Natalia; Hoffmann, Weronika; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek

    2016-01-01

    The ribonuclease Dicer is a multidomain enzyme that plays a fundamental role in the biogenesis of small regulatory RNAs (srRNAs), which control gene expression by targeting complementary transcripts and inducing their cleavage or repressing their translation. Recent studies of Dicer’s domains have permitted to propose their roles in srRNA biogenesis. For all of Dicer’s domains except one, called DUF283 (domain of unknown function), their involvement in RNA substrate recognition, binding or cleavage has been postulated. For DUF283, the interaction with Dicer’s protein partners has been the only function suggested thus far. In this report, we demonstrate that the isolated DUF283 domain from human Dicer is capable of binding single-stranded nucleic acids in vitro. We also show that DUF283 can act as a nucleic acid annealer that accelerates base-pairing between complementary RNA/DNA molecules in vitro. We further demonstrate an annealing activity of full length human Dicer. The overall results suggest that Dicer, presumably through its DUF283 domain, might facilitate hybridization between short RNAs and their targets. The presented findings reveal the complex nature of Dicer, whose functions may extend beyond the biogenesis of srRNAs. PMID:27045313

  7. Continuum Level Results from Particle Simulations of Active Suspensions

    NASA Astrophysics Data System (ADS)

    Delmotte, Blaise; Climent, Eric; Plouraboue, Franck; Keaveny, Eric

    2014-11-01

    Accurately simulating active suspensions on the lab scale is a technical challenge. It requires considering large numbers of interacting swimmers with well described hydrodynamics in order to obtain representative and reliable statistics of suspension properties. We have developed a computationally scalable model based on an extension of the Force Coupling Method (FCM) to active particles. This tool can handle the many-body hydrodynamic interactions between O (105) swimmers while also accounting for finite-size effects, steady or time-dependent strokes, or variable swimmer aspect ratio. Results from our simulations of steady-stroke microswimmer suspensions coincide with those given by continuum models, but, in certain cases, we observe collective dynamics that these models do not predict. We provide robust statistics of resulting distributions and accurately characterize the growth rates of these instabilities. In addition, we explore the effect of the time-dependent stroke on the suspension properties, comparing with those from the steady-stroke simulations. Authors acknowledge the ANR project Motimo for funding and the Calmip computing centre for technical support.

  8. A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

    PubMed Central

    2016-01-01

    Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex. PMID:25569308

  9. Io's Diverse Styles of Volcanic Activity: Results from Galileo NIMS

    NASA Technical Reports Server (NTRS)

    Lopes, R. M. C.; Smythe, W. D.; Kamp, L. W.; Doute, S.; Carlson, R.; McEwen, A.; Geissler, P.

    2001-01-01

    Observations by Galileo's Near-Infrared Mapping Spectrometer were used to map the thermal structure of several of Io's hot spots, revealing different styles of volcanism Additional information is contained in the original extended abstract..

  10. Active Spacecraft Potential Control: Results From the Double Star Project

    NASA Astrophysics Data System (ADS)

    Torkar, K.; Fazakerley, A.; Steiger, W.

    2006-10-01

    The ion emitter instrument "active spacecraft potential control" (ASPOC) has been used successfully in several magnetospheric missions including the European Space Agency Cluster Project. An improved version has been developed for the equatorial spacecraft of the Chinese-European Double Star mission (TC-1) launched in December 2003. The modifications include a new design of the ion emitter modules. As a result, higher currents than in previous missions can be achieved. The main objective of the investigation is the reduction of positive spacecraft potential in order to minimize perturbations to the plasma measurements onboard, in particular to the plasma electron instrument PEACE. These data show an almost complete suppression of photoelectrons when ASPOC is emitting at 30- to 50-muA beam current. The angular distribution of the electrons in the presence of the ion beam is investigated in detail. The measurement of ambient electron distributions is highly improved.

  11. Cues Resulting in Desire for Sexual Activity in Women

    PubMed Central

    McCall, Katie; Meston, Cindy

    2010-01-01

    Introduction A number of questionnaires have been created to assess levels of sexual desire in women, but to our knowledge, there are currently no validated measures for assessing cues that result in sexual desire. A questionnaire of this nature could be useful for both clinicians and researchers, because it considers the contextual nature of sexual desire and it draws attention to individual differences in factors that can contribute to sexual desire. Aim The aim of the present study was to create a multidimensional assessment tool of cues for sexual desire in women that is validated in women with and without hypoactive sexual desire disorder (HSDD). Methods Factor analyses conducted on both an initial sample (N = 874) and a community sample (N = 138) resulted in the Cues for Sexual Desire Scale (CSDS) which included four factors: (i) Emotional Bonding Cues; (ii) Erotic/ Explicit Cues; (iii) Visual/Proximity Cues; and (iv) Implicit/Romantic Cues. Main Outcome Measures Scale construction of cues associated with sexual desire and differences between women with and without sexual dysfunction. Results The CSDS demonstrated good reliability and validity and was able to detect significant differences between women with and without HSDD. Results from regression analyses indicated that both marital status and level of sexual functioning predicted scores on the CSDS. The CSDS provided predictive validity for the Female Sexual Function Index desire and arousal domain scores, and increased cues were related to a higher reported frequency of sexual activity in women. Conclusions The findings from the present study provide valuable information regarding both internal and external triggers that can result in sexual desire for women. We believe that the CSDS could be beneficial in therapeutic settings to help identify cues that do and do not facilitate sexual desire in women with clinically diagnosed desire difficulties. PMID:16942529

  12. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    PubMed Central

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group. PMID:26143919

  13. Results of mobile gamma scanning activities in Tonawanda, New York

    SciTech Connect

    Cottrell, W.D.; Witt, D.A.; Rodriguez, R.E.; Carrier, R.F.

    1990-12-01

    During the 1940s, the Linde Air Products Division of Union Carbide operated a plant in Tonawanda, New York, for the Manhattan Engineer District (MED) and the Atomic Energy Commission (AEC). Uranium production and some nickel processing were conducted at the site. It is the policy of the US Department of Energy (DOE) to verify that radiological conditions at such sites or facilities comply with current DOE guidelines. Guidelines for release and use of such sites have become more stringent as research has provided more information since previous cleanups. The Formerly Utilized Sites Remedial Action Program (FUSRAP) was established as part of that effort to confirm the closeout status of facilities under contract to agencies preceding DOE during early nuclear energy development. Under the FUSRAP program, the Linde site itself has been previously investigated to determine the extent of on-site radiological contamination. As a precaution to insure that no residual radioactive materials were transported off-site, the Department of Energy requested that ORNL survey the area in the vicinity of the Linde Plant, the waste water treatment facility on Tower Road, the Sheridan Park Fire Station (District 4), and the Tonawanda Landfill to assess whether any residual radioactive material could be detected. The survey was conducted the week of April 3, 1990. Results of analysis of soil samples from the Tonawanda Landfill revealed slightly elevated concentrations of {sup 238}U and {sup 226}Ra suggestive of residuals from former Linde Plant operations. Therefore, it is recommended that additional surveying of the landfill property and of Sheridan Creek from south of the Linde property to its confluence with the Niagara River be conducted. The survey should include the measurement of gamma radiation levels and radionuclide analysis of silt samples. 6 refs., 4 figs., 1 tab.

  14. Colonic perforation resulting from ingested chicken bone revealing previously undiagnosed colonic adenocarcinoma: report of a case and review of literature.

    PubMed

    McGregor, Douglas H; Liu, Xiaoying; Ulusarac, Ozlem; Ponnuru, Kimberly D; Schnepp, Stephanie L

    2011-02-18

    An 86 year old male with a four-day history of nonspecific gastrointestinal symptoms was found on colonoscopy to have evidence of sigmoid colon obstruction and possible perforation. Emergent operative exploration revealed diffuse peritonitis, sigmoid perforation, adjacent dense adhesions, and a foreign body protruding through the perforated area. Pathologic examination showed the foreign body to be a sliver of bone consistent with chicken bone and the sigmoid subacute perforation to be associated distally with a circumferential ulcerated obstructing mass, microscopically seen to be transmurally infiltrating adenocarcinoma, signet-ring cell type. There was extensive acute and organizing peritonitis, 100% Escherichia coli was cultured from peritoneal fluid, and the patient died two days postoperatively with sepsis and hypotension. This appears to be the fifth reported case of colonic perforation resulting from foreign body perforation due to previously undiagnosed adenocarcinoma. The four previously reported cases were all deeply invasive adenocarcinoma of sigmoid colon, and the foreign bodies included three chicken/poultry bones and a metallic staple. These five cases are highly unusual examples of a potentially lethal malignant neoplasm being clinically revealed by a usually (but not always) innocuous event, the ingestion of a small foreign body.

  15. Colonic perforation resulting from ingested chicken bone revealing previously undiagnosed colonic adenocarcinoma: report of a case and review of literature

    PubMed Central

    2011-01-01

    An 86 year old male with a four-day history of nonspecific gastrointestinal symptoms was found on colonoscopy to have evidence of sigmoid colon obstruction and possible perforation. Emergent operative exploration revealed diffuse peritonitis, sigmoid perforation, adjacent dense adhesions, and a foreign body protruding through the perforated area. Pathologic examination showed the foreign body to be a sliver of bone consistent with chicken bone and the sigmoid subacute perforation to be associated distally with a circumferential ulcerated obstructing mass, microscopically seen to be transmurally infiltrating adenocarcinoma, signet-ring cell type. There was extensive acute and organizing peritonitis, 100% Escherichia coli was cultured from peritoneal fluid, and the patient died two days postoperatively with sepsis and hypotension. This appears to be the fifth reported case of colonic perforation resulting from foreign body perforation due to previously undiagnosed adenocarcinoma. The four previously reported cases were all deeply invasive adenocarcinoma of sigmoid colon, and the foreign bodies included three chicken/poultry bones and a metallic staple. These five cases are highly unusual examples of a potentially lethal malignant neoplasm being clinically revealed by a usually (but not always) innocuous event, the ingestion of a small foreign body. PMID:21333012

  16. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  17. Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

    PubMed Central

    Hoskins, Aaron A; Rodgers, Margaret L; Friedman, Larry J; Gelles, Jeff; Moore, Melissa J

    2016-01-01

    The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation. DOI: http://dx.doi.org/10.7554/eLife.14166.001 PMID:27244240

  18. Magnetoencephalography reveals early activation of V4 in grapheme-color synesthesia.

    PubMed

    Brang, D; Hubbard, E M; Coulson, S; Huang, M; Ramachandran, V S

    2010-10-15

    Grapheme-color synesthesia is a neurological phenomenon in which letters and numbers (graphemes) consistently evoke particular colors (e.g. A may be experienced as red). The cross-activation theory proposes that synesthesia arises as a result of cross-activation between posterior temporal grapheme areas (PTGA) and color processing area V4, while the disinhibited feedback theory proposes that synesthesia arises from disinhibition of pre-existing feedback connections. Here we used magnetoencephalography (MEG) to test whether V4 and PTGA activate nearly simultaneously, as predicted by the cross-activation theory, or whether V4 activation occurs only after the initial stages of grapheme processing, as predicted by the disinhibited feedback theory. Using our high-resolution MEG source imaging technique (VESTAL), PTGA and V4 regions of interest (ROIs) were separately defined, and activity in response to the presentation of achromatic graphemes was measured. Activation levels in PTGA did not significantly differ between synesthetes and controls (suggesting similar grapheme processing mechanisms), whereas activation in V4 was significantly greater in synesthetes. In synesthetes, PTGA activation exceeded baseline levels beginning 105-109ms, and V4 activation did so 5ms later, suggesting nearly simultaneous activation of these areas. Results are discussed in the context of an updated version of the cross-activation model, the cascaded cross-tuning model of grapheme-color synesthesia.

  19. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    SciTech Connect

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  20. Optimal active vibration absorber: Design and experimental results

    NASA Technical Reports Server (NTRS)

    Lee-Glauser, Gina; Juang, Jer-Nan; Sulla, Jeffrey L.

    1992-01-01

    An optimal active vibration absorber can provide guaranteed closed-loop stability and control for large flexible space structures with collocated sensors/actuators. The active vibration absorber is a second-order dynamic system which is designed to suppress any unwanted structural vibration. This can be designed with minimum knowledge of the controlled system. Two methods for optimizing the active vibration absorber parameters are illustrated: minimum resonant amplitude and frequency matched active controllers. The Controls-Structures Interaction Phase-1 Evolutionary Model at NASA LaRC is used to demonstrate the effectiveness of the active vibration absorber for vibration suppression. Performance is compared numerically and experimentally using acceleration feedback.

  1. Whole-brain activity maps reveal stereotyped, distributed networks for visuomotor behavior.

    PubMed

    Portugues, Ruben; Feierstein, Claudia E; Engert, Florian; Orger, Michael B

    2014-03-19

    Most behaviors, even simple innate reflexes, are mediated by circuits of neurons spanning areas throughout the brain. However, in most cases, the distribution and dynamics of firing patterns of these neurons during behavior are not known. We imaged activity, with cellular resolution, throughout the whole brains of zebrafish performing the optokinetic response. We found a sparse, broadly distributed network that has an elaborate but ordered pattern, with a bilaterally symmetrical organization. Activity patterns fell into distinct clusters reflecting sensory and motor processing. By correlating neuronal responses with an array of sensory and motor variables, we find that the network can be clearly divided into distinct functional modules. Comparing aligned data from multiple fish, we find that the spatiotemporal activity dynamics and functional organization are highly stereotyped across individuals. These experiments systematically reveal the functional architecture of neural circuits underlying a sensorimotor behavior in a vertebrate brain.

  2. Whole-brain activity maps reveal stereotyped, distributed networks for visuomotor behavior

    PubMed Central

    Portugues, Ruben; Feierstein, Claudia E.; Engert, Florian; Orger, Michael B.

    2014-01-01

    Summary Most behaviors, even simple innate reflexes, are mediated by circuits of neurons spanning areas throughout the brain. However, in most cases, the distribution and dynamics of firing patterns of these neurons during behavior are not known. We imaged activity, with cellular resolution, throughout the whole brains of zebrafish performing the optokinetic response. We found a sparse, broadly distributed network that has an elaborate, but ordered, pattern, with a bilaterally symmetrical organization. Activity patterns fell into distinct clusters reflecting sensory and motor processing. By correlating neuronal responses with an array of sensory and motor variables, we find that the network can be clearly divided into distinct functional modules. Comparing aligned data from multiple fish, we find that the spatiotemporal activity dynamics and functional organization are highly stereotyped across individuals. These experiments reveal, for the first time in a vertebrate, the comprehensive functional architecture of the neural circuits underlying a sensorimotor behavior. PMID:24656252

  3. ASAR Revealing Hot-Spots of Internal Solitary Waves in the Arctic: Preliminary Results for the Laptev Sea

    NASA Astrophysics Data System (ADS)

    Kozlov, Igor E.; Zubkova, Evgenia V.; Kudryavtsev, Vladimir N.

    2016-08-01

    In this work we present first preliminary results of internal solitary waves' observations in the Laptev Sea using a set of ENVISAT Advanced Synthetic Aperture Radar (ASAR) images. Analysis of 354 ASAR images acquired between May-October 2011 helped to identify 91 distinct patterns of oceanic internal waves and reveal main regions of their occurrence. As found, the primary regions of IW observations are located north-west to Arctic Cape, over the continental slope regions east to Maly Taymyr Island and north-west to Kotelny Island, and east to the Gulf of Khatanga. Detailed maps of main internal wave parameters are presented, and some features of their propagation and implications for the vertical mixing in the Laptev Sea are also discussed.

  4. Interspecies activity correlations reveal functional correspondence between monkey and human brain areas.

    PubMed

    Mantini, Dante; Hasson, Uri; Betti, Viviana; Perrucci, Mauro G; Romani, Gian Luca; Corbetta, Maurizio; Orban, Guy A; Vanduffel, Wim

    2012-02-05

    Evolution-driven functional changes in the primate brain are typically assessed by aligning monkey and human activation maps using cortical surface expansion models. These models use putative homologous areas as registration landmarks, assuming they are functionally correspondent. For cases in which functional changes have occurred in an area, this assumption prohibits to reveal whether other areas may have assumed lost functions. Here we describe a method to examine functional correspondences across species. Without making spatial assumptions, we assessed similarities in sensory-driven functional magnetic resonance imaging responses between monkey (Macaca mulatta) and human brain areas by temporal correlation. Using natural vision data, we revealed regions for which functional processing has shifted to topologically divergent locations during evolution. We conclude that substantial evolution-driven functional reorganizations have occurred, not always consistent with cortical expansion processes. This framework for evaluating changes in functional architecture is crucial to building more accurate evolutionary models.

  5. High Fc Density Particles Result in Binary Complement Activation but Tunable Macrophage Phagocytosis

    NASA Astrophysics Data System (ADS)

    Sulchek, Todd; Pacheco, Patricia; White, David

    2014-03-01

    Macrophage phagocytosis and complement system activation represent two key components of the immune system and both can be activated through the presentation of multiple Fc domains of IgG antibodies. We have created functionalized micro- and nanoparticles with various densities of Fc domains to understand the modulation of the immune system for eventual use as a novel immunomodulation platform. Phagocytosis assays were carried out by adding functionalized particles to macrophage cells and quantitatively determined using fluorescent microscopy and flow cytometry. Complement system activation by the functionalized particles in human serum was quantified with an enzyme immunoassay. Our phagocytosis assay revealed a strong dependence on particle size and Fc density. For small particles, as the Fc density increased, the number of particles phagocytosed also increased. Large particles were phagocytosed at significantly lower levels and showed no dependency on Fc density. Complement was successfully activated at levels comparable to positive controls for small particles at high Fc densities. However at low Fc densities, there is a significant decrease in complement activation. This result suggests a binary response for complement system activation with a threshold density for successful activation. Therefore, varying the Fc density on micro/nanoparticles resulted in a tunable response in macrophage phagocytosis while a more binary response for complement activation.

  6. Active Region Oscillations: Results from SOHO JOP 097

    NASA Astrophysics Data System (ADS)

    O'Shea, E.; Fleck, B.; Muglach, K.; Sütterlin, P.

    2001-05-01

    We present here an analysis of data obtained in a sunspot region, using the Coronal Diagnostic Spectrometer (CDS) on SOHO. These data were obtained in the context of the Joint Observing Program (JOP) 97 which, together with CDS, included the Michelson Doppler Imaging (MDI) instrument on SOHO, the TRACE satellite and various ground based observatories, e.g. the DOT on La Palma. Using the lines of Fe XVI 335, Mg IX 368, He I 584, O III 599, Mg X 624 and O V 624 of CDS time series data were obtained in the pore and plage regions of sunspots associated with active regions AR 9166, 9166 and 9169 between September 19-29 2000. In addition to the time series datasets we also obtained 240 arcsec x 240 arcsec raster images of the sunspot regions examined. Using different time series analysis techniques we analyse the different periods of oscillation found in time series datasets and present the results here. This research is part of the European Solar Magnetometry Network supported by the EC through the TMR programme.

  7. ICARUS T600: physics results and future activities

    NASA Astrophysics Data System (ADS)

    Zani, Andrea

    2016-11-01

    The ICARUS T600 detector has proven the effectiveness of Liquid Argon TPC technology with a successful three-year long run at the INFN Gran Sasso National Laboratories (LNGS). ICARUS T600 LNGS data strongly contributed to the global effort in searching for neutrino oscillations mediated by sterile states. A definitive clarification of the sterile neutrino puzzle will come from the new multi-station, Short Baseline Neutrino (SBN) experiment at the Booster Neutrino Beam (BNB) at Fermilab, where a refurbished T600 will act as Far Detector. Presently the T600 detector is located at CERN, undergoing a major overhauling which will allow its participation in the 5 years program of the Fermilab upcoming SBN project. Moreover, ICARUS T600 is also foreseen to collect data with the NUMI Beam to be later exploited in the wider picture of the DUNE Long-Baseline project, recently undertaken by the United States community. This contribution will report the present physics results obtained by the ICARUS Collaboration, as well as describe the overhauling activities and the physics program for the detector in its future deployment at Fermilab.

  8. Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay

    SciTech Connect

    Kuo, Chun-Chen; Chu, Chi-Yuan; Lin, Jing-Jer; Lo, Lee-Chiang

    2010-01-01

    Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.

  9. Multichannel Detrended Fluctuation Analysis Reveals Synchronized Patterns of Spontaneous Spinal Activity in Anesthetized Cats

    PubMed Central

    Rodríguez, Erika E.; Hernández-Lemus, Enrique; Itzá-Ortiz, Benjamín A.; Jiménez, Ismael; Rudomín, Pablo

    2011-01-01

    The analysis of the interaction and synchronization of relatively large ensembles of neurons is fundamental for the understanding of complex functions of the nervous system. It is known that the temporal synchronization of neural ensembles is involved in the generation of specific motor, sensory or cognitive processes. Also, the intersegmental coherence of spinal spontaneous activity may indicate the existence of synaptic neural pathways between different pairs of lumbar segments. In this study we present a multichannel version of the detrended fluctuation analysis method (mDFA) to analyze the correlation dynamics of spontaneous spinal activity (SSA) from time series analysis. This method together with the classical detrended fluctuation analysis (DFA) were used to find out whether the SSA recorded in one or several segments in the spinal cord of the anesthetized cat occurs either in a random or in an organized manner. Our results are consistent with a non-random organization of the sets of neurons involved in the generation of spontaneous cord dorsum potentials (CDPs) recorded either from one lumbar segment (DFA- mean = 1.040.09) or simultaneously from several lumbar segments (mDFA- mean = 1.010.06), where  = 0.5 indicates randomness while 0.5 indicates long-term correlations. To test the sensitivity of the mDFA method we also examined the effects of small spinal lesions aimed to partially interrupt connectivity between neighboring lumbosacral segments. We found that the synchronization and correlation between the CDPs recorded from the L5 and L6 segments in both sides of the spinal cord were reduced when a lesion comprising the left dorsal quadrant was performed between the segments L5 and L6 (mDFA- = 0.992 as compared to initial conditions mDFA- = 1.186). The synchronization and correlation were reduced even further after a similar additional right spinal lesion (mDFA- = 0.924). In contrast to the classical methods, such as correlation

  10. Multichannel detrended fluctuation analysis reveals synchronized patterns of spontaneous spinal activity in anesthetized cats.

    PubMed

    Rodríguez, Erika E; Hernández-Lemus, Enrique; Itzá-Ortiz, Benjamín A; Jiménez, Ismael; Rudomín, Pablo

    2011-01-01

    The analysis of the interaction and synchronization of relatively large ensembles of neurons is fundamental for the understanding of complex functions of the nervous system. It is known that the temporal synchronization of neural ensembles is involved in the generation of specific motor, sensory or cognitive processes. Also, the intersegmental coherence of spinal spontaneous activity may indicate the existence of synaptic neural pathways between different pairs of lumbar segments. In this study we present a multichannel version of the detrended fluctuation analysis method (mDFA) to analyze the correlation dynamics of spontaneous spinal activity (SSA) from time series analysis. This method together with the classical detrended fluctuation analysis (DFA) were used to find out whether the SSA recorded in one or several segments in the spinal cord of the anesthetized cat occurs either in a random or in an organized manner. Our results are consistent with a non-random organization of the sets of neurons involved in the generation of spontaneous cord dorsum potentials (CDPs) recorded either from one lumbar segment (DFA-α mean = 1.04[Formula: see text]0.09) or simultaneously from several lumbar segments (mDFA-α mean = 1.01[Formula: see text]0.06), where α = 0.5 indicates randomness while α = 0.5 indicates long-term correlations. To test the sensitivity of the mDFA method we also examined the effects of small spinal lesions aimed to partially interrupt connectivity between neighboring lumbosacral segments. We found that the synchronization and correlation between the CDPs recorded from the L5 and L6 segments in both sides of the spinal cord were reduced when a lesion comprising the left dorsal quadrant was performed between the segments L5 and L6 (mDFA-[Formula: see text] = 0.992 as compared to initial conditions mDFA-α = 1.186). The synchronization and correlation were reduced even further after a similar additional right spinal lesion (mDFA-α = 0.924). In contrast

  11. Assessment Results Following Inquiry and Traditional Physics Laboratory Activities

    ERIC Educational Resources Information Center

    Bryan, Joel Arthur

    2006-01-01

    Preservice elementary teachers in a conceptual physics course were given multiple resources to use during several inquiry activities in order to investigate how materials were chosen, used, and valued. These students performed significantly better on assessment items related to the inquiry physics activities than on items related to traditional…

  12. Nuclear receptor engineering based on novel structure activity relationships revealed by farnesyl pyrophosphate.

    PubMed

    Goyanka, Ritu; Das, Sharmistha; Samuels, Herbert H; Cardozo, Timothy

    2010-11-01

    Nuclear receptors (NRs) comprise the second largest protein family targeted by currently available drugs, acting via specific ligand interactions within the ligand binding domain (LBD). Recently, farnesyl pyrophosphate (FPP) was shown to be a unique promiscuous NR ligand, activating a subset of NR family members and inhibiting wound healing in skin. The current study aimed at visualizing the unique basis of FPP interaction with multiple receptors in order to identify general structure-activity relationships that operate across the NR family. Docking of FPP to the 3D structures of the LBDs of a diverse set of NRs consistently revealed an electrostatic FPP pyrophosphate contact with an NR arginine conserved in the NR family, a hydrophobic farnesyl contact with NR helix-12 and a ligand binding pocket volume between 300 and 430 Å(3) as the minimal requirements for FPP activation of any NR. Lack of any of these structural features appears to render a given NR resistant to FPP activation. We used these structure-activity relationships to rationally design and successfully engineer several mutant human estrogen receptors that retain responsiveness to estradiol but no longer respond to FPP.

  13. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    PubMed

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

  14. Stellar activity and coronal heating: an overview of recent results

    PubMed Central

    Testa, Paola; Saar, Steven H.; Drake, Jeremy J.

    2015-01-01

    Observations of the coronae of the Sun and of solar-like stars provide complementary information to advance our understanding of stellar magnetic activity, and of the processes leading to the heating of their outer atmospheres. While solar observations allow us to study the corona at high spatial and temporal resolution, the study of stellar coronae allows us to probe stellar activity over a wide range of ages and stellar parameters. Stellar studies therefore provide us with additional tools for understanding coronal heating processes, as well as the long-term evolution of solar X-ray activity. We discuss how recent studies of stellar magnetic fields and coronae contribute to our understanding of the phenomenon of activity and coronal heating in late-type stars. PMID:25897087

  15. Stellar activity and coronal heating: an overview of recent results.

    PubMed

    Testa, Paola; Saar, Steven H; Drake, Jeremy J

    2015-05-28

    Observations of the coronae of the Sun and of solar-like stars provide complementary information to advance our understanding of stellar magnetic activity, and of the processes leading to the heating of their outer atmospheres. While solar observations allow us to study the corona at high spatial and temporal resolution, the study of stellar coronae allows us to probe stellar activity over a wide range of ages and stellar parameters. Stellar studies therefore provide us with additional tools for understanding coronal heating processes, as well as the long-term evolution of solar X-ray activity. We discuss how recent studies of stellar magnetic fields and coronae contribute to our understanding of the phenomenon of activity and coronal heating in late-type stars.

  16. Equilibrium physics breakdown reveals the active nature of red blood cell flickering

    NASA Astrophysics Data System (ADS)

    Turlier, Herve; Fedosov, Dmitry; Auth, Thorsten; Gov, Nir S.; Sykes, Cecile; Joanny, Jean-Francois; Gompper, Gerhard; Betz, Timo

    2015-03-01

    Red blood cell membrane flickering stimulated an abundant biological, biophysical and biochemical literature over the past 50 years. While the phenomenon has been interpreted as thermal fluctuations of the cell membrane, recent results suggest the involvement of metabolic processes. However, to date there is no direct and conclusive evidence that an active force drives membrane flickering. By comparing membrane undulations and active microrheology measurements on single human erythrocytes, we show that flickering is partly driven by an active metabolic process, as it does not satisfy the equilibrium fluctuation-dissipation relation on timescales slower than 100ms. Analytical and numerical models of the red blood cell reproduce experimental results. The analytical model assumes that membrane activity results from reversible binding of the elastic spectrin network to the lipid bilayer and predicts active fluctuations to increase with local curvature and extensional prestress in the cytoskeleton. Our mean-field calculation shows that the strength and kinetics of the binding activity regulates thereupon both passive and active mechanical properties of the red blood cell. Numerical simulations explore other possible origins of active forces on the membrane and predict coherent timescales for the molecular underlying metabolic processes.

  17. Charpy impact test results for low-activation ferritic alloys

    SciTech Connect

    Cannon, N.S.; Hu, W.L.; Gelles, D.S.

    1987-05-01

    The objective of this work is to evaluate the shift of the ductile to brittle transition temperature (DBTT) and the reduction of the upper shelf energy (USE) due to neutron irradiation of low activation ferritic alloys. Six low activation ferritic alloys have been tested following irradiation at 365/sup 0/C to 10 dpa and compared with control specimens in order to assess the effect of irradiation on Charpy impact properties.

  18. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells.

    PubMed

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-09-14

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation.

  19. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency.

  20. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells

    PubMed Central

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-01-01

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation. PMID:27624869

  1. Tc-99 Adsorption on Selected Activated Carbons - Batch Testing Results

    SciTech Connect

    Mattigod, Shas V.; Wellman, Dawn M.; Golovich, Elizabeth C.; Cordova, Elsa A.; Smith, Ronald M.

    2010-12-01

    CH2M HILL Plateau Remediation Company (CHPRC) is currently developing a 200-West Area groundwater pump-and-treat system as the remedial action selected under the Comprehensive Environmental Response, Compensation, and Liability Act Record of Decision for Operable Unit (OU) 200-ZP-1. This report documents the results of treatability tests Pacific Northwest National Laboratory researchers conducted to quantify the ability of selected activated carbon products (or carbons) to adsorb technetium-99 (Tc-99) from 200-West Area groundwater. The Tc-99 adsorption performance of seven activated carbons (J177601 Calgon Fitrasorb 400, J177606 Siemens AC1230AWC, J177609 Carbon Resources CR-1240-AW, J177611 General Carbon GC20X50, J177612 Norit GAC830, J177613 Norit GAC830, and J177617 Nucon LW1230) were evaluated using water from well 299-W19-36. Four of the best performing carbons (J177606 Siemens AC1230AWC, J177609 Carbon Resources CR-1240-AW, J177611 General Carbon GC20X50, and J177613 Norit GAC830) were selected for batch isotherm testing. The batch isotherm tests on four of the selected carbons indicated that under lower nitrate concentration conditions (382 mg/L), Kd values ranged from 6,000 to 20,000 mL/g. In comparison. Under higher nitrate (750 mg/L) conditions, there was a measureable decrease in Tc-99 adsorption with Kd values ranging from 3,000 to 7,000 mL/g. The adsorption data fit both the Langmuir and the Freundlich equations. Supplemental tests were conducted using the two carbons that demonstrated the highest adsorption capacity to resolve the issue of the best fit isotherm. These tests indicated that Langmuir isotherms provided the best fit for Tc-99 adsorption under low nitrate concentration conditions. At the design basis concentration of Tc 0.865 µg/L(14,700 pCi/L), the predicted Kd values from using Langmuir isotherm constants were 5,980 mL/g and 6,870 mL/g for for the two carbons. These Kd values did not meet the target Kd value of 9,000 mL/g. Tests

  2. Fifteen years of thermal activity at Vanuatu's volcanoes (2000-2015) revealed by MIROVA

    NASA Astrophysics Data System (ADS)

    Coppola, D.; Laiolo, M.; Cigolini, C.

    2016-08-01

    The Vanuatu archipelago consists of 80 islands and hosts 5 subaerial volcanoes (Yasur, Lopevi, Ambrym, Aoba and Gaua) that have shown sign of activity during the past decade. In this contribution we provide a 15 years-long datasets (2000-2015) of the thermal activity recorded at these active volcanoes by means of MIROVA (Middle InfraRed Observation of Volcanic Activity) a new volcanic hotspot detection system based on MODIS data. The analyzed volcanoes are characterized by a spectrum of volcanic activities whose thermal signature has been tracked and carefully analyzed. These include strombolian-vulcanian explosions at Yasur, lava flows at Lopevi, lava lakes at Ambrym, surtseyan-type eruptions within the Voui crater lake of Aoba and ash-dominated eruptions with strong degassing at Gaua. The collected data reveal several details of the long term eruptive dynamics at single sites such as a monthly long pulse in thermal emissions at Yasur volcano as well as at the two active craters of Ambrym (Benbow and Marum). Heating cycles within Aoba crater lake and intermittent pressurized eruptions at Lopevi volcano has also been detected and shed light in the eruptive dynamics of the analyzed volcanoes. In addition we were able to track a two years long intensification of thermal output at Benbow crater (Ambrym) that preceded the occurrence of the first intra-caldera eruptions of this volcano since 1989. We emphasize how the data provided by MIROVA represent a new, safe and affordable method for monitoring in near-real time a large spectrum of volcanic activities taking place at Vanuatu and other volcanic areas.

  3. Simultaneous PET-MRI reveals brain function in activated and resting state on metabolic, hemodynamic and multiple temporal scales.

    PubMed

    Wehrl, Hans F; Hossain, Mosaddek; Lankes, Konrad; Liu, Chih-Chieh; Bezrukov, Ilja; Martirosian, Petros; Schick, Fritz; Reischl, Gerald; Pichler, Bernd J

    2013-09-01

    Combined positron emission tomography (PET) and magnetic resonance imaging (MRI) is a new tool to study functional processes in the brain. Here we study brain function in response to a barrel-field stimulus simultaneously using PET, which traces changes in glucose metabolism on a slow time scale, and functional MRI (fMRI), which assesses fast vascular and oxygenation changes during activation. We found spatial and quantitative discrepancies between the PET and the fMRI activation data. The functional connectivity of the rat brain was assessed by both modalities: the fMRI approach determined a total of nine known neural networks, whereas the PET method identified seven glucose metabolism-related networks. These results demonstrate the feasibility of combined PET-MRI for the simultaneous study of the brain at activation and rest, revealing comprehensive and complementary information to further decode brain function and brain networks.

  4. Early activation of Broca's area in grammar processing as revealed by the syntactic mismatch negativity and distributed source analysis.

    PubMed

    Hanna, Jeff; Mejias, Sandrine; Schelstraete, Marie-Anne; Pulvermüller, Friedemann; Shtyrov, Yury; Van der Lely, Heather K J

    2014-01-01

    Though activation of Broca's region in the combinatorial processing of symbols (language, music) has been revealed by neurometabolic studies, most previous neurophysiological research found the earliest grammar indices in the temporal cortex, with inferior-frontal generators becoming active at relatively late stages. We use the attention- and task-free syntactic mismatch negativity (sMMN) event-related potential (ERP) to measure rapid and automatic sensitivity of the human brain to grammatical information in participants' native language (French). Further, sources underlying the MMN were estimated by applying the Parametrical Empirical Bayesian (PEB) approach, with the Multiple Sparse Priors (MSP) technique. Results showed reliable grammar-related activation focused on Broca's region already in the 150-190 ms time window, providing robust documentation of its involvement in the first stages of syntactic processing.

  5. Brain activity associated with omission of an aversive event reveals the effects of fear learning and generalization

    PubMed Central

    Dunsmoor, Joseph E.; LaBar, Kevin S.

    2012-01-01

    During fear learning, anticipation of an impending aversive stimulus increases defensive behaviors. Interestingly, omission of the aversive stimulus often produces another response around the time the event was expected. This omission response suggests that the subject detected a mismatch between what was predicted and what actually occurred, thereby providing an indirect measure of cognitive expectancy. Here, we used functional magnetic resonance imaging to investigate whether omission-related brain activity reflects fear expectancy during learning and generalization of conditioned fear. During conditioning, a face expressing a moderate amount of fear (conditioned stimulus, CS+) signaled delivery of an aversive shock unconditioned stimulus (US), whereas the same face with a neutral expression was unreinforced. In a subsequent generalization test, subjects were presented with faces expressing more or less fear intensity than the CS+. Psychophysiological results revealed an increase in the skin conductance response (SCR) during learning when the US was omitted. Omission-related SCRs were also observed during the generalization test following the offset of high-but not low-intensity face expressions. Neuroimaging results revealed omission-related neural activity during learning in the anterior cingulate cortex, parietal cortex, insula, and striatum. These same regions also showed omission-related responses during the generalization test following highly expressive fearful faces. Finally, regression analysis on omission responses during the generalization test revealed correlations in offset-related SCRs and neural activity in the dorsomedial prefrontal cortex and posterior parietal cortex. Thus, converging psychophysiological and neural activity upon omission of aversive stimulation provides a novel metric of US expectancy, even to generalized cues that had no prior history of reinforcement. PMID:22387662

  6. Benchmarking Evaluation Results for Prototype Extravehicular Activity Gloves

    NASA Technical Reports Server (NTRS)

    Aitchison, Lindsay; McFarland, Shane

    2012-01-01

    The Space Suit Assembly (SSA) Development Team at NASA Johnson Space Center has invested heavily in the advancement of rear-entry planetary exploration suit design but largely deferred development of extravehicular activity (EVA) glove designs, and accepted the risk of using the current flight gloves, Phase VI, for unique mission scenarios outside the Space Shuttle and International Space Station (ISS) Program realm of experience. However, as design reference missions mature, the risks of using heritage hardware have highlighted the need for developing robust new glove technologies. To address the technology gap, the NASA Game-Changing Technology group provided start-up funding for the High Performance EVA Glove (HPEG) Project in the spring of 2012. The overarching goal of the HPEG Project is to develop a robust glove design that increases human performance during EVA and creates pathway for future implementation of emergent technologies, with specific aims of increasing pressurized mobility to 60% of barehanded capability, increasing the durability by 100%, and decreasing the potential of gloves to cause injury during use. The HPEG Project focused initial efforts on identifying potential new technologies and benchmarking the performance of current state of the art gloves to identify trends in design and fit leading to establish standards and metrics against which emerging technologies can be assessed at both the component and assembly levels. The first of the benchmarking tests evaluated the quantitative mobility performance and subjective fit of four prototype gloves developed by Flagsuit LLC, Final Frontier Designs, LLC Dover, and David Clark Company as compared to the Phase VI. All of the companies were asked to design and fabricate gloves to the same set of NASA provided hand measurements (which corresponded to a single size of Phase Vi glove) and focus their efforts on improving mobility in the metacarpal phalangeal and carpometacarpal joints. Four test

  7. Geometry of the Farallon Slab Revealed by Joint Interpretation of Wavefield Imaging and Tomography Results from the Earthscope Transportable Array

    NASA Astrophysics Data System (ADS)

    Pavlis, G. L.; Wang, Y.

    2015-12-01

    A significant number of P and S wave tomography models have been produced in the past decade using various subsets of data from the Earthscope USArray and different inversion algorithms. We focus here on published tomography results that span large portions of the final footprint of the USArray. We use 3D visualization techniques to search for common features in different tomography models. We also compare tomography results to features seen in our current generation wavefield images. Recent innovations of our plane wave migration method have yielded what is arguably the highest resolution image ever produced of the mantle in the vicinity of the transition zone. The new results reveal a rich collection of coherent, dipping structures seen throughout the upper mantle and transition zone. These dipping interfaces are judged significant according to a coherence metric. We treat these surfaces as strain markers to assess proposed models for geometry of the 3D geometry of the Farallon Slab under North America. We find the following geologic interpretations are well supported by independent results: 1. The old Farallon under eastern North America and below the base of transition zone is universally seen as a high velocity anomaly. 2. All results support a simple, 3D kinematic model of the updip limit of the Farallon slab window that follows a track from Cape Mendocino, across Nevada, and northern Arizona and New Mexico. 3. All models show a strong low-velocity mantle under the southwestern U.S. 4. A low-velocity features is universally seen related to the Yellowstone-Snake River system. Shorter wavelength features observed in different tomography models are inconsistent showing that the theme of this session is very important to understand what features are in current results are real. Isopach maps of the thickness of the transition show a systematic difference in transition zone thickness in the western and eastern US. The transition zone thickens in the eastern US in

  8. Comparing GWAS Results of Complex Traits Using Full Genetic Model and Additive Models for Revealing Genetic Architecture

    PubMed Central

    Monir, Md. Mamun; Zhu, Jun

    2017-01-01

    Most of the genome-wide association studies (GWASs) for human complex diseases have ignored dominance, epistasis and ethnic interactions. We conducted comparative GWASs for total cholesterol using full model and additive models, which illustrate the impacts of the ignoring genetic variants on analysis results and demonstrate how genetic effects of multiple loci could differ across different ethnic groups. There were 15 quantitative trait loci with 13 individual loci and 3 pairs of epistasis loci identified by full model, whereas only 14 loci (9 common loci and 5 different loci) identified by multi-loci additive model. Again, 4 full model detected loci were not detected using multi-loci additive model. PLINK-analysis identified two loci and GCTA-analysis detected only one locus with genome-wide significance. Full model identified three previously reported genes as well as several new genes. Bioinformatics analysis showed some new genes are related with cholesterol related chemicals and/or diseases. Analyses of cholesterol data and simulation studies revealed that the full model performs were better than the additive-model performs in terms of detecting power and unbiased estimations of genetic variants of complex traits. PMID:28079101

  9. Rapid ester biosynthesis screening reveals a high activity alcohol-O-acyltransferase (AATase) from tomato fruit.

    PubMed

    Lin, Jyun-Liang; Zhu, Jie; Wheeldon, Ian

    2016-05-01

    Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl-CoA with an alcohol by alcohol-O-acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short- and medium-chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf-S.l). Atf1-S.l exhibited broad specificity towards acyl-CoAs with chain length from C4 to C10 and was specific towards 1-pentanol. The AATase screen also revealed new acyl-CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf-C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester-based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.

  10. Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center.

    PubMed

    Ruiz-Carrillo, David; Koch, Birgit; Parthier, Christoph; Wermann, Michael; Dambe, Tresfore; Buchholz, Mirko; Ludwig, Hans-Henning; Heiser, Ulrich; Rahfeld, Jens-Ulrich; Stubbs, Milton T; Schilling, Stephan; Demuth, Hans-Ulrich

    2011-07-19

    Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.

  11. Annoyance resulting from intrusion of aircraft sounds upon various activities

    NASA Technical Reports Server (NTRS)

    Gunn, W. J.; Shepherd, W. T.; Fletcher, J. L.

    1975-01-01

    An experiment was conducted in which subjects were engaged in TV viewing, telephone listening, or reverie (no activity) for a 1/2-hour session. During the session, they were exposed to a series of recorded aircraft sounds at the rate of one flight every 2 minutes. Within each session, four levels of flyover noise, separated by dB increments, were presented several times in a Latin Square balanced sequence. The peak level of the noisiest flyover in any session was fixed at 95, 90, 85, 75, or 70 dBA. At the end of the test session, subjects recorded their responses to the aircraft sounds, using a bipolar scale which covered the range from very pleasant to extremely annoying. Responses to aircraft noises were found to be significantly affected by the particular activity in which the subjects were engaged. Not all subjects found the aircraft sounds to be annoying.

  12. Comparative proteomic analysis reveals activation of mucosal innate immune signaling pathways during cholera.

    PubMed

    Ellis, Crystal N; LaRocque, Regina C; Uddin, Taher; Krastins, Bryan; Mayo-Smith, Leslie M; Sarracino, David; Karlsson, Elinor K; Rahman, Atiqur; Shirin, Tahmina; Bhuiyan, Taufiqur R; Chowdhury, Fahima; Khan, Ashraful Islam; Ryan, Edward T; Calderwood, Stephen B; Qadri, Firdausi; Harris, Jason B

    2015-03-01

    Vibrio cholerae O1 is a major cause of acute watery diarrhea in over 50 countries. Evidence suggests that V. cholerae O1 may activate inflammatory pathways, and a recent study of a Bangladeshi population showed that variants in innate immune genes play a role in mediating susceptibility to cholera. We analyzed human proteins present in the small intestine of patients infected with V. cholerae O1 to characterize the host response to this pathogen. We collected duodenal biopsy specimens from patients with acute cholera after stabilization and again 30 days after initial presentation. Peptides extracted from biopsy specimens were sequenced and quantified using label-free mass spectrometry and SEQUEST. Twenty-seven host proteins were differentially abundant between the acute and convalescent stages of infection; the majority of these have known roles in innate defense, cytokine production, and apoptosis. Immunostaining confirmed that two proteins, WARS and S100A8, were more abundant in lamina propria cells during the acute stage of cholera. Analysis of the differentially abundant proteins revealed the activation of key regulators of inflammation by the innate immune system, including Toll-like receptor 4, nuclear factor kappa-light-chain-enhancer of activated B cells, mitogen-activated protein kinases, and caspase-dependent inflammasomes. Interleukin-12β (IL-12β) was a regulator of several proteins that were activated during cholera, and we confirmed that IL-12β was produced by lymphocytes recovered from duodenal biopsy specimens of cholera patients. Our study shows that a broad inflammatory response is generated in the gut early after onset of cholera, which may be critical in the development of long-term mucosal immunity against V. cholerae O1.

  13. A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

    PubMed Central

    Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Stein, Richard A.; Bonner, Ross; Talley, Lauren; Parker, Mark D.; Mchaourab, Hassane S.; Yee, Vivien C.; Lodowski, David T.

    2015-01-01

    Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators. PMID:26415570

  14. Simultaneous fNIRS and thermal infrared imaging during cognitive task reveal autonomic correlates of prefrontal cortex activity

    NASA Astrophysics Data System (ADS)

    Pinti, Paola; Cardone, Daniela; Merla, Arcangelo

    2015-12-01

    Functional Near Infrared-Spectroscopy (fNIRS) represents a powerful tool to non-invasively study task-evoked brain activity. fNIRS assessment of cortical activity may suffer for contamination by physiological noises of different origin (e.g. heart beat, respiration, blood pressure, skin blood flow), both task-evoked and spontaneous. Spontaneous changes occur at different time scales and, even if they are not directly elicited by tasks, their amplitude may result task-modulated. In this study, concentration changes of hemoglobin were recorded over the prefrontal cortex while simultaneously recording the facial temperature variations of the participants through functional infrared thermal (fIR) imaging. fIR imaging provides touch-less estimation of the thermal expression of peripheral autonomic. Wavelet analysis revealed task-modulation of the very low frequency (VLF) components of both fNIRS and fIR signals and strong coherence between them. Our results indicate that subjective cognitive and autonomic activities are intimately linked and that the VLF component of the fNIRS signal is affected by the autonomic activity elicited by the cognitive task. Moreover, we showed that task-modulated changes in vascular tone occur both at a superficial and at larger depth in the brain. Combined use of fNIRS and fIR imaging can effectively quantify the impact of VLF autonomic activity on the fNIRS signals.

  15. Simultaneous fNIRS and thermal infrared imaging during cognitive task reveal autonomic correlates of prefrontal cortex activity

    PubMed Central

    Pinti, Paola; Cardone, Daniela; Merla, Arcangelo

    2015-01-01

    Functional Near Infrared-Spectroscopy (fNIRS) represents a powerful tool to non-invasively study task-evoked brain activity. fNIRS assessment of cortical activity may suffer for contamination by physiological noises of different origin (e.g. heart beat, respiration, blood pressure, skin blood flow), both task-evoked and spontaneous. Spontaneous changes occur at different time scales and, even if they are not directly elicited by tasks, their amplitude may result task-modulated. In this study, concentration changes of hemoglobin were recorded over the prefrontal cortex while simultaneously recording the facial temperature variations of the participants through functional infrared thermal (fIR) imaging. fIR imaging provides touch-less estimation of the thermal expression of peripheral autonomic. Wavelet analysis revealed task-modulation of the very low frequency (VLF) components of both fNIRS and fIR signals and strong coherence between them. Our results indicate that subjective cognitive and autonomic activities are intimately linked and that the VLF component of the fNIRS signal is affected by the autonomic activity elicited by the cognitive task. Moreover, we showed that task-modulated changes in vascular tone occur both at a superficial and at larger depth in the brain. Combined use of fNIRS and fIR imaging can effectively quantify the impact of VLF autonomic activity on the fNIRS signals. PMID:26632763

  16. Field-scale tracking of active methane-oxidizing communities in a landfill cover soil reveals spatial and seasonal variability.

    PubMed

    Henneberger, Ruth; Chiri, Eleonora; Bodelier, Paul E L; Frenzel, Peter; Lüke, Claudia; Schroth, Martin H

    2015-05-01

    Aerobic methane-oxidizing bacteria (MOB) in soils mitigate methane (CH4 ) emissions. We assessed spatial and seasonal differences in active MOB communities in a landfill cover soil characterized by highly variable environmental conditions. Field-based measurements of CH4 oxidation activity and stable-isotope probing of polar lipid-derived fatty acids (PLFA-SIP) were complemented by microarray analysis of pmoA genes and transcripts, linking diversity and function at the field scale. In situ CH4 oxidation rates varied between sites and were generally one order of magnitude lower in winter compared with summer. Results from PLFA-SIP and pmoA transcripts were largely congruent, revealing distinct spatial and seasonal clustering. Overall, active MOB communities were highly diverse. Type Ia MOB, specifically Methylomonas and Methylobacter, were key drivers for CH4 oxidation, particularly at a high-activity site. Type II MOB were mainly active at a site showing substantial fluctuations in CH4 loading and soil moisture content. Notably, Upland Soil Cluster-gamma-related pmoA transcripts were also detected, indicating concurrent oxidation of atmospheric CH4 . Spatial separation was less distinct in winter, with Methylobacter and uncultured MOB mediating CH4 oxidation. We propose that high diversity of active MOB communities in this soil is promoted by high variability in environmental conditions, facilitating substantial removal of CH4 generated in the waste body.

  17. Chimeric CD46/DAF molecules reveal a cryptic functional role for SCR1 of DAF in regulating complement activation.

    PubMed

    Christiansen, D; Loveland, B; Kyriakou, P; Lanteri, M; Rubinstein, E; Gerlier, D

    2000-01-01

    Chimeric proteins using membrane cofactor (CD46) and decay accelerating factor (DAF or CD55) were generated to further investigate the functional domains involved in the regulation of human serum complement. Following activation of the classical pathway, the isolated substitution of CD46 SCR III (x3DAF) exhibited a modest regulatory activity comparable to that of CD46. The isolated substitution of CD46 SCR IV (x4DAF), and the combined CD46 SCR III+IV substitutions (x3/4DAF) were essentially as efficient as DAF. No regulation of C3b deposition was observed with the combined CD46 SCR I+II substitutions (x1/2DAF). When tested after activation of the alternative pathway, both the x3DAF and x3/4DAF chimeras failed to regulate C3b deposition, while the x4DAF chimera still displayed some activity. In contrast to that observed following classical pathway activation, the x1/2DAF chimera exhibited a similar efficiency to wild type CD46 and DAF in controlling C3b deposition. Using SCR specific antibodies, the regulatory activity of the x1/2DAF chimera against the alternative pathway was mapped to the first three distal SCR (i.e. DAF 1, DAF 2 and CD46 III). These data demonstrate that several combinations of SCR domains from two related complement regulators can result in functional molecules, and reveal a novel and cryptic functional role for DAF SCR1.

  18. Experimental results using active control of traveling wave power flow

    NASA Technical Reports Server (NTRS)

    Miller, David W.; Hall, Steven R.

    1991-01-01

    Active structural control experiments conducted on a 24-ft pinned-free beam derived feedback compensators on the basis of a traveling-wave approach. A compensator is thus obtained which eliminates resonant behavior by absorbing all impinging power. A causal solution is derived for this noncausal compensator which mimics its behavior in a given frequency range, using the Wiener-Hopf. This optimal Wiener-Hopf compensator's structure-damping performance is found to exceed any obtainable by means of rate feedback. Performance limitations encompassed the discovery of frequencies above which the sensor and actuator were no longer dual and an inadvertent coupling of the control hardware to unmodeled structure torsion modes.

  19. Towards active capsular endoscopy: preliminary results on a legged platform.

    PubMed

    Menciassi, Arianna; Stefanini, Cesare; Orlandi, Giovanni; Quirini, Marco; Dario, Paolo

    2006-01-01

    This paper illustrates the problem of active locomotion in the gastrointestinal tract for endoscopic capsules. Authors analyze the problem of locomotion in unstructured, flexible and tubular environments and explain the reasons leading to the selection of a legged system. They present a theoretical simulation of legged capsule locomotion, which is used to define the optimal parameters for capsule design and gait selection. Finally, a legged capsule--about 3 cm3 in volume--is presented; it consists of 4 back legs whose actuation is achieved thanks to a miniaturized DC brushless motor. In vitro tests demonstrate good performance in terms of achievable speed (92 mm/min).

  20. The elastase-PK101 structure: Mechanism of an ultrasensitive activity-based probe revealed

    DOE PAGES

    Lechtenberg, Bernhard C.; Robinson, Howard R.; Kasperkiewicz, Paulina; ...

    2015-01-22

    Human neutrophil elastase (HNE) plays a central role in neutrophil host defense, but its broad specificity makes HNE a difficult target for both inhibitor and probe development. Recently, we identified the unnatural amino acid containing activity-based probe PK101, which exhibits astounding sensitivity and selectivity for HNE, yet completely lacks mechanistic explanation for its unique characteristics. Here, we present the crystal structure of the HNE-PK101 complex which not only reveals the basis for PK101 ultrasensitivity but also uncovers so far unrecognized HNE features. Strikingly, the Nle(O-Bzl) function in the P4 position of PK101 reveals and leverages an “exo-pocket” on HNE asmore » a critical factor for selectivity. Furthermore, the PK101 P3 position harbors a methionine dioxide function, which mimics a post-translationally oxidized methionine residue and forms a critical hydrogen bond to the backbone amide of Gly219 of HNE. Gly219 resides in a Gly–Gly motif that is unique to HNE, yet compulsory for this interaction. Consequently, this feature enables HNE to accommodate substrates that have undergone methionine oxidation, which constitutes a hallmark post-translational modification of neutrophil signaling.« less

  1. The elastase-PK101 structure: Mechanism of an ultrasensitive activity-based probe revealed

    SciTech Connect

    Lechtenberg, Bernhard C.; Robinson, Howard R.; Kasperkiewicz, Paulina; Drag, Marcin; Riedl, Stefan J.

    2015-01-22

    Human neutrophil elastase (HNE) plays a central role in neutrophil host defense, but its broad specificity makes HNE a difficult target for both inhibitor and probe development. Recently, we identified the unnatural amino acid containing activity-based probe PK101, which exhibits astounding sensitivity and selectivity for HNE, yet completely lacks mechanistic explanation for its unique characteristics. Here, we present the crystal structure of the HNE-PK101 complex which not only reveals the basis for PK101 ultrasensitivity but also uncovers so far unrecognized HNE features. Strikingly, the Nle(O-Bzl) function in the P4 position of PK101 reveals and leverages an “exo-pocket” on HNE as a critical factor for selectivity. Furthermore, the PK101 P3 position harbors a methionine dioxide function, which mimics a post-translationally oxidized methionine residue and forms a critical hydrogen bond to the backbone amide of Gly219 of HNE. Gly219 resides in a Gly–Gly motif that is unique to HNE, yet compulsory for this interaction. Consequently, this feature enables HNE to accommodate substrates that have undergone methionine oxidation, which constitutes a hallmark post-translational modification of neutrophil signaling.

  2. Spatially Defined EGF Receptor Activation Reveals an F-Actin-Dependent Phospho-Erk Signaling Complex

    PubMed Central

    Singhai, Amit; Wakefield, Devin L.; Bryant, Kirsten L.; Hammes, Stephen R.; Holowka, David; Baird, Barbara

    2014-01-01

    We investigated the association of signaling proteins with epidermal growth factor (EGF) receptors (EGFR) using biotinylated EGF bound to streptavidin that is covalently coupled in an ordered array of micron-sized features on silicon surfaces. Using NIH-3T3 cells stably expressing EGFR, we observe concentration of fluorescently labeled receptors and stimulated tyrosine phosphorylation that are spatially confined to the regions of immobilized EGF and quantified by cross-correlation analysis. We observe recruitment of phosphorylated paxillin to activated EGFR at these patterned features, as well as β1-containing integrins that preferentially localize to more peripheral EGF features, as quantified by radial fluorescence analysis. In addition, we detect recruitment of EGFP-Ras, MEK, and phosphorylated Erk to patterned EGF in a process that depends on F-actin and phosphoinositides. These studies reveal and quantify the coformation of multiprotein EGFR signaling complexes at the plasma membrane in response to micropatterned growth factors. PMID:25468343

  3. Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centre

    SciTech Connect

    Boucher, Ian W.; Kalliomaa, Anne K.; Levdikov, Vladimir M.; Blagova, Elena V.; Fogg, Mark J.; Brannigan, James A. Wilson, Keith S.; Wilkinson, Anthony J.

    2005-07-01

    The crystal structures of two manganese superoxide dismutases from B. anthracis were solved by X-ray crystallography using molecular replacement. The BA4499 and BA5696 genes of Bacillus anthracis encode proteins homologous to manganese superoxide dismutase, suggesting that this organism has an expanded repertoire of antioxidant proteins. Differences in metal specificity and quaternary structure between the dismutases of prokaryotes and higher eukaryotes may be exploited in the development of therapeutic antibacterial compounds. Here, the crystal structure of two Mn superoxide dismutases from B. anthracis solved to high resolution are reported. Comparison of their structures reveals that a highly conserved residue near the active centre is substituted in one of the proteins and that this is a characteristic feature of superoxide dismutases from the B. cereus/B. anthracis/B. thuringiensis group of organisms.

  4. Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

    PubMed Central

    He, Yufan; Haque, Mohammad Mahfuzul; Stuehr, Dennis J.; Lu, H. Peter

    2015-01-01

    Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS. PMID:26311846

  5. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.

    PubMed

    Jinek, Martin; Jiang, Fuguo; Taylor, David W; Sternberg, Samuel H; Kaya, Emine; Ma, Enbo; Anders, Carolin; Hauer, Michael; Zhou, Kaihong; Lin, Steven; Kaplan, Matias; Iavarone, Anthony T; Charpentier, Emmanuelle; Nogales, Eva; Doudna, Jennifer A

    2014-03-14

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

  6. Functional mapping of protein kinase A reveals its importance in adult Schistosoma mansoni motor activity.

    PubMed

    de Saram, Paulu S R; Ressurreição, Margarida; Davies, Angela J; Rollinson, David; Emery, Aidan M; Walker, Anthony J

    2013-01-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and 'smart' anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.

  7. The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

    SciTech Connect

    Chitnumsub, Penchit Ittarat, Wanwipa; Jaruwat, Aritsara; Noytanom, Krittikar; Amornwatcharapong, Watcharee; Pornthanakasem, Wichai; Chaiyen, Pimchai; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

    2014-06-01

    The crystal structure of P. falciparum SHMT revealed snapshots of an intriguing disulfide/sulfhydryl switch controlling the functional activity. Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme.

  8. The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on β-glucan.

    PubMed

    Miotto, Lis Schwartz; de Rezende, Camila Alves; Bernardes, Amanda; Serpa, Viviane Isabel; Tsang, Adrian; Polikarpov, Igor

    2014-01-01

    The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

  9. Differential brain activity states during the perception and nonperception of illusory motion as revealed by magnetoencephalography.

    PubMed

    Crowe, David A; Leuthold, Arthur C; Georgopoulos, Apostolos P

    2010-12-28

    We studied visual perception using an annular random-dot motion stimulus called the racetrack. We recorded neural activity using magnetoencephalography while subjects viewed variants of this stimulus that contained no inherent motion or various degrees of embedded motion. Subjects reported seeing rotary motion during viewing of all stimuli. We found that, in the absence of any motion signals, patterns of brain activity differed between states of motion perception and nonperception. Furthermore, when subjects perceived motion, activity states within the brain did not differ across stimuli of different amounts of embedded motion. In contrast, we found that during periods of nonperception brain-activity states varied with the amount of motion signal embedded in the stimulus. Taken together, these results suggest that during perception the brain may lock into a stable state in which lower-level signals are suppressed.

  10. Hellenic Amateur Astronomy Association's activities: Preliminary results on Perseids 2010

    NASA Astrophysics Data System (ADS)

    Maravelias, G.

    2011-01-01

    Preliminary results on the Perseids 2010 are presented. Visual and video observations were obtained by the author and a first reduction of the visual data shows that a maximum of ZHR ~120 was reached during the night 12-13 of August 2010. Moreover, a video setup was tested (DMK camera and UFO Capture v2) and the results show that, under some limitations, valuable data can be obtained.

  11. Palaeomagnetic results from Palaeocene basalts from Mongolia reveal no inclination shallowing at 60 Ma in Central Asia

    NASA Astrophysics Data System (ADS)

    Hankard, Fatim; Cogné, Jean-Pascal; Lagroix, France; Quidelleur, Xavier; Kravchinsky, Vadim A.; Bayasgalan, Amgalan; Lkhagvadorj, Purevdorj

    2008-01-01

    We present the results of a palaeomagnetic study of 277 cores drilled at 35 sites, in 32 basaltic flows from three Early Palaeocene volcanic regions in the Gobi Desert, Mongolia, at Sumber Uul (62.2 Ma; 42.6°N/104.0°E), Tulga (62.0 Ma; 43.2°N/104.1°E) and Khuts Uul (57.1 Ma; 43.2°N/104.6°E) localities. Samples from Sumber Uul (62.2 +/- 0.9 Ma) and Khuts Uul (57.1 +/- 0.8 Ma) localities were dated using the K-Ar Cassignol-Gillot technique. Stepwise thermal and alternating field demagnetizations isolated a stable A component of magnetization carried by single domain (SD) to nearly SD magnetite. We interpret this A component to be the primary magnetization of these basaltic lava flows.The Sumber Uul and Tulga data were combined and recomputed at the Sumber Uul locality because of their similar ages. The palaeopoles computed from the A components lie at λ = 85.2°N, φ = 92.5°E, dp/dm = 3.9/4.9 (n = 14 flows) for Sumber Uul-Tulga (average age: 62.1 +/- 5.9 Ma) and λ = 69.6°N, φ = 148.0°E, dp/dm = 6.3/7.3 (n = 14 flows) for Khuts Uul (average age: 57.1 +/- 0.8 Ma). The palaeomagnetic inclinations are steeper than expected at the sites and consequently our palaeopoles occupy a near-sided position with respect to the 60 Ma reference apparent polar wander path (APWP) pole for Europe (Besse & Courtillot 2002). However, they appear to fully conform to the new high-resolution APWP poles for the 65-42 Ma period of Moreau et al. (2007). Following these authors, we interpret this anomalous near-sided position of our poles as arising from a rapid true polar wander (TPW) event in the Palaeocene, highlighted by a cusp at anomalies 26-25 (61-56 Ma), inexistent in the European APWP of Besse & Courtillot (2002). We conclude that our new data do not reveal any anomalous shallow inclinations in the Central Asia Palaeocene effusive rocks which is consistent with the Late Jurassic/Early Cretaceous age of Mongol-Okhotsk ocean closure and amalgamation of Amuria and Siberia

  12. Summary of FY15 results of benchmark modeling activities

    SciTech Connect

    Arguello, J. Guadalupe

    2015-08-01

    Sandia is participating in the third phase of an is a contributing partner to a U.S.-German "Joint Project" entitled "Comparison of current constitutive models and simulation procedures on the basis of model calculations of the thermo-mechanical behavior and healing of rock salt." The first goal of the project is to check the ability of numerical modeling tools to correctly describe the relevant deformation phenomena in rock salt under various influences. Achieving this goal will lead to increased confidence in the results of numerical simulations related to the secure storage of radioactive wastes in rock salt, thereby enhancing the acceptance of the results. These results may ultimately be used to make various assertions regarding both the stability analysis of an underground repository in salt, during the operating phase, and the long-term integrity of the geological barrier against the release of harmful substances into the biosphere, in the post-operating phase.

  13. Generation of 2,000 breast cancer metabolic landscapes reveals a poor prognosis group with active serotonin production

    PubMed Central

    Leoncikas, Vytautas; Wu, Huihai; Ward, Lara T.; Kierzek, Andrzej M.; Plant, Nick J.

    2016-01-01

    A major roadblock in the effective treatment of cancers is their heterogeneity, whereby multiple molecular landscapes are classified as a single disease. To explore the contribution of cellular metabolism to cancer heterogeneity, we analyse the Metabric dataset, a landmark genomic and transcriptomic study of 2,000 individual breast tumours, in the context of the human genome-scale metabolic network. We create personalized metabolic landscapes for each tumour by exploring sets of active reactions that satisfy constraints derived from human biochemistry and maximize congruency with the Metabric transcriptome data. Classification of the personalized landscapes derived from 997 tumour samples within the Metabric discovery dataset reveals a novel poor prognosis cluster, reproducible in the 995-sample validation dataset. We experimentally follow mechanistic hypotheses resulting from the computational study and establish that active serotonin production is a major metabolic feature of the poor prognosis group. These data support the reconsideration of concomitant serotonin-specific uptake inhibitors treatment during breast cancer chemotherapy. PMID:26813959

  14. Results of Skylab medical experiment M171: Metabolic activity

    NASA Technical Reports Server (NTRS)

    Michel, E. L.; Rummel, J. A.; Sawin, C. F.; Buderer, M. C.; Lem, J. D.

    1974-01-01

    The experiment was conducted to establish whether man's ability to perform mechanical work would be progressively altered as a result of exposure to the weightless environment of space flight. The Skylab crewmen exercised on a bicycle ergometer at workloads approximating 25, 50, and 75 percent of their maximum aerobic capacity. The physiological parameters monitored were respiratory gas exchange, blood pressure, and vectorcardiogram/heart rate. The results of these tests indicate that the crewmen had no significant decrement in their responses to exercise during their exposure to zero gravity. The results of the third manned Skylab mission (Skylab 4) are presented and a comparison is made of the overall results obtained from the three successively longer Skylab manned missions. The Skylab 4 crewmembers' 84-day in-flight responses to exercise were no worse and were probably better than the responses of the crewmen on the first two Skylab missions. Indications that exercise was an important contributing factor in maintaining this response are discussed.

  15. (13)C ENDOR Spectroscopy of Lipoxygenase-Substrate Complexes Reveals the Structural Basis for C-H Activation by Tunneling.

    PubMed

    Horitani, Masaki; Offenbacher, Adam R; Carr, Cody A Marcus; Yu, Tao; Hoeke, Veronika; Cutsail, George E; Hammes-Schiffer, Sharon; Klinman, Judith P; Hoffman, Brian M

    2017-02-08

    In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn(2+) as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. (13)C ENDOR then reveals the locations of (13)C10 and reactive (13)C11 of linoleic acid relative to the metal; (1)H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

  16. Metabolic analysis revealed altered amino acid profiles in Lupinus albus organs as a result of boron deficiency.

    PubMed

    Alves, Marta; Chicau, Paula; Matias, Helena; Passarinho, José; Pinheiro, Carla; Ricardo, Cândido Pinto

    2011-07-01

    We analysed the changes in the metabolites of Lupinus albus organs (leaf-blades, petioles, apexes, hypocotyls and roots) as a consequence of B deficiency. The deficiency did not affect malate concentration and induced only minor changes in the sugar content, suggesting that the carbohydrate metabolism is little affected by the deficiency. Contrarily, marked changes in the content of free amino acids were observed, with some specific variations associated with the different organs. These changes indicate that various aspects of metabolism implicated in the amino acid accumulation were affected by B deficiency. Most of the detected changes appear to have implications with some stress responses or signalling processes. Asparagine and proline that increase in many stresses also accumulated in petioles, apexes and hypocotyls. Accumulation of γ-aminobutyric acid shunt amino acids, indicative of production of reactive oxygen species, occurs in the same three organs and also the roots. The increase in the branched-chain amino acids, observed in all organs, suggests the involvement of B with the cytoskeleton, whereas glycine decrease in leaf-blades and active growing organs (apexes and roots) could be associated with the proposed role of this amino acids in plant signalling in processes that might be associated with the decreased growth rates observed in B deficiency. Despite the admitted importance of free amino acids in plant metabolism, the available information on this matter is scarce. So our results bring new information concerning the effects of B deficiency in the metabolism of the several L. albus organs.

  17. Voltage-Sensitive Dyes And Imaging Techniques Reveal New Patterns Of Electrical Activity In Heart Cortex

    NASA Astrophysics Data System (ADS)

    Salama, Guy

    1988-04-01

    Voltage-sensitive dyes bind to the plasms membrane of excitable cells (ie., muscle or nerve cells) and exhibit fluorescence and/or absorption changes that vary linearly with changes in transmembrane electrical potential. These potentiometric optical probes can be used to measure local changes in transmembrane potential by monitoring optical signals from dye molecules bound to the surface membrane. Consequently, when excitable cells are stained with such a dye and are stimulated to fire an electrical impulse (ie., an action potential (AP)), the changes in dye fluorescence have the characteristic shape and time course of APs recorded with an intracellular micro-electrode. Potentiometric dyes in conjuction with imaging techniques can now be used to visualize complex patterns and propagation of electrical activity. With photodiode arrays on video imaging techniques, patterns of biological electrical activity can be obtained with high temporal and spatial resolution which could not be obtained by conventional micro-electrodes. These methods reveal new details and offer powerful approaches to study fundamental problem in cardiac electrophysiology, communication in nerve networks, and the organization of cortical neurons.

  18. Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

    PubMed Central

    Weinstock, Matthew T.; Jacobsen, Michael T.; Kay, Michael S.

    2014-01-01

    Mirror-image proteins (composed of d-amino acids) are promising therapeutic agents and drug discovery tools, but as synthesis of larger d-proteins becomes feasible, a major anticipated challenge is the folding of these proteins into their active conformations. In vivo, many large and/or complex proteins require chaperones like GroEL/ES to prevent misfolding and produce functional protein. The ability of chaperones to fold d-proteins is unknown. Here we examine the ability of GroEL/ES to fold a synthetic d-protein. We report the total chemical synthesis of a 312-residue GroEL/ES-dependent protein, DapA, in both l- and d-chiralities, the longest fully synthetic proteins yet reported. Impressively, GroEL/ES folds both l- and d-DapA. This work extends the limits of chemical protein synthesis, reveals ambidextrous GroEL/ES folding activity, and provides a valuable tool to fold d-proteins for drug development and mirror-image synthetic biology applications. PMID:25071217

  19. Quantitative proteomics reveals the induction of mitophagy in tumor necrosis factor-α-activated (TNFα) macrophages.

    PubMed

    Bell, Christina; English, Luc; Boulais, Jonathan; Chemali, Magali; Caron-Lizotte, Olivier; Desjardins, Michel; Thibault, Pierre

    2013-09-01

    Macrophages play an important role in innate and adaptive immunity as professional phagocytes capable of internalizing and degrading pathogens to derive antigens for presentation to T cells. They also produce pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that mediate local and systemic responses and direct the development of adaptive immunity. The present work describes the use of label-free quantitative proteomics to profile the dynamic changes of proteins from resting and TNF-α-activated mouse macrophages. These analyses revealed that TNF-α activation of macrophages led to the down-regulation of mitochondrial proteins and the differential regulation of several proteins involved in vesicle trafficking and immune response. Importantly, we found that the down-regulation of mitochondria proteins occurred through mitophagy and was specific to TNF-α, as other cytokines such as IL-1β and IFN-γ had no effect on mitochondria degradation. Furthermore, using a novel antigen presentation system, we observed that the induction of mitophagy by TNF-α enabled the processing and presentation of mitochondrial antigens at the cell surface by MHC class I molecules. These findings highlight an unsuspected role of TNF-α in mitophagy and expanded our understanding of the mechanisms responsible for MHC presentation of self-antigens.

  20. Activity-Based Protein Profiling Reveals Broad Reactivity of the Nerve Agent Sarin.

    PubMed

    Tuin, Adriaan W; Mol, Marijke A E; van den Berg, Roland M; Fidder, A; van der Marel, Gijs A; Overkleeft, Herman S; Noort, Daan

    2009-04-01

    Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.

  1. Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling

    NASA Astrophysics Data System (ADS)

    Mobberley, J. M.; Khodadad, C. L. M.; Visscher, P. T.; Reid, R. P.; Hagan, P.; Foster, J. S.

    2015-07-01

    Microbialites are sedimentary deposits formed by the metabolic interactions of microbes and their environment. These lithifying microbial communities represent one of the oldest ecosystems on Earth, yet the molecular mechanisms underlying the function of these communities are poorly understood. In this study, we used comparative metagenomic and metatranscriptomic analyses to characterize the spatial organization of the thrombolites of Highborne Cay, The Bahamas, an actively forming microbialite system. At midday, there were differences in gene expression throughout the spatial profile of the thrombolitic mat with a high abundance of transcripts encoding genes required for photosynthesis, nitrogen fixation and exopolymeric substance production in the upper three mm of the mat. Transcripts associated with denitrification and sulfate reduction were in low abundance throughout the depth profile, suggesting these metabolisms were less active during midday. Comparative metagenomics of the Bahamian thrombolites with other known microbialite ecosystems from across the globe revealed that, despite many shared core pathways, the thrombolites represented genetically distinct communities. This study represents the first time the metatranscriptome of living microbialite has been characterized and offers a new molecular perspective on those microbial metabolisms, and their underlying genetic pathways, that influence the mechanisms of carbonate precipitation in lithifying microbial mat ecosystems.

  2. Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling

    PubMed Central

    Mobberley, J. M.; Khodadad, C. L. M.; Visscher, P. T.; Reid, R. P.; Hagan, P.; Foster, J. S.

    2015-01-01

    Microbialites are sedimentary deposits formed by the metabolic interactions of microbes and their environment. These lithifying microbial communities represent one of the oldest ecosystems on Earth, yet the molecular mechanisms underlying the function of these communities are poorly understood. In this study, we used comparative metagenomic and metatranscriptomic analyses to characterize the spatial organization of the thrombolites of Highborne Cay, The Bahamas, an actively forming microbialite system. At midday, there were differences in gene expression throughout the spatial profile of the thrombolitic mat with a high abundance of transcripts encoding genes required for photosynthesis, nitrogen fixation and exopolymeric substance production in the upper three mm of the mat. Transcripts associated with denitrification and sulfate reduction were in low abundance throughout the depth profile, suggesting these metabolisms were less active during midday. Comparative metagenomics of the Bahamian thrombolites with other known microbialite ecosystems from across the globe revealed that, despite many shared core pathways, the thrombolites represented genetically distinct communities. This study represents the first time the metatranscriptome of living microbialite has been characterized and offers a new molecular perspective on those microbial metabolisms, and their underlying genetic pathways, that influence the mechanisms of carbonate precipitation in lithifying microbial mat ecosystems. PMID:26213359

  3. Structure analysis reveals the flexibility of the ADAMTS-5 active site

    SciTech Connect

    Shieh, Huey-Sheng; Tomasselli, Alfredo G.; Mathis, Karl J.; Schnute, Mark E.; Woodard, Scott S.; Caspers, Nicole; Williams, Jennifer M.; Kiefer, James R.; Munie, Grace; Wittwer, Arthur; Malfait, Anne-Marie; Tortorella, Micky D.

    2012-03-02

    A ((1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl) succinamide derivative (here referred to as Compound 12) shows significant activity toward many matrix metalloproteinases (MMPs), including MMP-2, MMP-8, MMP-9, and MMP-13. Modeling studies had predicted that this compound would not bind to ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) due to its shallow S1' pocket. However, inhibition analysis revealed it to be a nanomolar inhibitor of both ADAMTS-4 and -5. The observed inconsistency was explained by analysis of crystallographic structures, which showed that Compound 12 in complex with the catalytic domain of ADAMTS-5 (cataTS5) exhibits an unusual conformation in the S1' pocket of the protein. This first demonstration that cataTS5 can undergo an induced conformational change in its active site pocket by a molecule like Compound 12 should enable the design of new aggrecanase inhibitors with better potency and selectivity profiles.

  4. Recent tectonic activity on Mercury revealed by small thrust fault scarps

    NASA Astrophysics Data System (ADS)

    Watters, Thomas R.; Daud, Katie; Banks, Maria E.; Selvans, Michelle M.; Chapman, Clark R.; Ernst, Carolyn M.

    2016-10-01

    Large tectonic landforms on the surface of Mercury, consistent with significant contraction of the planet, were revealed by the flybys of Mariner 10 in the mid-1970s. The MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) mission confirmed that the planet's past 4 billion years of tectonic history have been dominated by contraction expressed by lobate fault scarps that are hundreds of kilometres long. Here we report the discovery of small thrust fault scarps in images from the low-altitude campaign at the end of the MESSENGER mission that are orders of magnitude smaller than the large-scale lobate scarps. These small scarps have tens of metres of relief, are only kilometres in length and are comparable in scale to small young scarps on the Moon. Their small-scale, pristine appearance, crosscutting of impact craters and association with small graben all indicate an age of less than 50 Myr. We propose that these scarps are the smallest members of a continuum in scale of thrust fault scarps on Mercury. The young age of the small scarps, along with evidence for recent activity on large-scale scarps, suggests that Mercury is tectonically active today and implies a prolonged slow cooling of the planet's interior.

  5. The uses and results of active tetanus immunization

    PubMed Central

    Scheibel, Inga

    1955-01-01

    Both in animal experiments and in the course of two world wars active immunization has proved a safe method of protection against tetanus, and a method superior to passive serum prophylaxis. The three types of vaccine—plain, combined, and precipitated or adsorbed—all have their advantages and disadvantages, and the choice between them must be left to individual national health authorities. They should, however, be administered in two or three doses to confer basic immunity. What amount of circulating antitoxin is necessary to give full protection has not been accurately determined, but it is clear that one recall dose should be given about a year after the first injections as part of the routine course of injections. This seems enough to provide a long-lasting immunity, but a dose of vaccine should also be given at the time of injury. General immunization of the population is not practicable, but children, who are among the groups most at risk, can be immunized relatively simply by combined diphtheria and tetanus vaccine; in many countries, indeed, this is being done on an ever-increasing scale. PMID:13270078

  6. Quantitative Proteomic Analysis Reveals Molecular Adaptations in the Hippocampal Synaptic Active Zone of Chronic Mild Stress-Unsusceptible Rats

    PubMed Central

    Zhou, Jian; Liu, Zhao; Yu, Jia; Han, Xin; Fan, Songhua; Shao, Weihua; Chen, Jianjun; Qiao, Rui

    2016-01-01

    Background: While stressful events are recognized as an important cause of major depressive disorder, some individuals exposed to life stressors maintain normal psychological functioning. The molecular mechanism(s) underlying this phenomenon remain unclear. Abnormal transmission and plasticity of hippocampal synapses have been implied to play a key role in the pathoetiology of major depressive disorder. Methods: A chronic mild stress protocol was applied to separate susceptible and unsusceptible rat subpopulations. Proteomic analysis using an isobaric tag for relative and absolute quantitation coupled with tandem mass spectrometry was performed to identify differential proteins in enriched hippocampal synaptic junction preparations. Results: A total of 4318 proteins were quantified, and 89 membrane proteins were present in differential amounts. Of these, SynaptomeDB identified 81 (91%) having a synapse-specific localization. The unbiased profiles identified several candidate proteins within the synaptic junction that may be associated with stress vulnerability or insusceptibility. Subsequent functional categorization revealed that protein systems particularly involved in membrane trafficking at the synaptic active zone exhibited a positive strain as potential molecular adaptations in the unsusceptible rats. Moreover, through STRING and immunoblotting analysis, membrane-associated GTP-bound Rab3a and Munc18-1 appear to coregulate syntaxin-1/SNAP25/VAMP2 assembly at the hippocampal presynaptic active zone of unsusceptible rats, facilitating SNARE-mediated membrane fusion and neurotransmitter release, and may be part of a stress-protection mechanism in actively maintaining an emotional homeostasis. Conclusions: The present results support the concept that there is a range of potential protein adaptations in the hippocampal synaptic active zone of unsusceptible rats, revealing new investigative targets that may contribute to a better understanding of stress

  7. An in vitro screening with emerging contaminants reveals inhibition of carboxylesterase activity in aquatic organisms.

    PubMed

    Solé, Montserrat; Sanchez-Hernandez, Juan C

    2015-12-01

    Pharmaceuticals and personal care products (PPCPs) form part of the new generation of pollutants present in many freshwater and marine ecosystems. Although environmental concentrations of these bioactive substances are low, they cause sublethal effects (e.g., enzyme inhibition) in non-target organisms. However, little is known on metabolism of PPCPs by non-mammal species. Herein, an in vitro enzyme trial was performed to explore sensitivity of carboxylesterase (CE) activity of aquatic organisms to fourteen PPCPs. The esterase activity was determined in the liver of Mediterranean freshwater fish (Barbus meridionalis and Squalius laietanus), coastal marine fish (Dicentrarchus labrax and Solea solea), middle-slope fish (Trachyrhynchus scabrus), deep-sea fish (Alepocephalus rostratus and Cataetix laticeps), and in the digestive gland of a decapod crustacean (Aristeus antennatus). Results showed that 100μM of the lipid regulators simvastatin and fenofibrate significantly inhibited (30-80% of controls) the CE activity of all target species. Among the personal care products, nonylphenol and triclosan were strong esterase inhibitors in most species (36-68% of controls). Comparison with literature data suggests that fish CE activity is as sensitive to inhibition by some PPCPs as that of mammals, although their basal activity levels are lower than in mammals. Pending further studies on the interaction between PPCPs and CE activity, we postulate that this enzyme may act as a molecular sink for certain PPCPs in a comparable way than that described for the organophosphorus pesticides.

  8. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    PubMed

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-09-15

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

  9. Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

    SciTech Connect

    Kiburu, Irene N.; LaRonde-LeBlanc, Nicole

    2012-10-10

    Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 {angstrom} and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.

  10. Active populations of rare microbes in oceanic environments as revealed by bromodeoxyuridine incorporation and 454 tag sequencing.

    PubMed

    Hamasaki, Koji; Taniguchi, Akito; Tada, Yuya; Kaneko, Ryo; Miki, Takeshi

    2016-02-01

    The "rare biosphere" consisting of thousands of low-abundance microbial taxa is important as a seed bank or a gene pool to maintain microbial functional redundancy and robustness of the ecosystem. Here we investigated contemporaneous growth of diverse microbial taxa including rare taxa and determined their variability in environmentally distinctive locations along a north-south transect in the Pacific Ocean in order to assess which taxa were actively growing and how environmental factors influenced bacterial community structures. A bromodeoxyuridine-labeling technique in combination with PCR amplicon pyrosequencing of 16S rRNA genes gave 215-793 OTUs from 1200 to 3500 unique sequences in the total communities and 175-299 OTUs nearly 860 to 1800 sequences in the active communities. Unexpectedly, many of the active OTUs were not detected in the total fractions. Among these active but rare OTUs, some taxa (2-4% of rare OTUs) showed much higher abundance (>0.10% of total reads) in the active fraction than in the total fraction, suggesting that their contribution to bacterial community productivity or growth was much larger than that expected from their standing stocks at each location. An ordination plot by the principal component analysis presented that bacterial community compositions among 4 sampling locations and between total and active fractions were distinctive with each other. A redundancy analysis revealed that the variability of community compositions significantly correlated to seawater temperature and dissolved oxygen concentration. Also, a variation partitioning analysis showed that the environmental factors explained 49% of the variability of community compositions and the distance only explained 4.0% of its variability. These results implied very dynamic change of community structures due to environmental filtering. The active bacterial populations are more diverse and spread further in rare biosphere than we have ever seen. This study implied that rare

  11. Interspecies cathelicidin comparison reveals divergence in antimicrobial activity, TLR modulation, chemokine induction and regulation of phagocytosis

    PubMed Central

    Coorens, Maarten; Scheenstra, Maaike R.; Veldhuizen, Edwin J. A.; Haagsman, Henk P.

    2017-01-01

    Cathelicidins are short cationic peptides initially described as antimicrobial peptides, which can also modulate the immune system. Because most findings have been described in the context of human LL-37 or murine CRAMP, or have been investigated under varying conditions, it is unclear which functions are cathelicidin specific and which functions are general cathelicidin properties. This study compares 12 cathelicidins from 6 species under standardized conditions to better understand the conservation of cathelicidin functions. Most tested cathelicidins had strong antimicrobial activity against E. coli and/or MRSA. Interestingly, while more physiological culture conditions limit the antimicrobial activity of almost all cathelicidins against E. coli, activity against MRSA is enhanced. Seven out of 12 cathelicidins were able to neutralize LPS and another 7 cathelicidins were able to neutralize LTA; however, there was no correlation found with LPS neutralization. In contrast, only 4 cathelicidins enhanced DNA-induced TLR9 activation. In conclusion, these results provide new insight in the functional differences of cathelicidins both within and between species. In addition, these results underline the importance not to generalize cathelicidin functions and indicates that caution should be taken in extrapolating results from LL-37- or CRAMP-related studies to other animal settings. PMID:28102367

  12. Proteomics analysis of dendritic cell activation by contact allergens reveals possible biomarkers regulated by Nrf2.

    PubMed

    Mussotter, Franz; Tomm, Janina Melanie; El Ali, Zeina; Pallardy, Marc; Kerdine-Römer, Saadia; Götz, Mario; von Bergen, Martin; Haase, Andrea; Luch, Andreas

    2016-12-15

    Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2(+/+)) and Nrf2 knockout (nrf2(-/-)) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100μM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100μM CA, respectively. For 5 and 10μM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2(-/-) BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization.

  13. Interspecies insertion polymorphism analysis reveals recent activity of transposable elements in extant coelacanths.

    PubMed

    Naville, Magali; Chalopin, Domitille; Volff, Jean-Nicolas

    2014-01-01

    Coelacanths are lobe-finned fish represented by two extant species, Latimeria chalumnae in South Africa and Comoros and L. menadoensis in Indonesia. Due to their intermediate phylogenetic position between ray-finned fish and tetrapods in the vertebrate lineage, they are of great interest from an evolutionary point of view. In addition, extant specimens look similar to 300 million-year-old fossils; because of their apparent slowly evolving morphology, coelacanths have been often described as « living fossils ». As an underlying cause of such a morphological stasis, several authors have proposed a slow evolution of the coelacanth genome. Accordingly, sequencing of the L. chalumnae genome has revealed a globally low substitution rate for protein-coding regions compared to other vertebrates. However, genome and gene evolution can also be influenced by transposable elements, which form a major and dynamic part of vertebrate genomes through their ability to move, duplicate and recombine. In this work, we have searched for evidence of transposition activity in coelacanth genomes through the comparative analysis of orthologous genomic regions from both Latimeria species. Comparison of 5.7 Mb (0.2%) of the L. chalumnae genome with orthologous Bacterial Artificial Chromosome clones from L. menadoensis allowed the identification of 27 species-specific transposable element insertions, with a strong relative contribution of CR1 non-LTR retrotransposons. Species-specific homologous recombination between the long terminal repeats of a new coelacanth endogenous retrovirus was also detected. Our analysis suggests that transposon activity is responsible for at least 0.6% of genome divergence between both Latimeria species. Taken together, this study demonstrates that coelacanth genomes are not evolutionary inert: they contain recently active transposable elements, which have significantly contributed to post-speciation genome divergence in Latimeria.

  14. Electrocorticography reveals the temporal dynamics of posterior parietal cortical activity during recognition memory decisions

    PubMed Central

    Gonzalez, Alex; Hutchinson, J. Benjamin; Uncapher, Melina R.; Chen, Janice; LaRocque, Karen F.; Foster, Brett L.; Rangarajan, Vinitha; Parvizi, Josef; Wagner, Anthony D.

    2015-01-01

    Theories of the neurobiology of episodic memory predominantly focus on the contributions of medial temporal lobe structures, based on extensive lesion, electrophysiological, and imaging evidence. Against this backdrop, functional neuroimaging data have unexpectedly implicated left posterior parietal cortex (PPC) in episodic retrieval, revealing distinct activation patterns in PPC subregions as humans make memory-related decisions. To date, theorizing about the functional contributions of PPC has been hampered by the absence of information about the temporal dynamics of PPC activity as retrieval unfolds. Here, we leveraged electrocorticography to examine the temporal profile of high gamma power (HGP) in dorsal PPC subregions as participants made old/new recognition memory decisions. A double dissociation in memory-related HGP was observed, with activity in left intraparietal sulcus (IPS) and left superior parietal lobule (SPL) differing in time and sign for recognized old items (Hits) and correctly rejected novel items (CRs). Specifically, HGP in left IPS increased for Hits 300–700 ms poststimulus onset, and decayed to baseline ∼200 ms preresponse. By contrast, HGP in left SPL increased for CRs early after stimulus onset (200−300 ms) and late in the memory decision (from 700 ms to response). These memory-related effects were unique to left PPC, as they were not observed in right PPC. Finally, memory-related HGP in left IPS and SPL was sufficiently reliable to enable brain-based decoding of the participant’s memory state at the single-trial level, using multivariate pattern classification. Collectively, these data provide insights into left PPC temporal dynamics as humans make recognition memory decisions. PMID:26283375

  15. Current Results and Proposed Activities in Microgravity Fluid Dynamics

    NASA Technical Reports Server (NTRS)

    Polezhaev, V. I.

    1996-01-01

    The Institute for Problems in Mechanics' Laboratory work in mathematical and physical modelling of fluid mechanics develops models, methods, and software for analysis of fluid flow, instability analysis, direct numerical modelling and semi-empirical models of turbulence, as well as experimental research and verification of these models and their applications in technological fluid dynamics, microgravity fluid mechanics, geophysics, and a number of engineering problems. This paper presents an overview of the results in microgravity fluid dynamics research during the last two years. Nonlinear problems of weakly compressible and compressible fluid flows are discussed.

  16. Preliminary results and future activities at the GARFIELD apparatus

    NASA Astrophysics Data System (ADS)

    Gramegna, F.; Mastinu, P. F.; Vannucci, L.; Bruno, M.; D'Agostino, M.; Fiandri, L.; Lanchais, A.; Vannini, G.; Casini, G.; Chiari, M.; Nannini, A.; Bonasera, A.; Cavallaro, S.; Cosmano, A.; Moroni, A.; Ordine, A.; Abbondanno, U.; Milazzo, P. M.; Margagliotti, G. M.

    2002-01-01

    A new apparatus has been designed and built to study reaction mechanisms in the energy regime of the ALPI linear accelerator of the Laboratori Nazionali di Legnaro (E/A = 5 - 20 MeV). In this paper the importance of studying these mechanisms will be underlined, no more as a problem limited to a narrow energy range or a single process, but as a continuous trend from low to high energies and from the physics of stable nuclei to that one regarding instabilities. With this remarks in mind, a first experiment has been performed studying the reaction 32S+58Ni at 11AMeV. Preliminary results show that important information can be derived on multi-body emission, which can contribute to renew the interest in this energy regime.

  17. Uncoupling Malt1 threshold function from paracaspase activity results in destructive autoimmune inflammation.

    PubMed

    Gewies, Andreas; Gorka, Oliver; Bergmann, Hanna; Pechloff, Konstanze; Petermann, Franziska; Jeltsch, Katharina M; Rudelius, Martina; Kriegsmann, Mark; Weichert, Wilko; Horsch, Marion; Beckers, Johannes; Wurst, Wolfgang; Heikenwalder, Mathias; Korn, Thomas; Heissmeyer, Vigo; Ruland, Jürgen

    2014-11-20

    The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.

  18. Modulation of fructokinase activity of potato (Solanum tuberosum) results in substantial shifts in tuber metabolism.

    PubMed

    Davies, Howard V; Shepherd, Louise V T; Burrell, Michael M; Carrari, Fernando; Urbanczyk-Wochniak, Ewa; Leisse, Andrea; Hancock, Robert D; Taylor, Mark; Viola, Roberto; Ross, Heather; McRae, Diane; Willmitzer, Lothar; Fernie, Alisdair R

    2005-07-01

    Potato plants (Solanum tuberosum L. cvs Desiree and Record) transformed with sense and antisense constructs of a cDNA encoding the potato fructokinase StFK1 exhibited altered transcription of this gene, altered amount of protein and altered enzyme activities. Measurement of the maximal catalytic activity of fructokinase revealed a 2-fold variation in leaf (from 90 to 180% of wild type activity) and either a 10- or 30-fold variation in tuber (from 10 or 30% to 300% in Record and Desiree, respectively) activity. The comparative effect of the antisense construct in leaf and tuber tissue suggests that this isoform is only a minor contributor to the total fructokinase activity in the leaf but the predominant isoform in the tuber. Antisense inhibition of the fructokinase resulted in a reduced tuber yield; however, its overexpression had no impact on this parameter. The modulation of fructokinase activity had few, consistent effects on carbohydrate levels, with the exception of a general increase in glucose content in the antisense lines, suggesting that this enzyme is not important for the control of starch synthesis. However, when metabolic fluxes were estimated, it became apparent that the transgenic lines display a marked shift in metabolism, with the rate of redistribution of radiolabel to sucrose markedly affected by the activity of fructokinase. These data suggest an important role for fructokinase, acting in concert with sucrose synthase, in maintaining a balance between sucrose synthesis and degradation by a mechanism independent of that controlled by the hexose phosphate-mediated activation of sucrose phosphate synthase.

  19. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.

    PubMed

    Cho, Joo-Youn; Matsubara, Tsutomu; Kang, Dong Wook; Ahn, Sung-Hoon; Krausz, Kristopher W; Idle, Jeffrey R; Luecke, Hans; Gonzalez, Frank J

    2010-05-01

    Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3alpha,6,7alpha,12alpha-tetrol (3alpha,6,7alpha,12alpha-tetrahydroxy-5beta-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.

  20. Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of GPCR Activation

    PubMed Central

    Staus, Dean P; Strachan, Ryan T; Manglik, Aashish; Pani, Biswaranjan; Kahsai, Alem W; Kim, Tae Hun; Wingler, Laura M; Ahn, Seungkirl; Chatterjee, Arnab; Masoudi, Ali; Kruse, Andrew C; Pardon, Els; Steyaert, Jan; Weis, William I; Prosser, R. Scott; Kobilka, Brian K; Costa, Tommaso; Lefkowitz, Robert J

    2016-01-01

    G-protein coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies): a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60)6,7. We show that Nb60 stabilizes a previously unappreciated low affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the

  1. Determination of the physiological 2:2 TLR5:flagellin activation stoichiometry revealed by the activity of a fusion receptor

    SciTech Connect

    Ivičak-Kocjan, Karolina; Panter, Gabriela; Benčina, Mojca; Jerala, Roman

    2013-05-24

    Highlights: •The chimeric protein fusing flagellin to the TLR5 ectodomain is constitutively active. •Mutation P736H within the BB-loop of TLR5 TIR domain renders the receptor inactive. •The R90D mutation in flagellin inactivated autoactivation of the chimeric protein. •The 2:2 stoichiometry of the TLR5:flagellin complex is physiologically relevant. -- Abstract: Toll-like receptor 5 (TLR5) recognizes flagellin of most flagellated bacteria, enabling activation of the MyD88-dependent signaling pathway. The recently published crystal structure of a truncated zebrafish TLR5 ectodomain in complex with an inactive flagellin fragment indicated binding of two flagellin molecules to a TLR5 homodimer, however this complex did not dimerize in solution. In the present study, we aimed to determine the physiological stoichiometry of TLR5:flagellin activation by the use of a chimeric protein composed of an active flagellin fragment linked to the N-terminus of human TLR5 (SF-TLR5). This construct was constitutively active. Inactivation by the R90D mutation within flagellin demonstrated that autoactivation of the chimeric protein depended solely on the specific interaction between TLR5 and flagellin. Addition of wild-type hTLR5 substantially lowered autoactivation of SF-TLR5 in a concentration dependent manner, an effect which was reversible by the addition of exogenous Salmonella typhimurium flagellin, indicating the biological activity of a TLR5:flagellin complex with a 2:2 stoichiometry. These results, in addition to the combinations of inactive P736H mutation within the BB-loop of the TIR domain of TLR5 and SF-TLR5, further confirm the mechanism of TLR5 activation.

  2. Active nucleosome positioning beyond intrinsic biophysics is revealed by in vitro reconstitution.

    PubMed

    Korber, Philipp

    2012-04-01

    Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions ('genomic code for nucleosome positioning'). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called 'statistical positioning' may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5' ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.

  3. A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

    PubMed Central

    Hoggard, Timothy; Shor, Erika; Müller, Carolin A.; Nieduszynski, Conrad A.; Fox, Catherine A.

    2013-01-01

    Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time. PMID:24068963

  4. Cadmium-transformed cells in the in vitro cell transformation assay reveal different proliferative behaviours and activated pathways.

    PubMed

    Forcella, M; Callegaro, G; Melchioretto, P; Gribaldo, L; Frattini, M; Stefanini, F M; Fusi, P; Urani, C

    2016-10-01

    The in vitro Cell Transformation Assay (CTA) is a powerful tool for mechanistic studies of carcinogenesis. The endpoint is the classification of transformed colonies (foci) by means of standard morphological features. To increase throughput and reliability of CTAs, one of the suggested follow-up activities is to exploit the comprehension of the mechanisms underlying cell transformation. To this end, we have performed CTAs testing CdCl2, a widespread environmental contaminant classified as a human carcinogen with the underlying mechanisms of action not completely understood. We have isolated and re-seeded the cells at the end (6weeks) of in vitro CTAs to further identify the biochemical pathways underlying the transformed phenotype of foci. Morphological evaluations and proliferative assays confirmed the loss of contact-inhibition and the higher proliferative rate of transformed clones. The biochemical analysis of EGFR pathway revealed that, despite the same initial carcinogenic stimulus (1μM CdCl2 for 24h), transformed clones are characterized by the activation of two different molecular pathways: proliferation (Erk activation) or survival (Akt activation). Our preliminary results on molecular characterization of cell clones from different foci could be exploited for CTAs improvement, supporting the comprehension of the in vivo process and complementing the morphological evaluation of foci.

  5. Spatio-Temporal Analysis of Micro Economic Activities in Rome Reveals Patterns of Mixed-Use Urban Evolution.

    PubMed

    Fiasconaro, Alessandro; Strano, Emanuele; Nicosia, Vincenzo; Porta, Sergio; Latora, Vito

    2016-01-01

    Understanding urban growth is one with understanding how society evolves to satisfy the needs of its individuals in sharing a common space and adapting to the territory. We propose here a quantitative analysis of the historical development of a large urban area by investigating the spatial distribution and the age of commercial activities in the whole city of Rome. We find that the age of activities of various categories presents a very interesting double exponential trend, with a transition possibly related to the long-term economical effects determined by the oil crisis of the Seventies. The diversification of commercial categories, studied through various measures of entropy, shows, among other interesting features, a saturating behaviour with the density of activities. Moreover, different couples of commercial categories exhibit over the years a tendency to attract in space. Our results demonstrate that the spatio-temporal distribution of commercial activities can provide important insights on the urbanisation processes at work, revealing specific and non trivial socio-economical dynamics, as the presence of crisis periods and expansion trends, and contributing to the characterisation of the maturity of urban areas.

  6. Spatio-Temporal Analysis of Micro Economic Activities in Rome Reveals Patterns of Mixed-Use Urban Evolution

    PubMed Central

    Fiasconaro, Alessandro; Strano, Emanuele; Nicosia, Vincenzo; Porta, Sergio; Latora, Vito

    2016-01-01

    Understanding urban growth is one with understanding how society evolves to satisfy the needs of its individuals in sharing a common space and adapting to the territory. We propose here a quantitative analysis of the historical development of a large urban area by investigating the spatial distribution and the age of commercial activities in the whole city of Rome. We find that the age of activities of various categories presents a very interesting double exponential trend, with a transition possibly related to the long-term economical effects determined by the oil crisis of the Seventies. The diversification of commercial categories, studied through various measures of entropy, shows, among other interesting features, a saturating behaviour with the density of activities. Moreover, different couples of commercial categories exhibit over the years a tendency to attract in space. Our results demonstrate that the spatio-temporal distribution of commercial activities can provide important insights on the urbanisation processes at work, revealing specific and non trivial socio-economical dynamics, as the presence of crisis periods and expansion trends, and contributing to the characterisation of the maturity of urban areas. PMID:26982028

  7. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors

    PubMed Central

    Levis, Mark; Brown, Patrick; Smith, B. Douglas; Stine, Adam; Pham, Rosalyn; Stone, Richard; DeAngelo, Daniel; Galinsky, Ilene; Giles, Frank; Estey, Elihu; Kantarjian, Hagop; Cohen, Pamela; Wang, Yanfeng; Roesel, Johannes; Karp, Judith E.; Small, Donald

    2006-01-01

    We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML. PMID:16857987

  8. Working memory and lexical ambiguity resolution as revealed by ERPs: a difficult case for activation theories.

    PubMed

    Gunter, Thomas C; Wagner, Susanne; Friederici, Angela D

    2003-07-01

    This series of three event-related potential experiments explored the issue of whether the underlying mechanism of working memory (WM) supporting language processing is inhibitory or activational in nature. These different cognitive mechanisms have been proposed to explain the more efficient processing of subjects with a high WM span compared to those with a low WM span. Participants with high and low WM span were presented with sentences containing a homonym followed three words later by a nominal disambiguation cue and a final disambiguation using a verb. At the position of the disambiguation cue, inhibitory or activational WM mechanisms predict contrasting results. When activation is the underlying mechanism for efficient processing, the prediction is that high memory span persons activate both meanings of the homonym equally in WM, whereas low memory span persons only have one meaning present. When inhibition is the underlying mechanism, the predictions are the reverse. The ERP data, in particular, the variations of the meaning related N400 component, showed clear evidence for inhibition as the underlying cognitive mechanism in high-span readers. For low-span participants the cueing towards the dominant or the subordinate meaning elicited an equivalently large N400 component suggesting that both meanings are active in WM. In high-span subjects, the dominant disambiguation cue elicited a smaller N400 than the subordinate one, indicating that for these subjects particularly the dominant meaning is active. The experiments showed that inhibitory processes are probably underlying WM used during language comprehension in high-span subjects. Moreover, they demonstrate that these subjects can use their inhibition in a more flexible manner than low-span subjects. The effects that these processing differences have on the efficiency of language parsing are discussed.

  9. Results.

    ERIC Educational Resources Information Center

    Zemsky, Robert; Shaman, Susan; Shapiro, Daniel B.

    2001-01-01

    Describes the Collegiate Results Instrument (CRI), which measures a range of collegiate outcomes for alumni 6 years after graduation. The CRI was designed to target alumni from institutions across market segments and assess their values, abilities, work skills, occupations, and pursuit of lifelong learning. (EV)

  10. Ribosome•RelA structures reveal the mechanism of stringent response activation

    PubMed Central

    Loveland, Anna B; Bah, Eugene; Madireddy, Rohini; Zhang, Ying; Brilot, Axel F; Grigorieff, Nikolaus; Korostelev, Andrei A

    2016-01-01

    Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 Å wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding. DOI: http://dx.doi.org/10.7554/eLife.17029.001 PMID:27434674

  11. High throughput sequencing reveals alterations in the recombination signatures with diminishing Spo11 activity.

    PubMed

    Rockmill, Beth; Lefrançois, Philippe; Voelkel-Meiman, Karen; Oke, Ashwini; Roeder, G Shirleen; Fung, Jennifer C

    2013-10-01

    Spo11 is the topoisomerase-like enzyme responsible for the induction of the meiosis-specific double strand breaks (DSBs), which initiates the recombination events responsible for proper chromosome segregation. Nineteen PCR-induced alleles of SPO11 were identified and characterized genetically and cytologically. Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants. Arrest by ndt80 restored these events in two severe hypomorphic mutants, suggesting that ndt80-arrested nuclei are capable of extended DSB activity. While crossing-over, spore viability and synaptonemal complex (SC) formation defects correlated, the extent of such defects was not predictive of the level of heteroallelic gene conversions (prototrophs) exhibited by each mutant. High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type. By modeling the extent of these tract changes, we could account for the discrepancy in genetic measurements of prototrophy and crossover association. These findings provide an explanation for the unexpectedly low prototroph levels exhibited by spo11 hypomorphs and have important implications for genetic studies that assume an unbiased recovery of prototrophs, such as measurements of CO homeostasis. Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis).

  12. Transcriptional profiling of Chinese medicinal formula Si-Wu-Tang on breast cancer cells reveals phytoestrogenic activity

    PubMed Central

    2013-01-01

    Background Si-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women’s diseases. In our previous study, the microarray gene expression profiles of SWT on breast cancer cell line MCF-7 were found similar to the effect of β-estradiol (E2) on MCF-7 cells in the Connectivity Map database. Methods Further data analysis was conducted to find the main similarities and differences between the effects of SWT and E2 on MCF-7 gene expression. The cell proliferation assay on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells were used to examine such estrogenic activity. The estrogenic potency of SWT was further confirmed by estrogen-responsive element (ERE) luciferase reporter assay in MCF-7 cells. Results Many estrogen regulated genes strongly up-regulated by E2 were similarly up-regulated by SWT, e.g., GREB1, PGR and EGR3. Of interest with regard to safety of SWT, the oncogenes MYBL1 and RET were strongly induced by E2 but not by SWT. Quantitative RT-PCR analysis revealed a highly concordant expression change in selected genes with data obtained by microarrays. Further supporting SWT’s estrogenic activity, in MCF-7 but not in MDA-MB-231 cells, SWT stimulated cell growth at lower concentrations (< 3.0 mg/ml), while at high concentrations, it inhibits the growth of both cell lines. The growth inhibitory potency of SWT was significantly higher in MDA-MB-231 than in MCF-7 cells. The SWT-induced cell growth of MCF-7 could be blocked by addition of the estrogen receptor antagonist tamoxifen. In addition, SWT was able to activate the ERE activity at lower concentrations. The herbal components Angelicae, Chuanxiong and Rehmanniae at lower concentrations (< 3.0 mg/ml) also showed growth-inducing and ERE-activating activity in MCF-7 cells. Conclusions These results revealed a new mechanism to support the clinical use of SWT for estrogen related diseases

  13. Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing.

    PubMed

    Guo, Feng; Zhang, Tong

    2013-05-01

    Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also sequencing results, using two activated sludge samples (two sub-samples each, i.e. ethanol-fixed and fresh, as-is). The results indicate that the bead-beating step is necessary for DNA extraction from activated sludge. The two kits without the bead-beating step yielded very low amounts of DNA, and the least abundant operational taxonomic units (OTUs), and significantly underestimated the Gram-positive Actinobacteria, Nitrospirae, Chloroflexi, and Alphaproteobacteria and overestimated Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and the rare phyla whose cell walls might have been readily broken. Among the other five kits, FastDNA(@) SPIN Kit for Soil extracted the most and the purest DNA. Although the number of total OTUs obtained using this kit was not the highest, the abundant OTUs and abundance of Actinobacteria demonstrated its efficiency. The three MoBio kits and one ZR kit produced fair results, but had a relatively low DNA yield and/or less Actinobacteria-related sequences. Moreover, the 50 % ethanol fixation increased the DNA yield, but did not change the sequenced microbial community in a significant way. Based on the present study, the FastDNA SPIN kit for Soil is recommended for DNA extraction of activated sludge samples. More importantly, the selection of the DNA extraction kit must be done carefully if the samples contain dominant lysing-resistant groups, such as Actinobacteria and Nitrospirae.

  14. Response inhibition results in the emotional devaluation of faces: neural correlates as revealed by fMRI.

    PubMed

    Doallo, Sonia; Raymond, Jane E; Shapiro, Kimron L; Kiss, Monika; Eimer, Martin; Nobre, Anna C

    2012-08-01

    Although it is well established that prior experience with faces determines their subsequent social-emotional evaluation, recent work shows that top-down inhibitory mechanisms, including response inhibition, can lead to social devaluation after even a single, brief exposure. These rapidly induced effects indicate interplay among perceptual, attentional, response-selection and social-emotional networks; yet, the brain mechanisms underlying this are not well understood. This study used functional magnetic resonance imaging (fMRI) to investigate the neural mechanism mediating the relationship between inhibitory control and emotional devaluation. Participants performed two tasks: (i) a Go/No-Go task in response to faces and (ii) a trustworthiness rating task involving the previously seen faces. No-Go faces were rated as significantly less trustworthy than Go faces. By examining brain activations during Task 1, behavioral measures and brain activations obtained in Task 2 could be predicted. Specifically, activity in brain areas during Task 1 associated with (i) executive control and response suppression (i.e. lateral prefrontal cortex) and (ii) affective responses and value representation (i.e. orbitofrontal cortex), systematically covaried with behavioral ratings and amygdala activity obtained during Task 2. The present findings offer insights into the neural mechanisms linking inhibitory processes to affective responses.

  15. Activities and geochronology of (137)Cs in lake sediments resulting from sediment resuspension.

    PubMed

    Matisoff, Gerald

    2017-02-01

    In lakes with a large surface area to watershed ratio (137)Cs delivery is primarily by direct atmospheric fallout to the lake surface, where its activity in the sediments has been used to estimate the exposure to organisms and sediment mass deposition rates. Comparison of (137)Cs in the historical atmospheric fallout record with (137)Cs activity profiles in sediment cores reveals that although the general features of a maxima in the fallout deposition can be matched to activity peaks in the core, the general shape of the (137)Cs profile is not an exact replica of the fallout history. Instead, the sediment reflects post-depositional processes such as resuspension, bioturbation, partitioning of (137)Cs between the sediment solids and the pore fluids, and molecular diffusion of (137)Cs through the pore fluids. Presented here is a model that couples these processes to a system time averaging (STA) model that accounts for the time history of (137)Cs fallout and the particle residence time in the water column or in the 'active' surface sediment subject to resuspension. Sediment profiles are examined by comparing reasonable ranges of each of the coefficients of each of these major processes and by applying the model to cores collected from two large, shallow lakes, Lake Erie (USA/Canada) and Lake Winnipeg (Canada). The results indicate that the STA model with molecular diffusion and sediment resuspension best describes the data from these large, shallow lakes.

  16. FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca(2+)/PKA crosstalk.

    PubMed

    Schott, Micah B; Gonowolo, Faith; Maliske, Benjamin; Grove, Bryon

    2016-04-01

    Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca(2+)]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium "switch" that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca(2+) and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca(2+)/PKA crosstalk was not observed in cells expressing a gravin mutant that resisted calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca(2+) and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may

  17. Revealing the Origin of Activity in Nitrogen-Doped Nanocarbons towards Electrocatalytic Reduction of Carbon Dioxide.

    PubMed

    Xu, Junyuan; Kan, Yuhe; Huang, Rui; Zhang, Bingsen; Wang, Bolun; Wu, Kuang-Hsu; Lin, Yangming; Sun, Xiaoyan; Li, Qingfeng; Centi, Gabriele; Su, Dangsheng

    2016-05-23

    Carbon nanotubes (CNTs) are functionalized with nitrogen atoms for reduction of carbon dioxide (CO2 ). The investigation explores the origin of the catalyst's activity and the role of nitrogen chemical states therein. The catalysts show excellent performances, with about 90 % current efficiency for CO formation and stability over 60 hours. The Tafel analyses and density functional theory calculations suggest that the reduction of CO2 proceeds through an initial rate-determining transfer of one electron to CO2 , which leads to the formation of carbon dioxide radical anion (CO2 (.-) ). The initial reduction barrier is too high on pristine CNTs, resulting in a very high overpotentials at which the hydrogen evolution reaction dominates over CO2 reduction. The doped nitrogen atoms stabilize the radical anion, thereby lowering the initial reduction barrier and improving the intrinsic activity. The most efficient nitrogen chemical state for this reaction is quaternary nitrogen, followed by pyridinic and pyrrolic nitrogen.

  18. Cingulate seizure-like activity reveals neuronal avalanche regulated by network excitability and thalamic inputs

    PubMed Central

    2014-01-01

    Background Cortical neurons display network-level dynamics with unique spatiotemporal patterns that construct the backbone of processing information signals and contribute to higher functions. Recent years have seen a wealth of research on the characteristics of neuronal networks that are sufficient conditions to activate or cease network functions. Local field potentials (LFPs) exhibit a scale-free and unique event size distribution (i.e., a neuronal avalanche) that has been proven in the cortex across species, including mice, rats, and humans, and may be used as an index of cortical excitability. In the present study, we induced seizure activity in the anterior cingulate cortex (ACC) with medial thalamic inputs and evaluated the impact of cortical excitability and thalamic inputs on network-level dynamics. We measured LFPs from multi-electrode recordings in mouse cortical slices and isoflurane-anesthetized rats. Results The ACC activity exhibited a neuronal avalanche with regard to avalanche size distribution, and the slope of the power-law distribution of the neuronal avalanche reflected network excitability in vitro and in vivo. We found that the slope of the neuronal avalanche in seizure-like activity significantly correlated with cortical excitability induced by γ-aminobutyric acid system manipulation. The thalamic inputs desynchronized cingulate seizures and affected the level of cortical excitability, the modulation of which could be determined by the slope of the avalanche size. Conclusions We propose that the neuronal avalanche may be a tool for analyzing cortical activity through LFPs to determine alterations in network dynamics. PMID:24387299

  19. Modulation of the hormone setting by Rhodococcus fascians results in ectopic KNOX activation in Arabidopsis.

    PubMed

    Depuydt, Stephen; Dolezal, Karel; Van Lijsebettens, Mieke; Moritz, Thomas; Holsters, Marcelle; Vereecke, Danny

    2008-03-01

    The biotrophic actinomycete Rhodococcus fascians has a profound impact on plant development and a common aspect of the symptomatology is the deformation of infected leaves. In Arabidopsis (Arabidopsis thaliana), the serrated leaf margins formed upon infection resemble the leaf phenotype of transgenic plants with ectopic expression of KNOTTED-like homeobox (KNOX) genes. Through transcript profiling, we demonstrate that class-I KNOX genes are transcribed in symptomatic leaves. Functional analysis revealed that BREVIPEDICELLUS/KNOTTED-LIKE1 and mainly SHOOT MERISTEMLESS were essential for the observed leaf dissection. However, these results also positioned the KNOX genes downstream in the signaling cascade triggered by R. fascians infection. The much faster activation of ARABIDOPSIS RESPONSE REGULATOR5 and the establishment of homeostatic and feedback mechanisms to control cytokinin (CK) levels support the overrepresentation of this hormone in infected plants due to the secretion by the pathogen, thereby placing the CK response high up in the cascade. Hormone measurements show a net decrease of tested CKs, indicating either that secretion by the bacterium and degradation by the plant are in balance, or, as suggested by the strong reaction of 35S:CKX plants, that other CKs are at play. At early time points of the interaction, activation of gibberellin 2-oxidase presumably installs a local hormonal setting favorable for meristematic activity that provokes leaf serrations. The results are discussed in the context of symptom development, evasion of plant defense, and the establishment of a specific niche by R. fascians.

  20. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    SciTech Connect

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  1. Structures of BmrR-drug complexes reveal a rigid multidrug binding pocket and transcription activation through tyrosine expulsion.

    PubMed

    Newberry, Kate J; Huffman, Joy L; Miller, Marshall C; Vazquez-Laslop, Nora; Neyfakh, Alex A; Brennan, Richard G

    2008-09-26

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  2. DOPAMINE RECEPTOR ACTIVATION REVEALS A NOVEL, KYNURENATE-SENSITIVE COMPONENT OF STRIATAL NMDA NEUROTOXICITY

    PubMed Central

    Poeggeler, Burkhard; Rassoulpour, Arash; Wu, Hui-Qiu; Guidetti, Paolo; Roberts, Rosalinda C.; Schwarcz, Robert

    2007-01-01

    The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays an important role in brain physiology, but excessive receptor stimulation results in seizures and excitotoxic nerve cell death. NMDA receptor-mediated neuronal excitation and injury can be prevented by high, non-physiological concentrations of the neuroinhibitory tryptophan metabolite kynurenic acid (KYNA). Here we report that endogenous KYNA, which is formed in and released from astrocytes, controls NMDA receptors in vivo. This was revealed with the aid of the dopaminergic drugs d-amphetamine and apomorphine, which cause rapid, transient decreases in striatal KYNA levels in rats. Intrastriatal injections of the excitotoxins NMDA or quinolinate (but not the non-NMDA receptor agonist kainate) at the time of maximal KYNA reduction resulted in 2-3-fold increases in excitotoxic lesion size. Pre-treatment with kynurenine 3-hydroxylase inhibitors or dopamine receptor antagonists, two classes of pharmacological agents that prevented the reduction in brain KYNA caused by dopaminergic stimulation, abolished the potentiation of neurotoxicity. Thus, the present study identifies a previously unappreciated role of KYNA as a functional link between dopamine receptor stimulation and NMDA neurotoxicity in the striatum. PMID:17629627

  3. Extensive hydrothermal activity revealed by multi-tracer survey in the Wallis and Futuna region (SW Pacific)

    NASA Astrophysics Data System (ADS)

    Konn, C.; Fourré, E.; Jean-Baptiste, P.; Donval, J. P.; Guyader, V.; Birot, D.; Alix, A. S.; Gaillot, A.; Perez, F.; Dapoigny, A.; Pelleter, E.; Resing, J. A.; Charlou, J. L.; Fouquet, Y.

    2016-10-01

    The study area is close to the Wallis and Futuna Islands in the French EEZ. It exists on the western boundary of the fastest tectonic area in the world at the junction of the Lau and North-Fiji basins. At this place, the unstable back-arc accommodates the plate motion in three ways: (i) the north Fiji transform fault, (ii) numerous unstable spreading ridges, and (iii) large areas of recent volcanic activity. This instability creates bountiful opportunity for hydrothermal discharge to occur. Based on geochemical (CH4, TDM, 3He) and geophysical (nephelometry) tracer surveys: (1) no hydrothermal activity could be found on the Futuna Spreading Centre (FSC) which sets the western limit of hydrothermal activity; (2) four distinct hydrothermal active areas were identified: Kulo Lasi Caldera, Amanaki Volcano, Fatu Kapa and Tasi Tulo areas; (3) extensive and diverse hydrothermal manifestations were observed and especially a 2D distribution of the sources. At Kulo Lasi, our data and especially tracer ratios (CH4/3He 50×106 and CH4/TDM 4.5) reveal a transient CH4 input, with elevated levels of CH4 measured in 2010, that had vanished in 2011, most likely caused by an eruptive magmatic event. By contrast at Amanaki, vertical tracer profiles and tracer ratios point to typical seawater/basalt interactions. Fatu Kapa is characterised by a substantial spatial variability of the hydrothermal water column anomalies, most likely due to widespread focused and diffuse hydrothermal discharge in the area. In the Tasi Tulo zone, the hydrothermal signal is characterised by a total lack of turbidity, although other tracer anomalies are in the same range as in nearby Fatu Kapa. The background data set revealed the presence of a Mn and 3He chronic plume due to the extensive and cumulative venting over the entire area. To that respect, we believe that the joined domain composed of our active area and the nearby active area discovered in the East by Lupton et al. (2012) highly contribute to the

  4. Analysis of Ankyrin Repeats Reveals How a Single Point Mutation in RFXANK Results in Bare Lymphocyte Syndrome

    PubMed Central

    Nekrep, Nada; Geyer, Matthias; Jabrane-Ferrat, Nabila; Peterlin, B. Matija

    2001-01-01

    Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed. PMID:11463838

  5. Digital Health: Tracking Physiomes and Activity Using Wearable Biosensors Reveals Useful Health-Related Information

    PubMed Central

    Zhou, Gao; Zhou, Wenyu; Schüssler-Fiorenza Rose, Sophia Miryam; Perelman, Dalia; Colbert, Elizabeth; Runge, Ryan; Rego, Shannon; Sonecha, Ria; Datta, Somalee; McLaughlin, Tracey; Snyder, Michael P.

    2017-01-01

    A new wave of portable biosensors allows frequent measurement of health-related physiology. We investigated the use of these devices to monitor human physiological changes during various activities and their role in managing health and diagnosing and analyzing disease. By recording over 250,000 daily measurements for up to 43 individuals, we found personalized circadian differences in physiological parameters, replicating previous physiological findings. Interestingly, we found striking changes in particular environments, such as airline flights (decreased peripheral capillary oxygen saturation [SpO2] and increased radiation exposure). These events are associated with physiological macro-phenotypes such as fatigue, providing a strong association between reduced pressure/oxygen and fatigue on high-altitude flights. Importantly, we combined biosensor information with frequent medical measurements and made two important observations: First, wearable devices were useful in identification of early signs of Lyme disease and inflammatory responses; we used this information to develop a personalized, activity-based normalization framework to identify abnormal physiological signals from longitudinal data for facile disease detection. Second, wearables distinguish physiological differences between insulin-sensitive and -resistant individuals. Overall, these results indicate that portable biosensors provide useful information for monitoring personal activities and physiology and are likely to play an important role in managing health and enabling affordable health care access to groups traditionally limited by socioeconomic class or remote geography. PMID:28081144

  6. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases.

  7. Digital Health: Tracking Physiomes and Activity Using Wearable Biosensors Reveals Useful Health-Related Information.

    PubMed

    Li, Xiao; Dunn, Jessilyn; Salins, Denis; Zhou, Gao; Zhou, Wenyu; Schüssler-Fiorenza Rose, Sophia Miryam; Perelman, Dalia; Colbert, Elizabeth; Runge, Ryan; Rego, Shannon; Sonecha, Ria; Datta, Somalee; McLaughlin, Tracey; Snyder, Michael P

    2017-01-01

    A new wave of portable biosensors allows frequent measurement of health-related physiology. We investigated the use of these devices to monitor human physiological changes during various activities and their role in managing health and diagnosing and analyzing disease. By recording over 250,000 daily measurements for up to 43 individuals, we found personalized circadian differences in physiological parameters, replicating previous physiological findings. Interestingly, we found striking changes in particular environments, such as airline flights (decreased peripheral capillary oxygen saturation [SpO2] and increased radiation exposure). These events are associated with physiological macro-phenotypes such as fatigue, providing a strong association between reduced pressure/oxygen and fatigue on high-altitude flights. Importantly, we combined biosensor information with frequent medical measurements and made two important observations: First, wearable devices were useful in identification of early signs of Lyme disease and inflammatory responses; we used this information to develop a personalized, activity-based normalization framework to identify abnormal physiological signals from longitudinal data for facile disease detection. Second, wearables distinguish physiological differences between insulin-sensitive and -resistant individuals. Overall, these results indicate that portable biosensors provide useful information for monitoring personal activities and physiology and are likely to play an important role in managing health and enabling affordable health care access to groups traditionally limited by socioeconomic class or remote geography.

  8. A Sleeping Beauty screen reveals NF-kB activation in CLL mouse model.

    PubMed

    Zanesi, Nicola; Balatti, Veronica; Riordan, Jesse; Burch, Aaron; Rizzotto, Lara; Palamarchuk, Alexey; Cascione, Luciano; Lagana, Alessandro; Dupuy, Adam J; Croce, Carlo M; Pekarsky, Yuri

    2013-05-23

    TCL1 oncogene is overexpressed in aggressive form of human chronic lymphocytic leukemia (CLL) and its dysregulation in mouse B cells causes a CD5-positive leukemia similar to the aggressive form of human CLLs. To identify oncogenes that cooperate with Tcl1, we performed genetic screen in Eμ-TCL1 mice using Sleeping Beauty transposon-mediated mutagenesis. Analysis of transposon common insertion sites identified 7 genes activated by transposon insertions. Overexpression of these genes in mouse CLL was confirmed by real time reverse transcription-polymerase chain reaction. Interestingly, the main known function of 4 of 7 genes (Nfkb1, Tab2, Map3K14, and Nfkbid) is participation in or activation of the nuclear factor-kB (NF-kB) pathway. In addition, activation of the NF-kB is 1 of main functions of Akt2, also identified in the screen. These findings demonstrate cooperation of Tcl1 and the NF-kB pathway in the pathogenesis of aggressive CLL. Identification cooperating cancer genes will result in the development of combinatorial therapies to treat CLL.

  9. A Rodent Model of Schizophrenia Reveals Increase in Creatine Kinase Activity with Associated Behavior Changes

    PubMed Central

    Canever, Leila; Oliveira, Larissa; de Luca, Renata D'Altoé; Correa, Paulo T. F.; Fraga, Daiane de B.; Matos, Maria Paula; Scaini, Giselli; Quevedo, João; Streck, Emílio L.; Zugno, Alexandra I.

    2010-01-01

    Schizophrenia is a debilitating mental disorder characterized by positive (delusions, hallucinations, disorganized speech) and negative (affective flattering, avolition and social withdrawal) symptoms as well as cognitive deficits. The frequency, severity and topography characterize the disorder as heterogeneous, the pathophysiology of schizophrenia is poorly understood. Sub-anesthetic doses of ketamine produce hyperactivity, stereotypy and abnormal social interaction and it is used as a model of schizophrenia. In this study, we induced an animal model by acute sub-anesthetic doses of ketamine and tested different behavioral parameters. We also evaluated the activity of creatine kinase (CK) in brain of rats treated with ketamine. Our results demonstrated that administration of 10, 25 and 50 mg/kg of ketamine induced an increase of covered distance in habituated and non-habituated rats to the behavioral apparatus. Ketamine administration induced significant social deficits and stereotypic behavioral in all doses tested. Finally we evaluated the effect of different doses of ketamine on creatinine kinase (CK) activity and we observed that CK activity is increased inspecific regions of the brain. Our study suggests that our animal model may be used as a model of schizophrenia and that cerebral energy metabolism might be altered in the brain of schizophrenic patients, probably leading to alterations that might be involved in the pathogenesis of schizophrenia. PMID:21270541

  10. 10Be surface exposure dating reveals strong active deformation in the central Andean backarc interior

    NASA Astrophysics Data System (ADS)

    García Morabito, Ezequiel; Terrizzano, Carla; Zech, Roland; Willett, Sean; Yamin, Marcela; Haghipour, Negar; Wuethrich, Lorenz; Christl, Marcus; María Cortes, José; Ramos, Victor

    2016-04-01

    Understanding the deformation associated with active thrust wedges is essential to evaluate seismic hazard. How is active faulting distributed throughout the wedge, and how much deformation is taken up by individual structures? We address these questions for our study region, the central Andean backarc of Argentina. We combined a structural and geomorphological approach with surface exposure dating (10Be) of alluvial fans and strath terraces in two key localities at ~32° S: the Cerro Salinas, located in the active orogenic front of the Precordillera, and the Barreal block in the interior of the Andean mountain range. We analysed 22 surface samples and 6 depth profiles. At the thrust front, the oldest terrace (T1) yields an age of 100-130 ka, the intermediate terrace (T2) between 40-95 ka, and the youngest terrace (T3) an age of ~20 ka. In the Andean interior, T1´ dates to 117-146 ka, T2´ to ~70 ka, and T3´ to ~20 ka, all calculations assuming negligible erosion and using the scaling scheme for spallation based on Lal 1991, Stone 2000. Vertical slip rates of fault offsets are 0.3-0.5 mm/yr and of 0.6-1.2 mm/yr at the thrust front and in the Andean interior, respectively. Our results highlight: i) fault activity related to the growth of the Andean orogenic wedge is not only limited to a narrow thrust front zone. Internal structures have been active during the last 150 ka, ii) deformation rates in the Andean interior are comparable or even higher that those estimated and reported along the emerging thrust front, iii) distribution of active faulting seems to account for unsteady state conditions, and iv) seismic hazards may be more relevant in the internal parts of the Andean orogen than assumed so far. References Lal, D., 1991: Cosmic ray labeling of erosion surfaces: In situ nuclide production rates and erosion models. Earth and Planetary Science Letters 104: 424-439. Stone, J.O., 2000: Air pressure and cosmogenic isotope production. Journal of Geophysical

  11. Integrated Experimental and Model-based Analysis Reveals the Spatial Aspects of EGFR Activation Dynamics

    SciTech Connect

    Shankaran, Harish; Zhang, Yi; Chrisler, William B.; Ewald, Jonathan A.; Wiley, H. S.; Resat, Haluk

    2012-10-02

    The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models for deconvolving the contributions of receptor dimerization and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: 1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, 2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and 3) receptor dephosphorylation rates at the cell surface and early endosomes are comparable. We validated the last finding by measuring EGFR dephosphorylation rates at various times following ligand addition both in whole cells, and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. Further, the mathematical model described here can be extended to determine receptor dimer abundances in cells co-expressing various levels of ErbB receptors. This study demonstrates that an iterative cycle of

  12. Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.

    PubMed

    Czabotar, Peter E; Westphal, Dana; Dewson, Grant; Ma, Stephen; Hockings, Colin; Fairlie, W Douglas; Lee, Erinna F; Yao, Shenggen; Robin, Adeline Y; Smith, Brian J; Huang, David C S; Kluck, Ruth M; Adams, Jerry M; Colman, Peter M

    2013-01-31

    In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

  13. Protein profiling reveals antioxidant and signaling activities of NAP (Davunetide) in rodent hippocampus exposed to hypobaric hypoxia.

    PubMed

    Sethy, Niroj Kumar; Sharma, Narendra Kumar; Das, Mainak; Bhargava, Kalpana

    2014-11-01

    NAP (davunetide) is a clinical octapeptide and reportedly possesses neuroprotective, neurotrophic and cognitive protective properties. The information for NAP-mediated neuroproteome changes and associated signaling pathways during hypoxia will help in drug development programmes across the world. In the present study, we have evaluated the antioxidant activities of NAP in rat hippocampus exposed to hypobaric hypoxia (25,000 ft, 282 mm Hg) for 3, 6 and 12 h respectively. Using 2D-gel electrophoresis (2D-GE) with matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry, we have identified altered expression of 80 proteins in NAP-supplemented hippocampus after hypoxia. Pathway analysis revealed that NAP supplementation significantly regulated oxidative stress response, oxidoreductase activity and cellular response to stress pathways during hypoxia. Additionally, NAP supplementation also regulated energy production pathways along with AMP-activated protein kinase (AMPK) signaling and signaling by Rho family GTPases pathways. We observed higher expression of antioxidant Sod1, Eno1, Prdx2 and Prdx5 proteins that were subsequently validated by Western blotting. A higher level of Prdx2 was also observed by immunohistochemistry in NAP-supplemented hippocampus during hypoxia. In corroboration, we are able to detect significant lower level of protein carbonyls in NAP-supplemented hypoxic hippocampus suggesting amelioration of oxidant molecules by NAP supplementation. These results emphasize the antioxidant and signaling properties of NAP in rodent hippocampus during hypobaric hypoxia.

  14. Altered spontaneous activity in antisocial personality disorder revealed by regional homogeneity.

    PubMed

    Tang, Yan; Liu, Wangyong; Chen, Jingang; Liao, Jian; Hu, Dewen; Wang, Wei

    2013-08-07

    There is increasing evidence that antisocial personality disorder (ASPD) stems from brain abnormalities. However, there are only a few studies investigating brain structure in ASPD. The aim of this study was to find regional coherence abnormalities in resting-state functional MRI of ASPD. Thirty-two ASPD individuals and 34 controls underwent a resting-state functional MRI scan. The regional homogeneity (ReHo) approach was used to examine whether ASPD was related to alterations in resting-state neural activity. Support vector machine discriminant analysis was used to evaluate the sensitivity/specificity characteristics of the ReHo index in discriminating between the ASPD individuals and controls. The results showed that, compared with controls, ASPD individuals show lower ReHo in the right cerebellum posterior lobe (Crus1) and the right middle frontal gyrus, as well as higher ReHo in the right middle occipital gyrus (BA 19), left inferior temporal gyrus (BA 37), and right inferior occipital gyrus (cuneus, BA 18). All alternation regions reported a predictive accuracy above 70%. To our knowledge, this study was the first to study the change in regional activity coherence in the resting brain of ASPD individuals. These results not only elucidated the pathological mechanism of ASPD from a resting-state functional viewpoint but also showed that these alterations in ReHo may serve as potential markers for the detection of ASPD.

  15. Structure-activity relations of leucine derivatives reveal critical moieties for cellular uptake and activation of mTORC1-mediated signaling.

    PubMed

    Nagamori, Shushi; Wiriyasermkul, Pattama; Okuda, Suguru; Kojima, Naoto; Hari, Yoshiyuki; Kiyonaka, Shigeki; Mori, Yasuo; Tominaga, Hideyuki; Ohgaki, Ryuichi; Kanai, Yoshikatsu

    2016-04-01

    Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor.

  16. Expression and activity analysis reveal that heme oxygenase (decycling) 1 is associated with blue egg formation.

    PubMed

    Wang, Z P; Liu, R F; Wang, A R; Li, J Y; Deng, X M

    2011-04-01

    Biliverdin is responsible for the coloration of blue eggs and is secreted onto the eggshell by the shell gland. Previous studies confirmed that a significant difference exists in biliverdin content between blue eggs and brown eggs, although the reasons are still unknown. Because the pigment is derived from oxidative degradation of heme catalyzed by heme oxygenase (HO), this study compared heme oxygenase (decycling) 1 (HMOX1), the gene encoding HO expression and HO activity, in the shell glands of the Dongxiang blue-shelled chicken (n = 12) and the Dongxiang brown-shelled chicken (n = 12). Results showed that HMOX1 was highly expressed at the mRNA (1.58-fold; P < 0.05) and protein levels in blue-shelled chickens compared with brown-shelled chickens. At the functional level, blue-shelled chickens also showed 1.40-fold (P < 0.05) higher HO activity than brown-shelled chickens. To explore the reasons for the differential expression of HMOX1, an association study of 6 SNP capturing the majority of HMOX1 variants with the blue egg coloration was performed. Results showed no significant association between SNP and the blue egg coloration in HMOX1 (P > 0.05). Taken together, these results show that blue egg formation is associated with high expression of HMOX1 in the shell gland of Dongxiang blue-shelled chickens, and suggest that differential expression of HMOX1 in the 2 groups of chickens is most likely to arise from an alteration in the trans-acting factor.

  17. The role of disease perceptions and results sharing in psychological adaptation after genetic susceptibility testing: the REVEAL Study.

    PubMed

    Ashida, Sato; Koehly, Laura M; Roberts, J Scott; Chen, Clara A; Hiraki, Susan; Green, Robert C

    2010-12-01

    This study evaluates the extent to which psychological adaptation (validated measures of depressive symptoms, anxiety, and test-specific distress) after genetic susceptibility testing is influenced by changes in beliefs about Alzheimer's disease (AD) and sharing of test results with others. Adult children of AD patients (N=269) from a randomized clinical trial involving genetic testing for apolipoprotein E (APOE) provided information before, as well as 6 weeks and 12 months after results disclosure. The levels of adaptation varied highly among participants at 12-month assessment. Participants who learned that they were ε4 negative (lower risk) had a reduction in perceived risk and concern about developing AD compared with those who learned that they were ε4 positive. Those who received results through an extended educational protocol (three in-person visits) had a larger decline in AD concern than those in a condensed protocol (educational brochure and two in-person visits). Increase in AD concern 6 weeks after disclosure was associated with increase in depression scores (b=0.20, P<0.01) and anxiety levels (b=0.20, P<0.01), and higher distress associated with AD genetic testing (b=0.18, P=0.02) 1 year after testing. Increase in perceived risk (b=0.16, P=0.04) was also associated with higher AD genetic testing distress. Sharing the test results with health professionals and friends (but not family) was associated with decrease in depression (b=-0.11, P=0.05) and anxiety levels (b=-0.16, P<0.01), respectively after a year. Enhancing discussion with regard to risks and concerns about AD during pretesting counseling and obtaining support through sharing the results after testing may help facilitate test recipients' long-term psychological adaptation.

  18. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.

    PubMed

    Zhou, Hui; Lin-Wang, Kui; Wang, Huiliang; Gu, Chao; Dare, Andrew P; Espley, Richard V; He, Huaping; Allan, Andrew C; Han, Yuepeng

    2015-04-01

    Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors.

  19. Structure of protein O-mannose kinase reveals a unique active site architecture

    PubMed Central

    Zhu, Qinyu; Venzke, David; Walimbe, Ameya S; Anderson, Mary E; Fu, Qiuyu; Kinch, Lisa N; Wang, Wei; Chen, Xing; Grishin, Nick V; Huang, Niu; Yu, Liping; Dixon, Jack E; Campbell, Kevin P; Xiao, Junyu

    2016-01-01

    The ‘pseudokinase’ SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome. DOI: http://dx.doi.org/10.7554/eLife.22238.001 PMID:27879205

  20. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

    PubMed

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-11-20

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

  1. Early Category-Specific Cortical Activation Revealed by Visual Stimulus Inversion

    PubMed Central

    Meeren, Hanneke K. M.; Hadjikhani, Nouchine; Ahlfors, Seppo P.; Hämäläinen, Matti S.; de Gelder, Beatrice

    2008-01-01

    Visual categorization may already start within the first 100-ms after stimulus onset, in contrast with the long-held view that during this early stage all complex stimuli are processed equally and that category-specific cortical activation occurs only at later stages. The neural basis of this proposed early stage of high-level analysis is however poorly understood. To address this question we used magnetoencephalography and anatomically-constrained distributed source modeling to monitor brain activity with millisecond-resolution while subjects performed an orientation task on the upright and upside-down presented images of three different stimulus categories: faces, houses and bodies. Significant inversion effects were found for all three stimulus categories between 70–100-ms after picture onset with a highly category-specific cortical distribution. Differential responses between upright and inverted faces were found in well-established face-selective areas of the inferior occipital cortex and right fusiform gyrus. In addition, early category-specific inversion effects were found well beyond visual areas. Our results provide the first direct evidence that category-specific processing in high-level category-sensitive cortical areas already takes place within the first 100-ms of visual processing, significantly earlier than previously thought, and suggests the existence of fast category-specific neocortical routes in the human brain. PMID:18946504

  2. Transcriptomic profiling of microglia reveals signatures of cell activation and immune response, during experimental cerebral malaria

    PubMed Central

    Capuccini, Barbara; Lin, Jingwen; Talavera-López, Carlos; Khan, Shahid M.; Sodenkamp, Jan; Spaccapelo, Roberta; Langhorne, Jean

    2016-01-01

    Cerebral malaria is a pathology involving inflammation in the brain. There are many immune cell types activated during this process, but there is little information on the response of microglia, in this severe complication. We examined microglia by genome wide transcriptomic analysis in a model of experimental cerebral malaria (ECM), in which C57BL/6 mice are infected with Plasmodium berghei ANKA. Thousands of transcripts were differentially expressed in microglia at two different time points during infection. Proliferation of microglia was a dominant feature before the onset of ECM, and supporting this, we observed an increase in numbers of these cells in the brain. When cerebral malaria symptoms were manifest, genes involved in immune responses and chemokine production were upregulated, which were possibly driven by Type I Interferon. Consistent with this hypothesis, in vitro culture of a microglial cell line with Interferon-β, but not infected red blood cells, resulted in production of several of the chemokines shown to be upregulated in the gene expression analysis. It appears that these responses are associated with ECM, as microglia from mice infected with a mutant P. berghei parasite (ΔDPAP3), which does not cause ECM, did not show the same level of activation or proliferation. PMID:27991544

  3. Preserved hippocampus activation in normal aging as revealed by fMRI.

    PubMed

    Persson, Jonas; Kalpouzos, Grégoria; Nilsson, Lars-Göran; Ryberg, Mats; Nyberg, Lars

    2011-07-01

    The hippocampus is deteriorated in various pathologies such as Alzheimer's disease (AD) and such deterioration has been linked to memory impairment. By contrast, the structural and functional effects of normal aging on the hippocampus is a matter of debate, with some findings suggesting deterioration and others providing evidence of preservation. This constitutes a crucial question since many investigations on AD are based on the assumption that the deterioration of the hippocampus is the breaking point between normal and pathological aging. A growing number of fMRI studies specifically aimed at investigating hippocampal engagement in various cognitive tasks, notably memory tasks, but the results have been inconclusive. Here, we optimized the episodic face-name paired-associates task in order to test the functioning of the hippocampus in normal aging. Critically, we found no difference in the activation of the hippocampus between the young and a group of older participants. Analysis of individual patterns of activation substantiated this impression. Collectively, these findings provide evidence of preserved hippocampal functioning in normal aging.

  4. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  5. Time, space and emotion: fMRI reveals content-specific activation during text comprehension.

    PubMed

    Ferstl, Evelyn C; von Cramon, D Yves

    2007-11-12

    Story comprehension involves building a situation model of the text, i.e., a representation containing information on the who, where, when and why of the story. Using fMRI at 3T, domain-specific activations for three different information aspects were sought. Twenty participants read two sentence stories half of which contained inconsistencies concerning emotional, temporal or spatial information. Partly replicating previous results [E.C. Ferstl, M. Rinck, D.Y. von Cramon, Emotional and temporal aspects of situation model processing during text comprehension: an event-related fMRI study, J. Cogn. Neurosci. 17 (2005) 724-739], the anterior lateral prefrontal cortex/orbito-frontal cortex proved important for processing temporal information. The left anterior temporal lobe was particularly important during emotional stories. Most importantly, spatial information elicited bilateral activation in the collateral sulci and the posterior cingulate cortex, areas important for visuo-spatial cognition. These findings provide further evidence for content-specific processes during text comprehension.

  6. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance

    PubMed Central

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-01-01

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities. PMID:26584633

  7. Transcriptomic profiling of microglia reveals signatures of cell activation and immune response, during experimental cerebral malaria.

    PubMed

    Capuccini, Barbara; Lin, Jingwen; Talavera-López, Carlos; Khan, Shahid M; Sodenkamp, Jan; Spaccapelo, Roberta; Langhorne, Jean

    2016-12-19

    Cerebral malaria is a pathology involving inflammation in the brain. There are many immune cell types activated during this process, but there is little information on the response of microglia, in this severe complication. We examined microglia by genome wide transcriptomic analysis in a model of experimental cerebral malaria (ECM), in which C57BL/6 mice are infected with Plasmodium berghei ANKA. Thousands of transcripts were differentially expressed in microglia at two different time points during infection. Proliferation of microglia was a dominant feature before the onset of ECM, and supporting this, we observed an increase in numbers of these cells in the brain. When cerebral malaria symptoms were manifest, genes involved in immune responses and chemokine production were upregulated, which were possibly driven by Type I Interferon. Consistent with this hypothesis, in vitro culture of a microglial cell line with Interferon-β, but not infected red blood cells, resulted in production of several of the chemokines shown to be upregulated in the gene expression analysis. It appears that these responses are associated with ECM, as microglia from mice infected with a mutant P. berghei parasite (ΔDPAP3), which does not cause ECM, did not show the same level of activation or proliferation.

  8. Repeated surveys reveal nontectonic exposure of supposedly active normal faults in the central Apennines, Italy

    NASA Astrophysics Data System (ADS)

    Kastelic, Vanja; Burrato, Pierfrancesco; Carafa, Michele M. C.; Basili, Roberto

    2017-01-01

    We investigate the geomorphic processes that expose bedrock fault surfaces from under their slope-deposit cover in the central Apennines (Italy). These bedrock fault surfaces are generally located at various heights on mountain fronts above the local base level of glacio-fluvial valleys and intermountain fluvio-lacustrine basins and are laterally confined to the extent of related mountain fronts. The process that led to the exposure of fault surfaces has often been exclusively attributed to coseismic earthquake slip and used as proxy for tectonic slip rates and earthquake recurrence estimations. We present the results of monitoring the contact between the exposed fault surfaces and slope deposits at 23 measurement points on 12 different faults over 3.4 year long observation period. We detected either downward or upward movements of the slope deposit with respect to the fault surface between consecutive measurements. During the entire observation period all points, except one, registered a net downward movement in the 2.9-25.6 mm/yr range, resulting in the progressive exposure of the fault surface. During the monitoring period no major earthquakes occurred in the region, demonstrating that the measured exposure process is disconnected from seismic activity. Our results indicate that the fault surface exposure rates are rather due to gravitational and landsliding movements aided by weathering and slope degradation processes. The so far neglected slope degradation and other (sub)surface processes should thus be carefully taken into consideration before attempting to recover fault slip rates using surface gathered data.

  9. Impedance spectroscopy of micro-Droplets reveals activation of Bacterial Mechanosensitive Channels in Hypotonic Solutions

    NASA Astrophysics Data System (ADS)

    Ebrahimi, Aida; Alam, Muhammad A.

    Rapid detection of bacterial pathogens is of great importance in healthcare, food safety, environmental monitoring, and homeland security. Most bacterial detection platforms rely on binary fission (i.e. cell growth) to reach a threshold cell population that can be resolved by the sensing method. Since cell division depends on the bacteria type, the detection time of such methods can vary from hours to days. In contrast, in this work, we show that bacteria cells can be detected within minutes by relying on activation of specific protein channels, i.e. mechanosensitive channels (MS channels). When cells are exposed to hypotonic solutions, MS channels allow efflux of solutes to the external solution which leads to release the excessive membrane tension. Release of the cytoplasmic solutes, in turn, results in increase of the electrical conductance measured by droplet-based impedance sensing. The approach can be an effective technique for fast, pre-screening of bacterial contamination at ultra-low concentration.

  10. Changes in the cardiac muscle electric activity as a result of Coronary Artery Bypass Graft operation

    NASA Astrophysics Data System (ADS)

    Grajek, Magdalena; Krzyminiewski, Ryszard; Kalawski, Ryszard; Kulczak, Mariusz

    2008-01-01

    Many bioelectric signals have a complex internal structure that can be a rich source of information on the tissue or cell processes. The structure of such signals can be analysed in detail by applying digital methods of signal processing. Therefore, of substantial use in diagnosis of the coronary arterial disease is the method of digital enhancement of increasing signal resolution ECG (NURSE-ECG), permitting detection of temporary changes in the electric potentials in the cardiac muscle in the process of depolarisation. Thanks to the application of NURSE-ECG it has become possible to detect relatively small changes in the electric activity of particular fragments of the cardiac muscle undetectable by the standard ECG method, caused by ischemia, the effect of a drug or infarct. The aim of this study was to identify and analyse changes in the electric activity of the cardiac muscle as a result of the Coronary Artery Bypass Graft (CABG) operation. In this study the method of NURSE-ECG has been applied in order to identify and analyse changes in the electric activity of the cardiac muscle as a result of the CABG operation. In the study performed in cooperation of the Institute of Physics Adam Mickiewicz University and the Strus Hospital, Cardiac Surgery Ward, 37 patients with advanced coronary arterial disease were asked to participate. The patients were examined prior to the operation, on the day after the operation and two months after the operation and a year after the operation. The ECG recordings were subjected to a numerical procedure of resolution enhancement by a NURSE-ECG program to reveal the tentative changes in the electric potential of the cardiac muscle on its depolarisation. Results of the study have shown that the NURSE ECG method can be applied to monitor changes in the electric activity of the cardiac muscle occurring as a result of CABG operation. One the second day after the operation in the majority of patients (70%) a rapid decrease of the total

  11. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription.

    PubMed

    Hoffman, Elizabeth A; McCulley, Andrew; Haarer, Brian; Arnak, Remigiusz; Feng, Wenyi

    2015-03-01

    We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.

  12. Ocular-following responses to white noise stimuli in humans reveal a novel nonlinearity that results from temporal sampling

    PubMed Central

    Sheliga, Boris M.; Quaia, Christian; FitzGibbon, Edmond J.; Cumming, Bruce G.

    2016-01-01

    White noise stimuli are frequently used to study the visual processing of broadband images in the laboratory. A common goal is to describe how responses are derived from Fourier components in the image. We investigated this issue by recording the ocular-following responses (OFRs) to white noise stimuli in human subjects. For a given speed we compared OFRs to unfiltered white noise with those to noise filtered with band-pass filters and notch filters. Removing components with low spatial frequency (SF) reduced OFR magnitudes, and the SF associated with the greatest reduction matched the SF that produced the maximal response when presented alone. This reduction declined rapidly with SF, compatible with a winner-take-all operation. Removing higher SF components increased OFR magnitudes. For higher speeds this effect became larger and propagated toward lower SFs. All of these effects were quantitatively well described by a model that combined two factors: (a) an excitatory drive that reflected the OFRs to individual Fourier components and (b) a suppression by higher SF channels where the temporal sampling of the display led to flicker. This nonlinear interaction has an important practical implication: Even with high refresh rates (150 Hz), the temporal sampling introduced by visual displays has a significant impact on visual processing. For instance, we show that this distorts speed tuning curves, shifting the peak to lower speeds. Careful attention to spectral content, in the light of this nonlinearity, is necessary to minimize the resulting artifact when using white noise patterns undergoing apparent motion. PMID:26762277

  13. Atmosphere-Ionosphere Response to the M9 Tohoku Earthquake Revealed by Joined Satellite and Ground Observations. Preliminary Results

    NASA Technical Reports Server (NTRS)

    Ouzounov, Dimitar; Pulinets, Sergey; Romanov, Alexey; Tsybulya, Konstantin; Davidenko, Dimitri; Kafatos, Menas; Taylor, Patrick

    2011-01-01

    The recent M9 Tohoku Japan earthquake of March 11, 2011 was the largest recorded earthquake ever to hit this nation. We retrospectively analyzed the temporal and spatial variations of four different physical parameters - outgoing long wave radiation (OLR), GPS/TEC, Low-Earth orbit tomography and critical frequency foF2. These changes characterize the state of the atmosphere and ionosphere several days before the onset of this earthquake. Our first results show that on March 8th a rapid increase of emitted infrared radiation was observed from the satellite data and an anomaly developed near the epicenter. The GPS/TEC data indicate an increase and variation in electron density reaching a maximum value on March 8. Starting on this day in the lower ionospheric there was also confirmed an abnormal TEC variation over the epicenter. From March 3-11 a large increase in electron concentration was recorded at all four Japanese ground based ionosondes, which return to normal after the main earthquake. We found a positive correlation between the atmospheric and ionospheric anomalies and the Tohoku earthquake. This study may lead to a better understanding of the response of the atmosphere/ionosphere to the Great Tohoku earthquake.

  14. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription

    PubMed Central

    Hoffman, Elizabeth A.; McCulley, Andrew; Haarer, Brian; Arnak, Remigiusz

    2015-01-01

    We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites. PMID:25609572

  15. Ocular-following responses to white noise stimuli in humans reveal a novel nonlinearity that results from temporal sampling.

    PubMed

    Sheliga, Boris M; Quaia, Christian; FitzGibbon, Edmond J; Cumming, Bruce G

    2016-01-01

    White noise stimuli are frequently used to study the visual processing of broadband images in the laboratory. A common goal is to describe how responses are derived from Fourier components in the image. We investigated this issue by recording the ocular-following responses (OFRs) to white noise stimuli in human subjects. For a given speed we compared OFRs to unfiltered white noise with those to noise filtered with band-pass filters and notch filters. Removing components with low spatial frequency (SF) reduced OFR magnitudes, and the SF associated with the greatest reduction matched the SF that produced the maximal response when presented alone. This reduction declined rapidly with SF, compatible with a winner-take-all operation. Removing higher SF components increased OFR magnitudes. For higher speeds this effect became larger and propagated toward lower SFs. All of these effects were quantitatively well described by a model that combined two factors: (a) an excitatory drive that reflected the OFRs to individual Fourier components and (b) a suppression by higher SF channels where the temporal sampling of the display led to flicker. This nonlinear interaction has an important practical implication: Even with high refresh rates (150 Hz), the temporal sampling introduced by visual displays has a significant impact on visual processing. For instance, we show that this distorts speed tuning curves, shifting the peak to lower speeds. Careful attention to spectral content, in the light of this nonlinearity, is necessary to minimize the resulting artifact when using white noise patterns undergoing apparent motion.

  16. Laser speckle contrast reveals cerebral blood flow dynamics evoked by optogenetically controlled neuronal activity

    NASA Astrophysics Data System (ADS)

    Li, Nan; Thakor, Nitish V.; Pelled, Galit

    2013-03-01

    As a critical basis of functional brain imaging, neurovascular coupling describes the link between neuronal and hemodynamic changes. The majority of in vivo neurovascular coupling studies was performed by inducing sensory stimulation via afferent inputs. Unfortunately such an approach results in recruiting of multiple types of cells, which confounds the explanation of neuronal roles in stimulus evoked hemodynamic changes. Recently optogenetics has emerged to provide immediate control of neurons by exciting or inhibiting genetically engineered neurons expressing light sensitive proteins. However, there is a need for optical methods capable of imaging the concurrent hemodynamic changes. We utilize laser speckle contrast imaging (LSCI) to obtain high resolution display of cerebral blood flow (CBF) in the vicinity of the targeted neural population. LSCI is a minimally invasive method for imaging CBF in microvessels through thinned skull, and produces images with high spatiotemporal resolution, wide field of view. In the integrated system light sources with different wavelengths and band-passing/blocking filters were used to allow simultaneous optical manipulation of neuronal activities and optical imaging of corresponding CBF. Experimental studies were carried out in a rodent model expressing channalrhodopsin (ChR2) in excitatory neurons in the somatosensory cortex (S1). The results demonstrated significant increases of CBF in response to ChR2 stimulation (exciting neuronal firing) comparable to the CBF response to contralateral forepaw stimulation. The approach promises to be an exciting minimally invasive method to study neurovascular coupling. The complete system provides a novel approach for broad neuroscience applications.

  17. Small Conductance Ca2+-Activated K+ Channel Knock-Out Mice Reveal the Identity of Calcium-Dependent Afterhyperpolarization Currents

    PubMed Central

    Bond, Chris T.; Herson, Paco S.; Strassmaier, Timothy; Hammond, Rebecca; Stackman, Robert; Maylie, James; Adelman, John P.

    2010-01-01

    Action potentials in many central neurons are followed by a prolonged afterhyperpolarization (AHP) that influences firing frequency and affects neuronal integration. In hippocampal CA1 pyramidal neurons, the current ascribed to the AHP (IAHP) has three kinetic components. The IfastAHP is predominantly attributable to voltage-dependent K+ channels, whereas Ca2+-dependent and voltage-independent K+ channels contribute to the ImediumAHP (ImAHP) and IslowAHP (IsAHP). Apamin, which selectively suppresses a component of the mAHP, increases neuronal excitability and facilitates the induction of synaptic plasticity at Schaffer collateral synapses and hippocampal-dependent learning. The Ca2+-dependent components of the AHP have been attributed to the activity of small conductance Ca2+-activated K+(SK) channels. Examination of transgenic mice, each lacking one of the three SK channel genes expressed in the CNS, reveals that mice without the SK2 subunit completely lack the apamin-sensitive component of the ImAHP in CA1 neurons, whereas the IsAHP is not different in any of the SK transgenic mice. In each of the transgenic lines, the expression levels of the remaining SK genes are not changed. The results demonstrate that only SK2 channels are necessary for the ImAHP, and none of the SK channels underlie the IsAHP. PMID:15190101

  18. Metatranscriptomics Reveals the Active Bacterial and Eukaryotic Fibrolytic Communities in the Rumen of Dairy Cow Fed a Mixed Diet

    PubMed Central

    Comtet-Marre, Sophie; Parisot, Nicolas; Lepercq, Pascale; Chaucheyras-Durand, Frédérique; Mosoni, Pascale; Peyretaillade, Eric; Bayat, Ali R.; Shingfield, Kevin J.; Peyret, Pierre; Forano, Evelyne

    2017-01-01

    Ruminants have a unique ability to derive energy from the degradation of plant polysaccharides through the activity of the rumen microbiota. Although this process is well studied in vitro, knowledge gaps remain regarding the relative contribution of the microbiota members and enzymes in vivo. The present study used RNA-sequencing to reveal both the expression of genes encoding carbohydrate-active enzymes (CAZymes) by the rumen microbiota of a lactating dairy cow and the microorganisms forming the fiber-degrading community. Functional analysis identified 12,237 CAZymes, accounting for 1% of the transcripts. The CAZyme profile was dominated by families GH94 (cellobiose-phosphorylase), GH13 (amylase), GH43 and GH10 (hemicellulases), GH9 and GH48 (cellulases), PL11 (pectinase) as well as GH2 and GH3 (oligosaccharidases). Our data support the pivotal role of the most characterized fibrolytic bacteria (Prevotella, Ruminocccus and Fibrobacter), and highlight a substantial, although most probably underestimated, contribution of fungi and ciliate protozoa to polysaccharide degradation. Particularly these results may motivate further exploration of the role and the functions of protozoa in the rumen. Moreover, an important part of the fibrolytic bacterial community remains to be characterized since one third of the CAZyme transcripts originated from distantly related strains. These findings are used to highlight limitations of current metatranscriptomics approaches to understand the functional rumen microbial community and opportunities to circumvent them. PMID:28197133

  19. The Crystal Structure of Dehi Reveals a New A-Haloacid Dehalogenase Fold And Active Site Mechanism

    SciTech Connect

    Schmidberger, J.W.; Wilce, J.A.; Weightman, A.J.; Whisstock, J.C.; Wilce, M.C.J.

    2009-05-27

    Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I {alpha}-haloacid dehalogenase that can process both L- and D-substrates. The structure shows that the DehI monomer consists of two domains of {approx}130 amino acids that have {approx}16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both L- and D-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either L- or D-substrates in contrast to D-only enzymes. These details will assist with future bioengineering of dehalogenases.

  20. Bioinformatic analyses reveal a distinct Notch activation induced by STAT3 phosphorylation in the mesenchymal subtype of glioblastoma.

    PubMed

    Cheng, Wen; Zhang, Chuanbao; Ren, Xiufang; Jiang, Yang; Han, Sheng; Liu, Yang; Cai, Jinquan; Li, Mingyang; Wang, Kuanyu; Liu, Yanwei; Hu, Huimin; Li, Qingbin; Yang, Pei; Bao, Zhaoshi; Wu, Anhua

    2017-01-01

    OBJECTIVE Glioblastoma (GBM) is the most common and lethal type of malignant glioma. The Cancer Genome Atlas divides the gene expression-based classification of GBM into classical, mesenchymal, neural, and proneural subtypes, which is important for understanding GBM etiology and for designing effective personalized therapy. Signal transducer and activator of transcription 3 (STAT3), a critical transcriptional activator in tumorigenesis, is persistently phosphorylated and associated with an unfavorable prognosis in GBM. Although a set of specific targets has been identified, there have been no systematic analyses of STAT3 signaling based on GBM subtype. METHODS This study compared STAT3-associated messenger RNA, protein, and microRNA expression profiles across different subtypes of GBM. RESULTS The analyses revealed a prominent role for STAT3 in the mesenchymal but not in other GBM subtypes, which can be reliably used to classify patients with mesenchymal GBM into 2 groups according to phosphorylated STAT3 expression level. Differentially expressed genes suggest an association between Notch and STAT3 signaling in the mesenchymal subtype. Their association was validated in the U87 cell, a malignant glioma cell line annotated as mesenchymal subtype. Specific associated proteins and microRNAs further profile the STAT3 signaling among GBM subtypes. CONCLUSIONS These findings suggest a prominent role for STAT3 signaling in mesenchymal GBM and highlight the importance of identifying signaling pathways that contribute to specific cancer subtypes.

  1. Altered Spontaneous Activity in Patients with Persistent Somatoform Pain Disorder Revealed by Regional Homogeneity

    PubMed Central

    Yan, Chao; Lu, Jing; Li, Xuzhou; Tang, Chaozheng; Fan, Mingxia; Luo, Yanli

    2016-01-01

    Persistent somatoform pain disorder (PSPD) is a mental disorder un-associated with any somatic injury and can cause severe somatosensory and emotional impairments in patients. However, so far, the neuro-pathophysiological mechanism of the functional impairments in PSPD is still unclear. The present study assesses the difference in regional spontaneous activity between PSPD and healthy controls (HC) during a resting state, in order to elucidate the neural mechanisms underlying PSPD. Resting-state functional Magnetic Resonance Imaging data were obtained from 13 PSPD patients and 23 age- and gender-matched HC subjects in this study. Kendall’s coefficient of concordance was used to measure regional homogeneity (ReHo), and a two-sample t-test was subsequently performed to investigate the ReHo difference between PSPD and HC. Additionally, the correlations between the mean ReHo of each survived area and the clinical assessments were further analyzed. Compared with the HC group, patients with PSPD exhibited decreased ReHo in the bilateral primary somatosensory cortex, posterior cerebellum, and occipital lobe, while increased ReHo in the prefrontal cortex (PFC) and default mode network (including the medial PFC, right inferior parietal lobe (IPL), and left supramarginal gyrus). In addition, significant positive correlations were found between the mean ReHo of both right IPL and left supramarginal gyrus and participants’ Self-Rating Anxiety Scale (SAS) scores, and between the mean ReHo of the left middle frontal gyrus and Visual Analogue Scale (VAS) scores. Our results suggest that abnormal spontaneous brain activity in specific brain regions during a resting state may be associated with the dysfunctions in pain, memory and emotional processing commonly observed in patients with PSPD. These findings help us to understand the neural mechanisms underlying PSPD and suggest that the ReHo metric could be used as a clinical marker for PSPD. PMID:26977802

  2. Dissection of a Ciona regulatory element reveals complexity of cross-species enhancer activity

    PubMed Central

    Chen, Wei-Chung; Pauls, Stefan; Bacha, Jamil; Elgar, Greg; Loose, Matthew; Shimeld, Sebastian M.

    2014-01-01

    Vertebrate genomes share numerous conserved non-coding elements, many of which function as enhancer elements and are hypothesised to be under evolutionary constraint due to a need to be bound by combinations of sequence-specific transcription factors. In contrast, few such conserved elements can be detected between vertebrates and their closest invertebrate relatives. Despite this lack of sequence identity, cross-species transgenesis has identified some cases where non-coding DNA from invertebrates drives reporter gene expression in transgenic vertebrates in patterns reminiscent of the expression of vertebrate orthologues. Such instances are presumed to reflect the presence of conserved suites of binding sites in the regulatory regions of invertebrate and vertebrate orthologues, such that both regulatory elements can correctly interpret the trans-activating environment. Shuffling of binding sites has been suggested to lie behind loss of sequence conservation; however this has not been experimentally tested. Here we examine the underlying basis of enhancer activity for the Ciona intestinalis βγ-crystallin gene, which drives expression in the lens of transgenic vertebrates despite the Ciona lineage predating the evolution of the lens. We construct an interactive gene regulatory network (GRN) for vertebrate lens development, allowing network interactions to be robustly catalogued and conserved network components and features to be identified. We show that a small number of binding motifs are necessary for Ciona βγ-crystallin expression, and narrow down the likely factors that bind to these motifs. Several of these overlap with the conserved core of the vertebrate lens GRN, implicating these sites in cross species function. However when we test these motifs in a transgenic vertebrate they prove to be dispensable for reporter expression in the lens. These results show that current models depicting cross species enhancer function as dependent on conserved binding

  3. Climate impacts on human settlement and agricultural activities in northern Norway revealed through sediment biogeochemistry.

    PubMed

    D'Anjou, Robert M; Bradley, Raymond S; Balascio, Nicholas L; Finkelstein, David B

    2012-12-11

    Disentangling the effects of climate change and anthropogenic activities on the environment is a major challenge in paleoenvironmental research. Here, we used fecal sterols and other biogeochemical compounds in lake sediments from northern Norway to identify both natural and anthropogenic signals of environmental change during the late Holocene. The area was first occupied by humans and their grazing animals at ∼2,250 ± 75 calendar years before 1950 AD (calendar years before present). The arrival of humans is indicated by an abrupt increase in coprostanol (and its epimer epicoprostanol) in the sediments and an associated increase in 5β-stigmastanol (and 5β-epistigmastanol), which resulted from human and animal feces washing into the lake. Human settlement was accompanied by an abrupt increase in landscape fires (indicated by the rise in pyrolytic polycyclic aromatic hydrocarbons) and a decline in woodland (registered by a change in n-alkane chain lengths from leaf waxes), accelerating a process that began earlier in the Holocene. Human activity and associated landscape changes in the region over the last two millennia were mainly driven by summer temperatures, as indicated by independent tree-ring reconstructions, although there were periods when socioeconomic factors played an equally important role. In this study, fecal sterols in lake sediments have been used to provide a record of human occupancy through time. This approach may be useful in many archeological studies, both to confirm the presence of humans and grazing animals, and to distinguish between anthropogenic and natural factors that have influenced the environment in the past.

  4. Young asteroidal fluid activity revealed by absolute age from apatite in carbonaceous chondrite.

    PubMed

    Zhang, Ai-Cheng; Li, Qiu-Li; Yurimoto, Hisayoshi; Sakamoto, Naoya; Li, Xian-Hua; Hu, Sen; Lin, Yang-Ting; Wang, Ru-Cheng

    2016-09-29

    Chondritic meteorites, consisting of the materials that have formed in the early solar system (ESS), have been affected by late thermal events and fluid activity to various degrees. Determining the timing of fluid activity in ESS is of fundamental importance for understanding the nature, formation, evolution and significance of fluid activity in ESS. Previous investigations have determined the relative ages of fluid activity with short-lived isotope systematics. Here we report an absolute (207)Pb/(206)Pb isochron age (4,450±50 Ma) of apatite from Dar al Gani (DaG) 978, a type ∼3.5, ungrouped carbonaceous chondrite. The petrographic, mineralogical and geochemical features suggest that the apatite in DaG 978 should have formed during metamorphism in the presence of a fluid. Therefore, the apatite age represents an absolute age for fluid activity in an asteroidal setting. An impact event could have provided the heat to activate this young fluid activity in ESS.

  5. Young asteroidal fluid activity revealed by absolute age from apatite in carbonaceous chondrite

    NASA Astrophysics Data System (ADS)

    Zhang, Ai-Cheng; Li, Qiu-Li; Yurimoto, Hisayoshi; Sakamoto, Naoya; Li, Xian-Hua; Hu, Sen; Lin, Yang-Ting; Wang, Ru-Cheng

    2016-09-01

    Chondritic meteorites, consisting of the materials that have formed in the early solar system (ESS), have been affected by late thermal events and fluid activity to various degrees. Determining the timing of fluid activity in ESS is of fundamental importance for understanding the nature, formation, evolution and significance of fluid activity in ESS. Previous investigations have determined the relative ages of fluid activity with short-lived isotope systematics. Here we report an absolute 207Pb/206Pb isochron age (4,450+/-50 Ma) of apatite from Dar al Gani (DaG) 978, a type ~3.5, ungrouped carbonaceous chondrite. The petrographic, mineralogical and geochemical features suggest that the apatite in DaG 978 should have formed during metamorphism in the presence of a fluid. Therefore, the apatite age represents an absolute age for fluid activity in an asteroidal setting. An impact event could have provided the heat to activate this young fluid activity in ESS.

  6. Experimentally activated immune defence in female pied flycatchers results in reduced breeding success.

    PubMed

    Ilmonen, P; Taarna, T; Hasselquist, D

    2000-04-07

    Traditional explanations for the negative fitness consequences of parasitism have focused on the direct pathogenic effects of infectious agents. However, because of the high selection pressure by the parasites, immune defences are likely to be costly and trade off with other fitness-related traits, such as reproductive effort. In a field experiment, we immunized breeding female flycatchers with non-pathogenic antigens (diphtheria-tetanus vaccine), which excluded the direct negative effects of parasites, in order to test the consequences of activated immune defence on hosts' investment in reproduction and self-maintenance. Immunized females decreased their feeding effort and investment in self-maintenance (rectrix regrowth) and had lower reproductive output (fledgling quality and number) than control females injected with saline. Our results reveal the phenotypic cost of immune defence by showing that an activated immune system per se can lower the host's breeding success. This may be caused by an energetic or nutritional trade-off between immune function and physical workload when feeding young or be an adaptive response to 'infection' to avoid physiological disorders such as oxidative stress and immunopathology.

  7. A biosensor for the protease TACE reveals actin damage induced TACE activation

    PubMed Central

    Chapnick, Douglas A.; Bunker, Eric; Liu, Xuedong

    2016-01-01

    Ligand shedding has gained increased attention as a major posttranslational modification mechanism used by cells to respond to diverse environmental conditions. The TACEadam17 protease is a critical mediator of such ligand shedding, regulating the maturation and release of an impressive range of extracellular substrates that drive diverse cellular responses. Exactly how this protease is itself activated remains unclear, in part due to the lack of available tools to measure TACE activity with temporal and spatial resolution in live cells. We have developed a FRET based biosensor for TACE activity (TSen), which is capable of reporting TACE activation kinetics in live cells with a high degree of specificity. TSen was used in combination with chemical biology to probe the dependence of various means of TACE activation on p38 and Erk kinase activities, as well as to identify a novel connection between actin cytoskeletal disruption and TACE activation. Such cytoskeletal disruption leads to rapid and robust TACE activation in some cell types and accumulation of TACE at the plasma membrane, allowing for increased cleavage of endogenous substrates. Our study highlights both the versatility of TSen as a tool to understand the mechanisms of TACE activation in live cells and the importance of actin cytoskeletal integrity as a modulator of TACE activity. PMID:25714465

  8. Electromyographic activation reveals cortical and sub-cortical dissociation during emergence from general anesthesia.

    PubMed

    Hight, Darren F; Voss, Logan J; García, Paul S; Sleigh, Jamie W

    2016-07-21

    During emergence from anesthesia patients regain their muscle tone (EMG). In a typical population of surgical patients the actual volatile gas anesthetic concentrations in the brain (CeMAC) at which EMG activation occurs remains unknown, as is whether EMG activation at higher CeMACs is correlated with subsequent severe pain, or with cortical activation. Electroencephalographic (EEG) and EMG activity was recorded from the forehead of 273 patients emerging from general anesthesia following surgery. We determined CeMAC at time of EMG activation and at return of consciousness. Pain was assessed immediately after return of consciousness using an 11 point numerical rating scale. The onset of EMG activation during emergence was associated with neither discernible muscle movement nor with the presence of exogenous stimulation in half the patients. EMG activation could be modelled as two distinct processes; termed high- and low-CeMAC (occurring higher or lower than 0.07 CeMAC). Low-CeMAC activation was typically associated with simultaneous EMG activation and consciousness, and the presence of a laryngeal mask. In contrast, high-CeMAC EMG activation occurred independently of return of consciousness, and was not associated with severe post-operative pain, but was more common in the presence of an endotracheal tube. Patients emerging from general anesthesia with an endotracheal tube in place are more likely to have an EMG activation at higher CeMAC concentrations. These activations are not associated with subsequent high-pain, nor with cortical arousal, as evidenced by continuing delta waves in the EEG. Conversely, patients emerging from general anesthesia with a laryngeal mask demonstrate marked neural inertia-EMG activation occurs at a low CeMAC, and is closely temporally associated with return of consciousness.

  9. National level promotion of physical activity: results from England's ACTIVE for LIFE campaign

    PubMed Central

    Hillsdon, M; Cavill, N; Nanchahal, K; Diamond, A; White, I

    2001-01-01

    STUDY OBJECTIVE—To assess the impact of a national campaign on awareness of the campaign, change in knowledge of physical activity recommendations and self reported physical activity.
DESIGN—three year prospective longitudinal survey using a multi-stage, cluster random probability design to select participants.
SETTING—England.
PARTICIPANTS—A nationally representative sample of 3189 adults aged 16-74 years.
MAIN OUTCOME MEASURES—Awareness of the advertising element of the campaign, changes in knowledge of physical activity recommendations for health and self reported physical activity.
RESULTS—38% of participants were aware of the main advertising images, assessed six to eight months after the main television advertisement. The proportion of participants knowledgeable about moderate physical activity recommendations increased by 3.0% (95% CI: 1.4%, 4.5%) between waves 1 and 2 and 3.7% (95% CI: 2.1%, 5.3%) between waves 1 and 3. The change in proportion of active people between baseline and waves 1 and 2 was
−0.02 (95% CI: −2.0 to +1.7) and between waves 1 and 3 was −9.8 (−7.9 to −11.7).
CONCLUSION—The proportion of participants who were knowledgeable about the new recommendations, increased significantly after the campaign. There was however, no significant difference in knowledge by awareness of the main campaign advertisement. There is no evidence that ACTIVE for LIFE improved physical activity, either overall or in any subgroup.


Keywords: exercise; mass media; follow up studies; health promotion; physical activity PMID:11553661

  10. What's in a name? Brain activity reveals categorization processes differ across languages.

    PubMed

    Liu, Chao; Tardif, Twila; Mai, Xiaoqin; Gehring, William J; Simms, Nina; Luo, Yue-Jia

    2010-11-01

    The linguistic relativity hypothesis proposes that speakers of different languages perceive and conceptualize the world differently, but do their brains reflect these differences? In English, most nouns do not provide linguistic clues to their categories, whereas most Mandarin Chinese nouns provide explicit category information, either morphologically (e.g., the morpheme "vehicle" che1 in the noun "train" huo3che1) or orthographically (e.g., the radical "bug" chong2 in the character for the noun "butterfly" hu2die2). When asked to judge the membership of atypical (e.g., train) vs. typical (e.g., car) pictorial exemplars of a category (e.g., vehicle), English speakers (N = 26) showed larger N300 and N400 event-related potential (ERP) component differences, whereas Mandarin speakers (N = 27) showed no such differences. Further investigation with Mandarin speakers only (N = 22) found that it was the morphologically transparent items that did not show a typicality effect, whereas orthographically transparent items elicited moderate N300 and N400 effects. In a follow-up study with English speakers only (N = 25), morphologically transparent items also showed different patterns of N300 and N400 activation than nontransparent items even for English speakers. Together, these results demonstrate that even for pictorial stimuli, how and whether category information is embedded in object names affects the extent to which typicality is used in category judgments, as shown in N300 and N400 responses.

  11. Optical Coherence Tomography angiography reveals laminar microvascular hemodynamics in the rat somatosensory cortex during activation.

    PubMed

    Srinivasan, Vivek J; Radhakrishnan, Harsha

    2014-11-15

    The BOLD (blood-oxygen-level dependent) fMRI (functional Magnetic Resonance Imaging) signal is shaped, in part, by changes in red blood cell (RBC) content and flow across vascular compartments over time. These complex dynamics have been challenging to characterize directly due to a lack of appropriate imaging modalities. In this study, making use of infrared light scattering from RBCs, depth-resolved Optical Coherence Tomography (OCT) angiography was applied to image laminar functional hyperemia in the rat somatosensory cortex. After defining and validating depth-specific metrics for changes in RBC content and speed, laminar hemodynamic responses in microvasculature up to cortical depths of >1mm were measured during a forepaw stimulus. The results provide a comprehensive picture of when and where changes in RBC content and speed occur during and immediately following cortical activation. In summary, the earliest and largest microvascular RBC content changes occurred in the middle cortical layers, while post-stimulus undershoots were most prominent superficially. These laminar variations in positive and negative responses paralleled known distributions of excitatory and inhibitory synapses, suggesting neuronal underpinnings. Additionally, the RBC speed response consistently returned to baseline more promptly than RBC content after the stimulus across cortical layers, supporting a "flow-volume mismatch" of hemodynamic origin.

  12. Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation

    SciTech Connect

    Liu, Haijun; Zhang, Hao; King, Jeremy D.; Wolf, Nathan R.; Prado, Mindy; Gross, Michael L.; Blankenship, Robert E.

    2014-12-01

    The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.

  13. Potentially novel copper resistance genes in copper-enriched activated sludge revealed by metagenomic analysis.

    PubMed

    Li, Li-Guan; Cai, Lin; Zhang, Xu-Xiang; Zhang, Tong

    2014-12-01

    In this study, we utilized the Illumina high-throughput metagenomic approach to investigate diversity and abundance of both microbial community and copper resistance genes (CuRGs) in activated sludge (AS) which was enriched under copper selective stress up to 800 mg/L. The raw datasets (~3.5 Gb for each sample, i.e., the copper-enriched AS and the control AS) were merged and normalized for the BLAST analyses against the SILVA SSU rRNA gene database and self-constructed copper resistance protein database (CuRD). Also, the raw metagenomic sequences were assembled into contigs and analyzed based on Open Reading Frames (ORFs) to identify potentially novel copper resistance genes. Among the different resistance systems for copper detoxification under the high copper stress condition, the Cus system was the most enriched system. The results also indicated that genes encoding multi-copper oxidase played a more important role than those encoding efflux proteins. More significantly, several potentially novel copper resistance ORFs were identified by Pfam search and phylogenic analysis. This study demonstrated a new understanding of microbial-mediated copper resistance under high copper stress using high-throughput shotgun sequencing technique.

  14. Analysis of Culex and Aedes mosquitoes in southwestern Nigeria revealed no West Nile virus activity

    PubMed Central

    Sule, Waidi Folorunso; Oluwayelu, Daniel Oladimeji

    2016-01-01

    Introduction Amplification and transmission of West Nile virus (WNV) by mosquitoes are driven by presence and number of viraemic/susceptible avian hosts. Methods In order to predict risk of WNV infection to humans, we collected mosquitoes from horse stables in Lagos and Ibadan, southwestern Nigeria. The mosquitoes were sorted and tested in pools with real-time RT-PCR to detect WNV (or flavivirus) RNA using WNV-specific primers and probes, as well as, pan-flavivirus-specific primers in two-step real-time RT-PCR. Minimum infection rate (MIR) was used to estimate mosquito infection rate. Results Only two genera of mosquitoes were caught (Culex, 98.9% and Aedes, 1.0%) totalling 4,112 females. None of the 424 mosquito pools tested was positive for WNV RNA; consequently the MIR was zero. Sequencing and BLAST analysis of amplicons detected in pan-flavivirus primer-mediated RT-PCR gave a consensus sequence of 28S rRNA of Culex quinquefasciatus suggesting integration of flaviviral RNA into mosquito genome. Conclusion While the latter finding requires further investigation, we conclude there was little or no risk of human infection with WNV in the study areas during sampling. There was predominance of Culex mosquito, a competent WNV vector, around horse stables in the study areas. However, mosquito surveillance needs to continue for prompt detection of WNV activity in mosquitoes. PMID:27279943

  15. Intratympanic manganese administration revealed sound intensity and frequency dependent functional activity in rat auditory pathway.

    PubMed

    Jin, Seong-Uk; Lee, Jae-Jun; Hong, Kwan Soo; Han, Mun; Park, Jang-Woo; Lee, Hui Joong; Lee, Sangheun; Lee, Kyu-Yup; Shin, Kyung Min; Cho, Jin Ho; Cheong, Chaejoon; Chang, Yongmin

    2013-09-01

    The cochlear plays a vital role in the sense and sensitivity of hearing; however, there is currently a lack of knowledge regarding the relationships between mechanical transduction of sound at different intensities and frequencies in the cochlear and the neurochemical processes that lead to neuronal responses in the central auditory system. In the current study, we introduced manganese-enhanced MRI (MEMRI), a convenient in vivo imaging method, for investigation of how sound, at different intensities and frequencies, is propagated from the cochlear to the central auditory system. Using MEMRI with intratympanic administration, we demonstrated differential manganese signal enhancements according to sound intensity and frequencies in the ascending auditory pathway of the rat after administration of intratympanic MnCl2.Compared to signal enhancement without explicit sound stimuli, auditory structures in the ascending auditory pathway showed stronger signal enhancement in rats who received sound stimuli of 10 and 40 kHz. In addition, signal enhancement with a stimulation frequency of 40 kHz was stronger than that with 10 kHz. Therefore, the results of this study seem to suggest that, in order to achieve an effective response to high sound intensity or frequency, more firing of auditory neurons, or firing of many auditory neurons together for the pooled neural activity is needed.

  16. The Truth Before and After: Brain Potentials Reveal Automatic Activation of Event Knowledge during Sentence Comprehension.

    PubMed

    Nieuwland, Mante S

    2015-11-01

    How does knowledge of real-world events shape our understanding of incoming language? Do temporal terms like "before" and "after" impact the online recruitment of real-world event knowledge? These questions were addressed in two ERP experiments, wherein participants read sentences that started with "before" or "after" and contained a critical word that rendered each sentence true or false (e.g., "Before/After the global economic crisis, securing a mortgage was easy/harder"). The critical words were matched on predictability, rated truth value, and semantic relatedness to the words in the sentence. Regardless of whether participants explicitly verified the sentences or not, false-after-sentences elicited larger N400s than true-after-sentences, consistent with the well-established finding that semantic retrieval of concepts is facilitated when they are consistent with real-world knowledge. However, although the truth judgments did not differ between before- and after-sentences, no such sentence N400 truth value effect occurred in before-sentences, whereas false-before-sentences elicited an enhanced subsequent positive ERPs. The temporal term "before" itself elicited more negative ERPs at central electrode channels than "after." These patterns of results show that, irrespective of ultimate sentence truth value judgments, semantic retrieval of concepts is momentarily facilitated when they are consistent with the known event outcome compared to when they are not. However, this inappropriate facilitation incurs later processing costs as reflected in the subsequent positive ERP deflections. The results suggest that automatic activation of event knowledge can impede the incremental semantic processes required to establish sentence truth value.

  17. Characterization of the Active Microbiotas Associated with Honey Bees Reveals Healthier and Broader Communities when Colonies are Genetically Diverse

    PubMed Central

    Mattila, Heather R.; Rios, Daniela; Walker-Sperling, Victoria E.; Roeselers, Guus; Newton, Irene L. G.

    2012-01-01

    Recent losses of honey bee colonies have led to increased interest in the microbial communities that are associated with these important pollinators. A critical function that bacteria perform for their honey bee hosts, but one that is poorly understood, is the transformation of worker-collected pollen into bee bread, a nutritious food product that can be stored for long periods in colonies. We used 16S rRNA pyrosequencing to comprehensively characterize in genetically diverse and genetically uniform colonies the active bacterial communities that are found on honey bees, in their digestive tracts, and in bee bread. This method provided insights that have not been revealed by past studies into the content and benefits of honey bee-associated microbial communities. Colony microbiotas differed substantially between sampling environments and were dominated by several anaerobic bacterial genera never before associated with honey bees, but renowned for their use by humans to ferment food. Colonies with genetically diverse populations of workers, a result of the highly promiscuous mating behavior of queens, benefited from greater microbial diversity, reduced pathogen loads, and increased abundance of putatively helpful bacteria, particularly species from the potentially probiotic genus Bifidobacterium. Across all colonies, Bifidobacterium activity was negatively correlated with the activity of genera that include pathogenic microbes; this relationship suggests a possible target for understanding whether microbes provide protective benefits to honey bees. Within-colony diversity shapes microbiotas associated with honey bees in ways that may have important repercussions for colony function and health. Our findings illuminate the importance of honey bee-bacteria symbioses and examine their intersection with nutrition, pathogen load, and genetic diversity, factors that are considered key to understanding honey bee decline. PMID:22427917

  18. Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity.

    PubMed

    Offman, Marc N; Krol, Marcin; Patel, Naina; Krishnan, Shekhar; Liu, JiZhong; Saha, Vaskar; Bates, Paul A

    2011-02-03

    Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginase to resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

  19. fMRI reveals neural activity overlap between adult and infant pain.

    PubMed

    Goksan, Sezgi; Hartley, Caroline; Emery, Faith; Cockrill, Naomi; Poorun, Ravi; Moultrie, Fiona; Rogers, Richard; Campbell, Jon; Sanders, Michael; Adams, Eleri; Clare, Stuart; Jenkinson, Mark; Tracey, Irene; Slater, Rebeccah

    2015-04-21

    Limited understanding of infant pain has led to its lack of recognition in clinical practice. While the network of brain regions that encode the affective and sensory aspects of adult pain are well described, the brain structures involved in infant nociceptive processing are completely unknown, meaning we cannot infer anything about the nature of the infant pain experience. Using fMRI we identified the network of brain regions that are active following acute noxious stimulation in newborn infants, and compared the activity to that observed in adults. Significant infant brain activity was observed in 18 of the 20 active adult brain regions but not in the infant amygdala or orbitofrontal cortex. Brain regions that encode sensory and affective components of pain are active in infants, suggesting that the infant pain experience closely resembles that seen in adults. This highlights the importance of developing effective pain management strategies in this vulnerable population.

  20. Transcriptional response of zebrafish embryos exposed to neurotoxic compounds reveals a muscle activity dependent hspb11 expression.

    PubMed

    Klüver, Nils; Yang, Lixin; Busch, Wibke; Scheffler, Katja; Renner, Patrick; Strähle, Uwe; Scholz, Stefan

    2011-01-01

    Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sop(fixe) mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.

  1. RNA Profiles of Porcine Embryos during Genome Activation Reveal Complex Metabolic Switch Sensitive to In Vitro Conditions

    PubMed Central

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben; Hyttel, Poul; Collas, Philippe; Cabot, Ryan

    2013-01-01

    Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA). While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell stage) EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery), protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism), different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos

  2. Optogenetic activation reveals distinct roles of PIP3 and Akt in adipocyte insulin action.

    PubMed

    Xu, Yingke; Nan, Di; Fan, Jiannan; Bogan, Jonathan S; Toomre, Derek

    2016-05-15

    Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.

  3. Revealing a strongly reddened, faint active galactic nucleus population by stacking deep co-added images

    NASA Astrophysics Data System (ADS)

    Varga, József; Csabai, István.; Dobos, László

    2012-10-01

    More than half of the sources identified by recent radio sky surveys have not been detected by wide-field optical surveys. We present a study, based on our co-added image stacking technique, in which our aim is to detect the optical emission from unresolved, isolated radio sources of the Very Large Array (VLA) Faint Images of the Radio Sky at Twenty-cm (FIRST) survey that have no identified optical counterparts in the Sloan Digital Sky Survey (SDSS) Stripe 82 co-added data set. From the FIRST catalogue, 2116 such radio point sources were selected, and cut-out images, centred on the FIRST coordinates, were generated from the Stripe 82 images. The already co-added cut-outs were stacked once again to obtain images of high signal-to-noise ratio, in the hope that optical emission from the radio sources would become detectable. Multiple stacks were generated, based on the radio luminosity of the point sources. The resulting stacked images show central peaks similar to point sources. The peaks have very red colours with steep optical spectral energy distributions. We have found that the optical spectral index αν falls in the range -2.9 ≤ αν ≤ -2.2 (Sν∝ναν), depending only weakly on the radio flux. The total integration times of the stacks are between 270 and 300 h, and the corresponding 5σ detection limit is estimated to be about mr ≃ 26.6 mag. We argue that the detected light is mainly from the central regions of dust-reddened Type 1 active galactic nuclei. Dust-reddened quasars might represent an early phase of quasar evolution, and thus they can also give us an insight into the formation of massive galaxies. The data used in the paper are available on-line at http://www.vo.elte.hu/doublestacking.

  4. Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography.

    PubMed Central

    Weisbrod, S T

    1982-01-01

    Nucleosomes from actively transcribed genes (active nucleosomes) contain nonhistone proteins HMG 14 and 17 and are preferentially sensitive to digestion by DNAse I. Active nucleosomes isolated by chromatography on an HMG 14 and 17 glass bead affinity column were analyzed with respect to overall structure, accessory nonhistone components and modifications to the DNA and histones. The experiments lead to the following conclusions: the DNA in the active nucleosome is undermethylated compared to bulk DNA; topoisomerase I is a non-stoichiometric component of the active nucleosome fraction; the level of histone acetylation is enriched in active nucleosomes, but the extent of enrichment cannot account for HMG binding; and the two histone H3 molecules in the active nucleosome can dimerize more readily and are, therefore, probably closer together than those in the bulk of the nucleosomes. Additionally it is shown that HMG 14 and 17 prefer to bind to single- vs. double-stranded nucleic acids. The role of HMG 14 and 17 in producing a highly DNAse I sensitive structure and correspondingly helping to facilitate transcription is discussed in terms of these properties. Images PMID:6210882

  5. Equilibrium physics breakdown reveals the active nature of red blood cell flickering

    NASA Astrophysics Data System (ADS)

    Turlier, H.; Fedosov, D. A.; Audoly, B.; Auth, T.; Gov, N. S.; Sykes, C.; Joanny, J.-F.; Gompper, G.; Betz, T.

    2016-05-01

    Red blood cells, or erythrocytes, are seen to flicker under optical microscopy, a phenomenon initially described as thermal fluctuations of the cell membrane. But recent studies have suggested the involvement of non-equilibrium processes, without definitively ruling out equilibrium interpretations. Using active and passive microrheology to directly compare the membrane response and fluctuations on single erythrocytes, we report here a violation of the fluctuation-dissipation relation, which is a direct demonstration of the non-equilibrium nature of flickering. With an analytical model of the composite erythrocyte membrane and realistic stochastic simulations, we show that several molecular mechanisms may explain the active fluctuations, and we predict their kinetics. We demonstrate that tangential metabolic activity in the network formed by spectrin, a cytoskeletal protein, can generate curvature-mediated active membrane motions. We also show that other active membrane processes represented by direct normal force dipoles may explain the observed membrane activity. Our findings provide solid experimental and theoretical frameworks for future investigations of the origin and function of active motion in cells.

  6. Young asteroidal fluid activity revealed by absolute age from apatite in carbonaceous chondrite

    PubMed Central

    Zhang, Ai-Cheng; Li, Qiu-Li; Yurimoto, Hisayoshi; Sakamoto, Naoya; Li, Xian-Hua; Hu, Sen; Lin, Yang-Ting; Wang, Ru-Cheng

    2016-01-01

    Chondritic meteorites, consisting of the materials that have formed in the early solar system (ESS), have been affected by late thermal events and fluid activity to various degrees. Determining the timing of fluid activity in ESS is of fundamental importance for understanding the nature, formation, evolution and significance of fluid activity in ESS. Previous investigations have determined the relative ages of fluid activity with short-lived isotope systematics. Here we report an absolute 207Pb/206Pb isochron age (4,450±50 Ma) of apatite from Dar al Gani (DaG) 978, a type ∼3.5, ungrouped carbonaceous chondrite. The petrographic, mineralogical and geochemical features suggest that the apatite in DaG 978 should have formed during metamorphism in the presence of a fluid. Therefore, the apatite age represents an absolute age for fluid activity in an asteroidal setting. An impact event could have provided the heat to activate this young fluid activity in ESS. PMID:27682449

  7. Single-Molecule Nanocatalysis Reveals Catalytic Activation Energy of Single Nanocatalysts.

    PubMed

    Chen, Tao; Zhang, Yuwei; Xu, Weilin

    2016-09-28

    By monitoring the temperature-dependent catalytic activity of single Au nanocatalysts for a fluorogenic reaction, we derive the activation energies via multiple methods for two sequential catalytic steps (product formation and dissociation) on single nanocatalysts. The wide distributions of activation energies across multiple individual nanocatalysts indicate a huge static heterogeneity among the individual nanocatalysts. The compensation effect and isokinetic relationship of catalytic reactions are observed at the single particle level. This study exemplifies another function of single-molecule nanocatalysis and improves our understanding of heterogeneous catalysis.

  8. The crystal structure of the cysteine protease Xylellain from Xylella fastidiosa reveals an intriguing activation mechanism.

    PubMed

    Leite, Ney Ribeiro; Faro, Aline Regis; Dotta, Maria Amélia Oliva; Faim, Livia Maria; Gianotti, Andreia; Silva, Flavio Henrique; Oliva, Glaucius; Thiemann, Otavio Henrique

    2013-02-14

    Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator.

  9. Spores of most common airborne fungi reveal no ice nucleation activity

    NASA Astrophysics Data System (ADS)

    Pummer, B. G.; Atanasova, L.; Bauer, H.; Bernardi, J.; Druzhinina, I. S.; Grothe, H.

    2013-06-01

    Fungal spores are ubiquitous biological aerosols, which are considered to show ice nucleation (IN) activity. In this study the respective IN activity was tested in oil emulsion in the immersion freezing mode. The focus was laid on species of economical, ecological or sanitary significance. For the first time, not only common moulds, but also edible mushrooms (Basidiomycota, Agaricomycetes) were investigated, as they contribute massively to the total amount of fungal spores in the atmosphere. Only Fusarium avenaceum showed freezing events at low subzero-temperatures, while the other investigated fungal spores showed no significant IN activity. Furthermore, we selected a set of fungal strains from different sites and exposed them to occasional freezing stress during cultivation. Although the total protein expression was altered by this treatment, it had no significant impact on the IN activity.

  10. Activation pathway of Src kinase reveals intermediate states as targets for drug design

    NASA Astrophysics Data System (ADS)

    Shukla, Diwakar; Meng, Yilin; Roux, Benoît; Pande, Vijay S.

    2014-03-01

    Unregulated activation of Src kinases leads to aberrant signalling, uncontrolled growth and differentiation of cancerous cells. Reaching a complete mechanistic understanding of large-scale conformational transformations underlying the activation of kinases could greatly help in the development of therapeutic drugs for the treatment of these pathologies. In principle, the nature of conformational transition could be modelled in silico via atomistic molecular dynamics simulations, although this is very challenging because of the long activation timescales. Here we employ a computational paradigm that couples transition pathway techniques and Markov state model-based massively distributed simulations for mapping the conformational landscape of c-src tyrosine kinase. The computations provide the thermodynamics and kinetics of kinase activation for the first time, and help identify key structural intermediates. Furthermore, the presence of a novel allosteric site in an intermediate state of c-src that could be potentially used for drug design is predicted.

  11. Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways

    PubMed Central

    Zeng, Hui; Wu, Huixia; Sloane, Valerie; Jones, Rheinallt; Yu, Yimin; Lin, Patricia; Gewirtz, Andrew T.; Neish, Andrew S.

    2015-01-01

    Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-α, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-α, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-κB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections. PMID:16179598

  12. A systems study reveals concurrent activation of AMPK and mTOR by amino acids

    PubMed Central

    Pezze, Piero Dalle; Ruf, Stefanie; Sonntag, Annika G.; Langelaar-Makkinje, Miriam; Hall, Philip; Heberle, Alexander M.; Navas, Patricia Razquin; van Eunen, Karen; Tölle, Regine C.; Schwarz, Jennifer J.; Wiese, Heike; Warscheid, Bettina; Deitersen, Jana; Stork, Björn; Fäßler, Erik; Schäuble, Sascha; Hahn, Udo; Horvatovich, Peter; Shanley, Daryl P.; Thedieck, Kathrin

    2016-01-01

    Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational–experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes. PMID:27869123

  13. Activity Patterns of Free-Ranging Koalas (Phascolarctos cinereus) Revealed by Accelerometry

    PubMed Central

    Ryan, Michelle A.; Whisson, Desley A.; Holland, Greg J.; Arnould, John P. Y.

    2013-01-01

    An understanding of koala activity patterns is important for measuring the behavioral response of this species to environmental change, but to date has been limited by the logistical challenges of traditional field methodologies. We addressed this knowledge gap by using tri-axial accelerometer data loggers attached to VHF radio collars to examine activity patterns of adult male and female koalas in a high-density population at Cape Otway, Victoria, Australia. Data were obtained from 27 adult koalas over two 7-d periods during the breeding season: 12 in the early-breeding season in November 2010, and 15 in the late-breeding season in January 2011. Multiple 15 minute observation blocks on each animal were used for validation of activity patterns determined from the accelerometer data loggers. Accelerometry was effective in distinguishing between inactive (sleeping, resting) and active (grooming, feeding and moving) behaviors. Koalas were more active during the early-breeding season with a higher index of movement (overall dynamic body acceleration [ODBA]) for both males and females. Koalas showed a distinct temporal pattern of behavior, with most activity occurring from mid-afternoon to early morning. Accelerometry has potential for examining fine-scale behavior of a wide range of arboreal and terrestrial species. PMID:24224050

  14. Structure of Escherichia coli tyrosine Kinase Etk Reveals a Novel Activation Mechanism

    SciTech Connect

    Lee,D.; Zheng, J.; She, Y.; Jia, Z.

    2008-01-01

    While protein tyrosine (Tyr) kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. The inner-membrane Wzc/Etk protein belongs to the bacterial PTK family, which has an important function in regulating the polymerization and transport of virulence-determining capsular polysaccharide (CPS). The kinase uses a unique two-step activation process involving intra-phosphorylation of a Tyr residue, although the molecular mechanism remains unknown. Herein, we report the first crystal structure of a bacterial PTK, the C-terminal kinase domain of Escherichia coli Tyr kinase (Etk) at 2.5-Angstroms resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting mass spectrometric evidence of Etk, a unique activation mechanism is proposed that involves the phosphorylated Tyr residue, Y574, at the active site and its specific interaction with a previously unidentified key Arg residue, R614, to unblock the active site. Both in vitro kinase activity and in vivo antibiotics resistance studies using structure-guided mutants further support the novel activation mechanism.

  15. Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation

    PubMed Central

    Ikeh, Mélanie A. C.; Haider, Mohammed; Brown, Alistair J. P.; Morgan, Brian A.; Erwig, Lars P.; Quinn, Janet

    2017-01-01

    The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the ‘p38-related’ Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation. PMID:28135328

  16. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle

    PubMed Central

    Nuber, Susanne; Zabel, Ulrike; Lorenz, Kristina; Nuber, Andreas; Milligan, Graeme; Tobin, Andrew B.; Lohse, Martin J.; Hoffmann, Carsten

    2016-01-01

    (β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs)1–3. They bind to active, phosphorylated GPCRs and thereby shut off ‘classical’ signalling to G proteins3,4, trigger internalization of GPCRs via interaction with the clathrin machinery5–7 and mediate signalling via ‘non-classical’ pathways1,2. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs)1,3,4. The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography8–10. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor–β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling. PMID:27007855

  17. How community environment shapes physical activity: perceptions revealed through the PhotoVoice method.

    PubMed

    Belon, Ana Paula; Nieuwendyk, Laura M; Vallianatos, Helen; Nykiforuk, Candace I J

    2014-09-01

    A growing body of evidence shows that community environment plays an important role in individuals' physical activity engagement. However, while attributes of the physical environment are widely investigated, sociocultural, political, and economic aspects of the environment are often neglected. This article helps to fill these knowledge gaps by providing a more comprehensive understanding of multiple dimensions of the community environment relative to physical activity. The purpose of this study was to qualitatively explore how people's experiences and perceptions of their community environments affect their abilities to engage in physical activity. A PhotoVoice method was used to identify barriers to and opportunities for physical activity among residents in four communities in the province of Alberta, Canada, in 2009. After taking pictures, the thirty-five participants shared their perceptions of those opportunities and barriers in their community environments during individual interviews. Using the Analysis Grid for Environments Linked to Obesity (ANGELO) framework, themes emerging from these photo-elicited interviews were organized in four environment types: physical, sociocultural, economic, and political. The data show that themes linked to the physical (56.6%) and sociocultural (31.4%) environments were discussed more frequently than the themes of the economic (5.9%) and political (6.1%) environments. Participants identified nuanced barriers and opportunities for physical activity, which are illustrated by their quotes and photographs. The findings suggest that a myriad of factors from physical, sociocultural, economic, and political environments influence people's abilities to be physically active in their communities. Therefore, adoption of a broad, ecological perspective is needed to address the barriers and build upon the opportunities described by participants to make communities more healthy and active.

  18. Precursory Activity Before Larger Events in Greece Revealed by Aggregated Seismicity Data

    NASA Astrophysics Data System (ADS)

    Adamaki, Angeliki K.; Roberts, Roland G.

    2017-03-01

    We investigate the seismicity rate behaviour in and around Greece during 2009, seeking significant changes in rate preceding larger events. For individual larger events it is difficult to clearly distinguish precursory rate changes from other, possibly unrelated, variations in seismicity. However, when we aggregate seismicity data occurring within a radius of 10 km and in a 50-day window prior to earthquakes with, e.g. magnitude ≥3.5, the resulting aggregated time series show a clearly increasing trend starting 2-3 weeks prior to the "mainshock" time. We apply statistical tests to investigate if the observed behaviour may be simply consistent with random (poissonian) variations, or, as some earlier studies suggest, with clustering in the sense that high activity rates at some time may imply increased rates later, and thus (randomly) greater probability of larger coming events than for periods of lower seismicity. In this case, rate increases have little useful predictive power. Using data from the entire catalogue, the aggregated rate changes before larger events are clearly and strongly statistically significant and cannot be explained by such clustering. To test this we choose events at random from the catalogue as potential "mainshocks". The events preceding the randomly chosen earthquakes show less pronounced rate increases compared to the observed rate changes prior to larger events. Similar behaviour is observed in data sub-sets. However, statistical confidence decreases for geographical subsets containing few "mainshocks" as it does when data are weighted such that "mainshocks" with many preceding events are strongly downweighted relative to those with fewer. The analyses suggest that genuine changes in aggregated rate do occur prior to larger events and that this behaviour is not due to a small number of mainshocks with many preceding events dominating the analysis. It does not automatically follow that it will be possible to routinely observe precursory

  19. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    PubMed

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  20. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

  1. From Blame to Punishment: Disrupting Prefrontal Cortex Activity Reveals Norm Enforcement Mechanisms.

    PubMed

    Buckholtz, Joshua W; Martin, Justin W; Treadway, Michael T; Jan, Katherine; Zald, David H; Jones, Owen; Marois, René

    2015-09-23

    The social welfare provided by cooperation depends on the enforcement of social norms. Determining blameworthiness and assigning a deserved punishment are two cognitive cornerstones of norm enforcement. Although prior work has implicated the dorsolateral prefrontal cortex (DLPFC) in norm-based judgments, the relative contribution of this region to blameworthiness and punishment decisions remains poorly understood. Here, we used repetitive transcranial magnetic stimulation (rTMS) and fMRI to determine the specific role of DLPFC function in norm-enforcement behavior. DLPFC rTMS reduced punishment for wrongful acts without affecting blameworthiness ratings, and fMRI revealed punishment-selective DLPFC recruitment, suggesting that these two facets of norm-based decision making are neurobiologically dissociable. Finally, we show that DLPFC rTMS affects punishment decision making by altering the integration of information about culpability and harm. Together, these findings reveal a selective, causal role for DLPFC in norm enforcement: representational integration of the distinct information streams used to make punishment decisions.

  2. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

    PubMed

    Irani, Seema; Yogesha, S D; Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L; Nie, Grace; Prescott, Nicholas A; Zhang, Yan Jessie

    2016-03-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

  3. Recent and episodic volcanic and glacial activity on Mars revealed by the High Resolution Stereo Camera.

    PubMed

    Neukum, G; Jaumann, R; Hoffmann, H; Hauber, E; Head, J W; Basilevsky, A T; Ivanov, B A; Werner, S C; van Gasselt, S; Murray, J B; McCord, T

    2004-12-23

    The large-area coverage at a resolution of 10-20 metres per pixel in colour and three dimensions with the High Resolution Stereo Camera Experiment on the European Space Agency Mars Express Mission has made it possible to study the time-stratigraphic relationships of volcanic and glacial structures in unprecedented detail and give insight into the geological evolution of Mars. Here we show that calderas on five major volcanoes on Mars have undergone repeated activation and resurfacing during the last 20 per cent of martian history, with phases of activity as young as two million years, suggesting that the volcanoes are potentially still active today. Glacial deposits at the base of the Olympus Mons escarpment show evidence for repeated phases of activity as recently as about four million years ago. Morphological evidence is found that snow and ice deposition on the Olympus construct at elevations of more than 7,000 metres led to episodes of glacial activity at this height. Even now, water ice protected by an insulating layer of dust may be present at high altitudes on Olympus Mons.

  4. Small-angle neutron and X-ray scattering reveal conformational changes in rhodopsin activation

    NASA Astrophysics Data System (ADS)

    Shrestha, Utsab R.; Bhowmik, Debsindhu; Perera, Suchitrhanga M. C. D.; Chawla, Udeep; Struts, Andrey V.; Graziono, Vito; Pingali, Sai Venkatesh; Heller, William T.; Qian, Shuo; Brown, Michael F.; Chu, Xiang-Qiang

    2015-03-01

    Understanding G-protein-coupled receptor (GPCR) activation plays a crucial role in the development of novel improved molecular drugs. During photo-activation, the retinal chromophore of the visual GPCR rhodopsin isomerizes from 11-cis to all-trans conformation, yielding an equilibrium between inactive Meta-I and active Meta-II states. The principal goals of this work are to address whether the activation of rhodopsin leads to a single state or a conformational ensemble, and how protein organizational structure changes with detergent environment in solution. We use both small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) techniques to answer the above questions. For the first time we observe the change in protein conformational ensemble upon photo-activation by SANS with contrast variation, which enables the separate study of the protein structure within the detergent assembly. In addition, SAXS study of protein structure within detergent assembly suggests that the detergent molecules form a belt of monolayer (micelle) around protein with different geometrical shapes to keep the protein in folded state.

  5. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

    PubMed Central

    Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L.; Nie, Grace; Prescott, Nicholas A.; Zhang, Yan Jessie

    2016-01-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families. PMID:26933063

  6. Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

    PubMed Central

    Jehuda-Cohen, T; Slade, B A; Powell, J D; Villinger, F; De, B; Folks, T M; McClure, H M; Sell, K W; Ahmed-Ansari, A

    1990-01-01

    Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals. Images PMID:2111024

  7. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    NASA Astrophysics Data System (ADS)

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-10-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.

  8. Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

    PubMed Central

    Rocio Servin-Vences, M; Moroni, Mirko; Lewin, Gary R; Poole, Kate

    2017-01-01

    The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes. DOI: http://dx.doi.org/10.7554/eLife.21074.001 PMID:28135189

  9. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    SciTech Connect

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  10. High throughput estimation of functional cell activities reveals disease mechanisms and predicts relevant clinical outcomes.

    PubMed

    Hidalgo, Marta R; Cubuk, Cankut; Amadoz, Alicia; Salavert, Francisco; Carbonell-Caballero, José; Dopazo, Joaquin

    2017-01-17

    Understanding the aspects of the cell functionality that account for disease or drug action mechanisms is a main challenge for precision medicine. Here we propose a new method that models cell signaling using biological knowledge on signal transduction. The method recodes individual gene expression values (and/or gene mutations) into accurate measurements of changes in the activity of signaling circuits, which ultimately constitute high-throughput estimations of cell functionalities caused by gene activity within the pathway. Moreover, such estimations can be obtained either at cohort-level, in case/control comparisons, or personalized for individual patients. The accuracy of the method is demonstrated in an extensive analysis involving 5640 patients from 12 different cancer types. Circuit activity measurements not only have a high diagnostic value but also can be related to relevant disease outcomes such as survival, and can be used to assess therapeutic interventions.

  11. Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity

    PubMed Central

    Stavrinides, Anna; Tatsis, Evangelos C.; Caputi, Lorenzo; Foureau, Emilien; Stevenson, Clare E. M.; Lawson, David M.; Courdavault, Vincent; O'Connor, Sarah E.

    2016-01-01

    Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold. PMID:27418042

  12. Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities

    SciTech Connect

    Gibson, S.; Jung, C.Y.; Takahashi, M.; Lenard, J.

    1986-10-07

    The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the monomer of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.

  13. In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins.

    PubMed

    Granstedt, Andrea E; Bosse, Jens B; Thiberge, Stephan Y; Enquist, Lynn W

    2013-09-10

    A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.

  14. Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

    PubMed

    Contreras-Vite, Juan A; Cruz-Rangel, Silvia; De Jesús-Pérez, José J; Figueroa, Iván A Aréchiga; Rodríguez-Menchaca, Aldo A; Pérez-Cornejo, Patricia; Hartzell, H Criss; Arreola, Jorge

    2016-07-01

    TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.

  15. Masked priming of conceptual features reveals differential brain activation during unconscious access to conceptual action and sound information.

    PubMed

    Trumpp, Natalie M; Traub, Felix; Kiefer, Markus

    2013-01-01

    Previous neuroimaging studies suggested an involvement of sensory-motor brain systems during conceptual processing in support of grounded cognition theories of conceptual memory. However, in these studies with visible stimuli, contributions of strategic imagery or semantic elaboration processes to observed sensory-motor activity cannot be entirely excluded. In the present study, we therefore investigated the electrophysiological correlates of unconscious feature-specific priming of action- and sound-related concepts within a novel feature-priming paradigm to specifically probe automatic processing of conceptual features without the contribution of possibly confounding factors such as orthographic similarity or response congruency. Participants were presented with a masked subliminal prime word and a subsequent visible target word. In the feature-priming conditions primes as well as targets belonged to the same conceptual feature dimension (action or sound, e.g., typewriter or radio) whereas in the two non-priming conditions, either the primes or the targets consisted of matched control words with low feature relevance (e.g., butterfly or candle). Event-related potential analyses revealed unconscious feature-specific priming effects at fronto-central electrodes within 100 to 180 ms after target stimulus onset that differed with regard to topography and underlying neural generators. In congruency with previous findings under visible stimulation conditions, these differential subliminal ERP feature-priming effects demonstrate an unconscious automatic access to action versus sound features of concepts. The present results therefore support grounded cognition theory suggesting that activity in sensory and motor areas during conceptual processing can also occur unconsciously and is not mandatorily accompanied by a vivid conscious experience of the conceptual content such as in imagery.

  16. Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

    PubMed Central

    Villar, Rina F.; Patel, Jinal; Weaver, Grant C.; Kanekiyo, Masaru; Wheatley, Adam K.; Yassine, Hadi M.; Costello, Catherine E.; Chandler, Kevin B.; McTamney, Patrick. M.; Nabel, Gary J.; McDermott, Adrian B.; Mascola, John R.; Carr, Steven A.; Lingwood, Daniel

    2016-01-01

    Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed. PMID:27796362

  17. How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

    PubMed Central

    Hytönen, Vesa P; Vogel, Viola

    2008-01-01

    Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

  18. Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

  19. Increasing AIP Macrocycle Size Reveals Key Features of agr Activation in Staphylococcus aureus.

    PubMed

    Johnson, Jeffrey G; Wang, Boyuan; Debelouchina, Galia T; Novick, Richard P; Muir, Tom W

    2015-05-04

    The agr locus in the commensal human pathogen, Staphylococcus aureus, is a two-promoter regulon with allelic variability that produces a quorum-sensing circuit involved in regulating virulence within the bacterium. Secretion of unique autoinducing peptides (AIPs) and detection of their concentrations by AgrC, a transmembrane receptor histidine kinase, coordinates local bacterial population density with global changes in gene expression. The finding that staphylococcal virulence can be inhibited through antagonism of this quorum-sensing pathway has fueled tremendous interest in understanding the structure-activity relationships underlying the AIP-AgrC interaction. The defining structural feature of the AIP is a 16-membered, thiolactone-containing macrocycle. Surprisingly, the importance of ring size on agr activation or inhibition has not been explored. In this study, we address this deficiency through the synthesis and functional analysis of AIP analogues featuring enlarged and reduced macrocycles. Notably, this study is the first to interrogate AIP function by using both established cell-based reporter gene assays and newly developed in vitro AgrC-I binding and autophosphorylation activity assays. Based on our data, we present a model for robust agr activation involving a cooperative, three-points-of-contact interaction between the AIP macrocycle and AgrC.

  20. Event-related potentials reveal early activation of body part representations in action concept comprehension.

    PubMed

    Lu, Aitao; Liu, Jing; Zhang, John X

    2012-03-09

    With tasks involving action concept comprehension, many fMRI studies have reported brain activations in sensori-motor regions specific to effectors of the referent action. There is relatively less evidence whether such activations reflect early semantic access or late conceptual re-processing. Here we recorded event-related potentials when participants recognized noun-verb pairs. For Congruent pairs, the verb was the one most commonly associated with the noun (e.g., football-kick). Compared with a control condition, verbs in Congruent pairs showed priming effects in the time windows of 100-150 ms and 210-260 ms. Such activation seems to be specific to body part but not other aspects of the action as similar priming effect was also found when the noun and verb involved different actions though sharing the same body part (e.g., football-jump), documenting for the first time the early activation of body part representations in action concept comprehension.

  1. Vector analysis of ecoenzyme activities reveal constraints on coupled C, N and P dynamics

    EPA Science Inventory

    We developed a quantitative method for estimating resource allocation strategies of microbial communities based on the proportional activities of four, key extracellular enzymes, 1,4-ß-glucosidase (BG), leucine amino-peptidase (LAP), 1,4-ß-N-acetylglucosaminidase (NAG...

  2. Ensemble Activation of G-Protein -Coupled Receptors Revealed by Small-Angle Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Chu, Xiang-Qiang; Perera, Suchithranga; Shrestha, Utsab; Chawla, Udeep; Struts, Andrey; Qian, Shuo; Brown, Michael

    2014-03-01

    Rhodopsin is a G-protein -coupled receptor (GPCR) involved in visual light perception and occurs naturally in a membrane lipid environment. Rhodopsin photoactivation yields cis-trans isomerization of retinal giving equilibrium between inactive Meta-I and active Meta-II states. Does photoactivation lead to a single Meta-II conformation, or do substates exist as described by an ensemble-activation mechanism (EAM)? We use small-angle neutron scattering (SANS) to investigate conformational changes in rhodopsin-detergent and rhodopsin-lipid complexes upon photoactivation. Meta-I state is stabilized in CHAPS-solubilized rhodopsin, while Meta-II is trapped in DDM-solubilized rhodopsin. SANS data are acquired from 80% D2O solutions and at contrast-matching points for both DDM and CHAPS samples. Our experiments demonstrate that for detergent-solubilized rhodopsin, SANS with contrast variation can detect structural differences between the rhodopsin dark-state, Meta-I, Meta-II, and ligand-free opsin states. Dark-state rhodopsin has more conformational flexibility in DDM micelles compared to CHAPS, which is consistent with an ensemble of activated Meta-II states. Furthermore, time-resolved SANS enables study of the time-dependent structural transitions between Meta-I and Meta-II, which is crucial to understanding the ensemble-based activation.

  3. Active role of the liquid phase of developer in revealing surface flaws by capillary methods

    SciTech Connect

    Prokhorenko, P.P.; Dezhkunov, N.V.; Stoicheva, I.V.

    1988-08-01

    The article investigates the interaction of two chemically nonreacting liquids after they have been brought into contact with each other in a capillary. It is established that the liquid phase of the developer is not only a passive carrier of the developing component but also exerts an active influence on the process of development, and consequently, on the detectability of flaws.

  4. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

    PubMed Central

    Beck, Florian; Geiger, Jörg; Gambaryan, Stepan; Solari, Fiorella A.; Dell’Aica, Margherita; Loroch, Stefan; Mattheij, Nadine J.; Mindukshev, Igor; Pötz, Oliver; Jurk, Kerstin; Burkhart, Julia M.; Fufezan, Christian; Heemskerk, Johan W. M.; Walter, Ulrich

    2017-01-01

    Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein–coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. PMID:28060719

  5. Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase.

    PubMed

    Burchett, Gina G; Folsom, Charles G; Lane, Kimberly T

    2015-01-01

    β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins.

  6. Balloon-related activities of the IMK/FZK: instruments, activities and results

    NASA Astrophysics Data System (ADS)

    Friedl-Vallon, Felix; Oelhaf, Hermann; Kleinert, Anne; Lengel, Anton; Maucher, Guido; Nordmeyer, Hans; Stowasser, Markus; Wetzel, Gerald; Fischer, Herbert

    2001-08-01

    The Institut für Meteorologie und Klimaforschung of the Forschungszentrum Karlsruhe (IMK/FZK) is operating a balloon-borne instrument for atmospheric research since the late eighties. The MIPAS-B (Michelson Interferometer for Passive Atmospheric Sounding - Balloon) instrument, a cryogenic Fourier spectrometer for limb emission measurements has flown 13 times in two different versions from France and Sweden. In this time the instrument has participated in three large international campaigns (EASOE, SESAME, THESEO) and several smaller field-activities (CHORUS, CHELOSBA, POSTA) concerning stratospheric chemistry from which important contributions to the understanding of the Arctic ozone depletion mechanisms have been achieved. Additionally, the instrument participated as core payload in the validation of the Japanese ILAS instrument on-board ADEOS and supported the validation of the American CLAES instrument onboard UARS. MIPAS-B data served also to test the level 2 algorithms for the MIPAS-instrument on-board ENVISAT. Present activities focus on the understanding of the composition and role of polar stratospheric clouds. The second centre of attention in the next years will be the validation of the European satellite sensors GOMOS, MIPAS, and SCIAMACHY with the established MIPAS-B version.

  7. Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

    PubMed

    Ponnuvel, Shobana; Subramanian, Balakumar; Ponnuraj, Karthe

    2015-10-01

    Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

  8. Fractionation of a Herbal Antidiarrheal Medicine Reveals Eugenol as an Inhibitor of Ca2+-Activated Cl− Channel TMEM16A

    PubMed Central

    Yao, Zhen; Namkung, Wan; Ko, Eun A.; Park, Jinhong; Tradtrantip, Lukmanee; Verkman, A. S.

    2012-01-01

    The Ca2+-activated Cl− channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl− conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl− conductance with single-site IC50∼150 µM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl− channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities. PMID:22666439

  9. A Global Genomic Screening Strategy Reveals Genetic and Chemical Activators ofPeroxisome Proliferator-Activated Receptor alpha (PPARalpha)

    EPA Science Inventory

    A comprehensive survey of chemical, diet and genetic perturbations that activate PPARalpha in the mouse liver has not been carried out but would be useful to identify the factors that may contribute to PPARalpha-dependent liver tumors. A gene signature dependent on PPARalpha ac...

  10. Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1.

    PubMed

    Goyette, Jesse; Salas, Citlali Solis; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel A; Allard, Jun; Dushek, Omer

    2017-03-01

    Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling.

  11. Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1

    PubMed Central

    Goyette, Jesse; Salas, Citlali Solis; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel A.; Allard, Jun; Dushek, Omer

    2017-01-01

    Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling. PMID:28378014

  12. Fluid escape structures in the Graham Bank region (Sicily Channel, Central Mediterranean) revealing volcanic and neotectonic activity.

    NASA Astrophysics Data System (ADS)

    Spatola, Daniele; Pennino, Valentina; Basilone, Luca; Interbartolo, Francesco; Micallef, Aaron; Sulli, Attilio; Basilone, Walter

    2016-04-01

    morphometric analysis of these volcanoes has been conducted: they are up to about 115-160 m high and 500-1500 m wide. Most of them show very strongly inclined flanks with 30° of average slope. The SCV2 and SCV3 form the Graham Bank, 3.5X2.8 km wide, elongated in the NW-SE direction. At the top of SCV2 focused seepage plumes were observed in the entire water column, through the CHIRP data, where we calculated that they release, a volume of about 10950 m3 and 43960 m3of gases, respectively. In this work, we present the first results of a data collection that have got as main result the identification and mapping of the fluid escape structures revealing the relationship between the active tectonic with migration of fluids, to be used to assess the Submarine Geo-Hazard in the Sicily Channel. We identified two fluid escape fields whose genesis and evolution appear linked to the neotectonic and volcanic activities respectively, that represent the main controlling factors for the migration of fluid; considering the good correlation between pockmarks and the main identified fault systems. In conclusion, our results suggest that the degassing of fluids in this region is rooted at depth, and is mainly aligned with the NW-SE dip/strike slip fault systems, repeatedly reactivated, and linked to the volcanic activity.

  13. Active Trans-Plasma Membrane Water Cycling in Yeast Is Revealed by NMR

    PubMed Central

    Zhang, Yajie; Poirier-Quinot, Marie; Springer, Charles S.; Balschi, James A.

    2011-01-01

    Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal 1H2O NMR relaxation time constant (T1) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τi), where τi−1 is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O2/5%CO2 (O2) or 95%N2/5%CO2 (N2). ATP was high during O2, and τi−1 was 3.1 s−1 at 25°C. After changing to N2, ATP decreased and τi−1 was 1.8 s−1. The principal active yeast ion transport protein is the plasma membrane H+-ATPase. Studies using the H+-ATPase inhibitor ebselen or a yeast genetic strain with reduced H+-ATPase found reduced τi−1, notwithstanding high ATP. Steady-state water exchange correlates with H+-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity. PMID:22261073

  14. A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A

    PubMed Central

    Mallorquí-Fernández, Noemí; Manandhar, Surya P.; Mallorquí-Fernández, Goretti; Usón, Isabel; Wawrzonek, Katarzyna; Kantyka, Tomasz; Solà, Maria; Thøgersen, Ida B.; Enghild, Jan J.; Potempa, Jan; Gomis-Rüth, F.Xavier

    2009-01-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defences and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and its self-processed mature form. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin and a latency flap in the zymogen. Dramatic rearrangement of up to 20Å of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain. PMID:17993455

  15. Spores of many common airborne fungi reveal no ice nucleation activity in oil immersion freezing experiments

    NASA Astrophysics Data System (ADS)

    Pummer, B. G.; Atanasova, L.; Bauer, H.; Bernardi, J.; Druzhinina, I. S.; Fröhlich-Nowoisky, J.; Grothe, H.

    2013-12-01

    Fungal spores are ubiquitous biological aerosols, which are considered to act as ice nuclei. In this study the ice nucleation (IN) activity of spores harvested from 29 fungal strains belonging to 21 different species was tested in the immersion freezing mode by microscopic observation of water-in-oil emulsions. Spores of 8 of these strains were also investigated in a microdroplet freezing array instrument. The focus was laid on species of economical, ecological or sanitary significance. Besides common molds (Ascomycota), some representatives of the widespread group of mushrooms (Basidiomycota) were also investigated. Fusarium avenaceum was the only sample showing IN activity at relatively high temperatures (about 264 K), while the other investigated fungal spores showed no freezing above 248 K. Many of the samples indeed froze at homogeneous ice nucleation temperatures (about 237 K). In combination with other studies, this suggests that only a limited number of species may act as atmospheric ice nuclei. This would be analogous to what is already known for the bacterial ice nuclei. Apart from that, we selected a set of fungal strains from different sites and exposed them to occasional freezing stress during their cultivation. This was in order to test if the exposure to a cold environment encourages the expression of ice nuclei during growth as a way of adaptation. Although the total protein expression was altered by this treatment, it had no significant impact on the IN activity.

  16. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  17. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  18. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  19. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    PubMed Central

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-01-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design. PMID:27708429

  20. A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A.

    PubMed

    Mallorquí-Fernández, Noemí; Manandhar, Surya P; Mallorquí-Fernández, Goretti; Usón, Isabel; Wawrzonek, Katarzyna; Kantyka, Tomasz; Solà, Maria; Thøgersen, Ida B; Enghild, Jan J; Potempa, Jan; Gomis-Rüth, F Xavier

    2008-02-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.

  1. Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation

    PubMed Central

    Kimata, Naoki; Pope, Andreyah; Eilers, Markus; Opefi, Chikwado A.; Ziliox, Martine; Hirshfeld, Amiram; Zaitseva, Ekaterina; Vogel, Reiner; Sheves, Mordechai; Reeves, Philip J.; Smith, Steven O.

    2016-01-01

    The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its β-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiff's base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation. PMID:27585742

  2. Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance

    PubMed Central

    Brooun, Alexei; Gajiwala, Ketan S.; Deng, Ya-Li; Liu, Wei; Bolaños, Ben; Bingham, Patrick; He, You-Ai; Diehl, Wade; Grable, Nicole; Kung, Pei-Pei; Sutton, Scott; Maegley, Karen A.; Yu, Xiu; Stewart, Al E.

    2016-01-01

    Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform. PMID:27122193

  3. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin.

    PubMed

    Lu, Bangmin; Zhang, Bin; Qi, Wei; Zhu, Yanan; Zhao, Yan; Zhou, Nan; Sun, Rong; Bao, Jinku; Wu, Chuanfang

    2014-11-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding.

  4. Active faults in the deformation zone off Noto Peninsula, Japan, revealed by high- resolution seismic profiles

    NASA Astrophysics Data System (ADS)

    Inoue, T.; Okamura, Y.; Murakami, F.; Kimura, H.; Ikehara, K.

    2008-12-01

    Recently, a lot of earthquakes occur in Japan. The deformation zone which many faults and folds have concentrated exists on the Japan Sea side of Japan. The 2007 Noto Hanto Earthquake (MJMA 6.9) and 2007 Chuetsu-oki Earthquake (MJMA 6.8) were caused by activity of parts of faults in this deformation zone. The Noto Hanto Earthquake occurred on 25 March, 2007 under the northwestern coast of Noto Peninsula, Ishikawa Prefecture, Japan. This earthquake is located in Quaternary deformation zone that is continued from northern margin of Noto Peninsula to southeast direction (Okamura, 2007a). National Institute of Advanced Industrial Science and Technology (AIST) carried out high-resolution seismic survey using Boomer and 12 channels short streamer cable in the northern part off Noto Peninsula, in order to clarify distribution and activities of active faults in the deformation zone. A twelve channels short streamer cable with 2.5 meter channel spacing developed by AIST and private corporation is designed to get high resolution seismic profiles in shallow sea area. The multi-channel system is possible to equip on a small fishing boat, because the data acquisition system is based on PC and the length of the cable is short and easy to handle. Moreover, because the channel spacing is short, this cable is very effective for a high- resolution seismic profiling survey in the shallow sea, and seismic data obtained by multi-channel cable can be improved by velocity analysis and CDP stack. In the northern part off Noto Peninsula, seismic profiles depicting geologic structure up to 100 meters deep under sea floor were obtained. The most remarkable reflection surface recognized in the seismic profiles is erosion surface at the Last Glacial Maximum (LGM). In the western part, sediments about 30 meters (40 msec) thick cover the erosional surface that is distributed under the shelf shallower than 100m in depth and the sediments thin toward offshore and east. Flexures like deformation in

  5. 76 FR 67142 - Certain Activated Carbon From the People's Republic of China: Final Results and Partial...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-31

    ...-activation chemical treatment (chemical or water washing, chemical impregnation or other treatment), or... dehydrates molecules in the raw material, and results in the formation of water that is removed from the raw... analysis memoranda. \\13\\ CCT submitted Active Carbon India Private Limited's (``Active Carbon'')...

  6. Stability of active mantle upwelling revealed by net characteristics of plate tectonics.

    PubMed

    Conrad, Clinton P; Steinberger, Bernhard; Torsvik, Trond H

    2013-06-27

    Viscous convection within the mantle is linked to tectonic plate motions and deforms Earth's surface across wide areas. Such close links between surface geology and deep mantle dynamics presumably operated throughout Earth's history, but are difficult to investigate for past times because the history of mantle flow is poorly known. Here we show that the time dependence of global-scale mantle flow can be deduced from the net behaviour of surface plate motions. In particular, we tracked the geographic locations of net convergence and divergence for harmonic degrees 1 and 2 by computing the dipole and quadrupole moments of plate motions from tectonic reconstructions extended back to the early Mesozoic era. For present-day plate motions, we find dipole convergence in eastern Asia and quadrupole divergence in both central Africa and the central Pacific. These orientations are nearly identical to the dipole and quadrupole orientations of underlying mantle flow, which indicates that these 'net characteristics' of plate motions reveal deeper flow patterns. The positions of quadrupole divergence have not moved significantly during the past 250 million years, which suggests long-term stability of mantle upwelling beneath Africa and the Pacific Ocean. These upwelling locations are positioned above two compositionally and seismologically distinct regions of the lowermost mantle, which may organize global mantle flow as they remain stationary over geologic time.

  7. Effective contractile response to voltage-gated Na+ channels revealed by a channel activator.

    PubMed

    Ho, W-S Vanessa; Davis, Alison J; Chadha, Preet S; Greenwood, Iain A

    2013-04-15

    This study investigated the molecular identity and impact of enhancing voltage-gated Na(+) (Na(V)) channels in the control of vascular tone. In rat isolated mesenteric and femoral arteries mounted for isometric tension recording, the vascular actions of the Na(V) channel activator veratridine were examined. Na(V) channel expression was probed by molecular techniques and immunocytochemistry. In mesenteric arteries, veratridine induced potent contractions (pEC(50) = 5.19 ± 0.20, E(max) = 12.0 ± 2.7 mN), which were inhibited by 1 μM TTX (a blocker of all Na(V) channel isoforms, except Na(V)1.5, Na(V)1.8, and Na(V)1.9), but not by selective blockers of Na(V)1.7 (ProTx-II, 10 nM) or Na(V)1.8 (A-80347, 1 μM) channels. The responses were insensitive to endothelium removal but were partly (~60%) reduced by chemical destruction of sympathetic nerves by 6-hydroxydopamine (2 mM) or antagonism at the α1-adrenoceptor by prazosin (1 μM). KB-R7943, a blocker of the reverse mode of the Na(+)/Ca(2+) exchanger (3 μM), inhibited veratridine contractions in the absence or presence of prazosin. T16A(inh)-A01, a Ca(2+)-activated Cl(-) channel blocker (10 μM), also inhibited the prazosin-resistant contraction to veratridine. Na(V) channel immunoreactivity was detected in freshly isolated mesenteric myocytes, with apparent colocalization with the Na(+)/Ca(2+) exchanger. Veratridine induced similar contractile effects in the femoral artery, and mRNA transcripts for Na(V)1.2 and Na(V)1.3 channels were evident in both vessel types. We conclude that, in addition to sympathetic nerves, NaV channels are expressed in vascular myocytes, where they are functionally coupled to the reverse mode of Na(+)/Ca(2+) exchanger and subsequent activation of Ca(2+)-activated Cl(-) channels, causing contraction. The TTX-sensitive Na(V)1.2 and Na(V)1.3 channels are likely involved in vascular control.

  8. The asymmetry of (-)α-pinene as revealed from its raman optical activity spectrum.

    PubMed

    Wang, Peijie; Fang, Yan; Wu, Guozhen

    2013-10-01

    An algorithm is employed to retrieve the differential bond polarizabilities of the C-C bonds from the Raman optical activity (ROA) spectrum of (-)α-pinene. (-)α-pinene possesses two asymmetric centers (carbon atoms) and a local mirror symmetry. These differential bond polarizabilities show the characteristics of the asymmetry around the asymmetric carbons with respect to the mirror reflection. This analysis is accompanied along with the ROA mode signatures and the calculated β(G ')(2) and β(A)(2) which show the ROA coupling mechanisms involving the electric/magnetic dipoles and the electric dipole/quadrupole, respectively.

  9. Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release.

    PubMed

    Held, Katharina; Kichko, Tatjana; De Clercq, Katrien; Klaassen, Hugo; Van Bree, Rieta; Vanherck, Jean-Christophe; Marchand, Arnaud; Reeh, Peter W; Chaltin, Patrick; Voets, Thomas; Vriens, Joris

    2015-03-17

    Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation.

  10. Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping.

    PubMed

    Roh, Tae-Young; Cuddapah, Suresh; Zhao, Keji

    2005-03-01

    The identity and developmental potential of a human cell is specified by its epigenome that is largely defined by patterns of chromatin modifications including histone acetylation. Here we report high-resolution genome-wide mapping of diacetylation of histone H3 at Lys 9 and Lys 14 in resting and activated human T cells by genome-wide mapping technique (GMAT). Our data show that high levels of the H3 acetylation are detected in gene-rich regions. The chromatin accessibility and gene expression of a genetic domain is correlated with hyperacetylation of promoters and other regulatory elements but not with generally elevated acetylation of the entire domain. Islands of acetylation are identified in the intergenic and transcribed regions. The locations of the 46,813 acetylation islands identified in this study are significantly correlated with conserved noncoding sequences (CNSs) and many of them are colocalized with known regulatory elements in T cells. TCR signaling induces 4045 new acetylation loci that may mediate the global chromatin remodeling and gene activation. We propose that the acetylation islands are epigenetic marks that allow prediction of functional regulatory elements.

  11. Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping

    PubMed Central

    Roh, Tae-Young; Cuddapah, Suresh; Zhao, Keji

    2005-01-01

    The identity and developmental potential of a human cell is specified by its epigenome that is largely defined by patterns of chromatin modifications including histone acetylation. Here we report high-resolution genome-wide mapping of diacetylation of histone H3 at Lys 9 and Lys 14 in resting and activated human T cells by genome-wide mapping technique (GMAT). Our data show that high levels of the H3 acetylation are detected in gene-rich regions. The chromatin accessibility and gene expression of a genetic domain is correlated with hyperacetylation of promoters and other regulatory elements but not with generally elevated acetylation of the entire domain. Islands of acetylation are identified in the intergenic and transcribed regions. The locations of the 46,813 acetylation islands identified in this study are significantly correlated with conserved noncoding sequences (CNSs) and many of them are colocalized with known regulatory elements in T cells. TCR signaling induces 4045 new acetylation loci that may mediate the global chromatin remodeling and gene activation. We propose that the acetylation islands are epigenetic marks that allow prediction of functional regulatory elements. PMID:15706033

  12. Phyllosilicate diversity and past aqueous activity revealed at Mawrth Vallis, Mars

    USGS Publications Warehouse

    Bishop, J.L.; Dobrea, E.Z.N.; McKeown, N.K.; Parente, M.; Ehlmann, B.L.; Michalski, J.R.; Milliken, R.E.; Poulet, F.; Swayze, G.A.; Mustard, J.F.; Murchie, S.L.; Bibring, J.-P.

    2008-01-01

    Observations by the Mars Reconnaissance Orbiter/Compact Reconnaissance Imaging Spectrometer for Mars in the Mawrth Vallis region show several phyllosilicate species, indicating a wide range of past aqueous activity. Iron/magnesium (Fe/Mg)-smectite is observed in light-toned outcrops that probably formed via aqueous alteration of basalt of the ancient cratered terrain. This unit is overlain by rocks rich in hydrated silica, montmorillonite, and kaolinite that may have formed via subsequent leaching of Fe and Mg through extended aqueous events or a change in aqueous chemistry. A spectral feature attributed to an Fe2+ phase is present in many locations in the Mawrth Vallis region at the transition from Fe/Mg-smectite to aluminum/silicon (Al/Si)-rich units. Fe2+-bearing materials in terrestrial sediments are typically associated with microorganisms or changes in pH or cations and could be explained here by hydrothermal activity. The stratigraphy of Fe/Mg-smectite overlain by a ferrous phase, hydrated silica, and then Al-phyllosilicates implies a complex aqueous history.

  13. Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces

    PubMed Central

    Orsi, William; Biddle, Jennifer F.; Edgcomb, Virginia

    2013-01-01

    The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface. PMID:23418556

  14. A validated tumorgraft model reveals activity of dovitinib against renal cell carcinoma.

    PubMed

    Sivanand, Sharanya; Peña-Llopis, Samuel; Zhao, Hong; Kucejova, Blanka; Spence, Patrick; Pavia-Jimenez, Andrea; Yamasaki, Toshinari; McBride, David J; Gillen, Jessica; Wolff, Nicholas C; Morlock, Lorraine; Lotan, Yair; Raj, Ganesh V; Sagalowsky, Arthur; Margulis, Vitaly; Cadeddu, Jeffrey A; Ross, Mark T; Bentley, David R; Kabbani, Wareef; Xie, Xian-Jin; Kapur, Payal; Williams, Noelle S; Brugarolas, James

    2012-06-06

    Most anticancer drugs entering clinical trials fail to achieve approval from the U.S. Food and Drug Administration. Drug development is hampered by the lack of preclinical models with therapeutic predictive value. Herein, we report the development and validation of a tumorgraft model of renal cell carcinoma (RCC) and its application to the evaluation of an experimental drug. Tumor samples from 94 patients were implanted in the kidneys of mice without additives or disaggregation. Tumors from 35 of these patients formed tumorgrafts, and 16 stable lines were established. Samples from metastatic sites engrafted at higher frequency than those from primary tumors, and stable engraftment of primary tumors in mice correlated with decreased patient survival. Tumorgrafts retained the histology, gene expression, DNA copy number alterations, and more than 90% of the protein-coding gene mutations of the corresponding tumors. As determined by the induction of hypercalcemia in tumorgraft-bearing mice, tumorgrafts retained the ability to induce paraneoplastic syndromes. In studies simulating drug exposures in patients, RCC tumorgraft growth was inhibited by sunitinib and sirolimus (the active metabolite of temsirolimus in humans), but not by erlotinib, which was used as a control. Dovitinib, a drug in clinical development, showed greater activity than sunitinib and sirolimus. The routine incorporation of models recapitulating the molecular genetics and drug sensitivities of human tumors into preclinical programs has the potential to improve oncology drug development.

  15. Nerve Growth Factor Promoter Activity Revealed in Mice Expressing Enhanced Green Fluorescent Protein

    PubMed Central

    Kawaja, Michael D.; Smithson, Laura J.; Elliott, Janet; Trinh, Gina; Crotty, Anne-Marie; Michalski, Bernadeta; Fahnestock, Margaret

    2012-01-01

    Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury. PMID:21456011

  16. A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities.

    PubMed

    Najdi, Rani; Proffitt, Kyle; Sprowl, Stephanie; Kaur, Simran; Yu, Jia; Covey, Tracy M; Virshup, David M; Waterman, Marian L

    2012-09-01

    Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. At least 14 out of the 19 Wnts activate β-catenin-dependent signaling, an activity that is cell type-dependent and tracks with the stabilization of β-catenin and LRP6 phosphorylation. We find that the core Wnt modification and secretion proteins Porcupine (PORCN) and Wntless (WLS) are essential for all Wnts to signal through β-catenin-dependent and independent pathways. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function.

  17. Phyllosilicate diversity and past aqueous activity revealed at Mawrth Vallis, Mars.

    PubMed

    Bishop, Janice L; Dobrea, Eldar Z Noe; McKeown, Nancy K; Parente, Mario; Ehlmann, Bethany L; Michalski, Joseph R; Milliken, Ralph E; Poulet, Francois; Swayze, Gregg A; Mustard, John F; Murchie, Scott L; Bibring, Jean-Pierre

    2008-08-08

    Observations by the Mars Reconnaissance Orbiter/Compact Reconnaissance Imaging Spectrometer for Mars in the Mawrth Vallis region show several phyllosilicate species, indicating a wide range of past aqueous activity. Iron/magnesium (Fe/Mg)-smectite is observed in light-toned outcrops that probably formed via aqueous alteration of basalt of the ancient cratered terrain. This unit is overlain by rocks rich in hydrated silica, montmorillonite, and kaolinite that may have formed via subsequent leaching of Fe and Mg through extended aqueous events or a change in aqueous chemistry. A spectral feature attributed to an Fe2+ phase is present in many locations in the Mawrth Vallis region at the transition from Fe/Mg-smectite to aluminum/silicon (Al/Si)-rich units. Fe2+-bearing materials in terrestrial sediments are typically associated with microorganisms or changes in pH or cations and could be explained here by hydrothermal activity. The stratigraphy of Fe/Mg-smectite overlain by a ferrous phase, hydrated silica, and then Al-phyllosilicates implies a complex aqueous history.

  18. X-ray structure of human aromatase reveals an androgen-specific active site.

    PubMed

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2010-02-28

    Aromatase is a unique cytochrome P450 that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens for the synthesis of estrogens. All human estrogens are synthesized via this enzymatic aromatization pathway. Aromatase inhibitors thus constitute a frontline therapy for estrogen-dependent breast cancer. Despite decades of intense investigation, this enzyme of the endoplasmic reticulum membrane has eluded all structure determination efforts. We have determined the crystal structure of the highly active aromatase purified from human placenta, in complex with its natural substrate androstenedione. The structure shows the binding mode of androstenedione in the catalytically active oxidized high-spin ferric state of the enzyme. Hydrogen bond-forming interactions and tight packing hydrophobic side chains that complement the puckering of the steroid backbone provide the molecular basis for the exclusive androgenic specificity of aromatase. Locations of catalytic residues and water molecules shed new light on the mechanism of the aromatization step. The structure also suggests a membrane integration model indicative of the passage of steroids through the lipid bilayer.

  19. Deep sequencing of subseafloor eukaryotic rRNA reveals active Fungi across marine subsurface provinces.

    PubMed

    Orsi, William; Biddle, Jennifer F; Edgcomb, Virginia

    2013-01-01

    The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.

  20. Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation

    PubMed Central

    Tang, Matthew Y.; Vranas, Marta; Krahn, Andrea I.; Pundlik, Shayal; Trempe, Jean- François; Fon, Edward A.

    2017-01-01

    Parkin and PINK1 function in a common pathway to clear damaged mitochondria. Parkin exists in an auto-inhibited conformation stabilized by multiple interdomain interactions. The binding of PINK1-generated phospho-ubiquitin and the phosphorylation of the ubiquitin-like (Ubl) domain of Parkin at Ser65 release its auto-inhibition, but how and when these events take place in cells remain to be defined. Here we show that mutations that we designed to activate Parkin by releasing the Repressor Element of Parkin (REP) domain, or by disrupting the interface between the RING0:RING2 domains, can completely rescue mutations in the Parkin Ubl that are defective in mitochondrial autophagy. Using a FRET reporter assay we show that Parkin undergoes a conformational change upon phosphorylation that can be mimicked by mutating Trp403 in the REP. We propose a hierarchical model whereby pUb binding on mitochondria enables Parkin phosphorylation, which, in turn, leads to REP removal, E3 ligase activation and mitophagy. PMID:28276439

  1. Coseismic landslides reveal near-surface rock strength in a high-relief tectonically active setting

    USGS Publications Warehouse

    Gallen, Sean F; Clark, Marin K; Godt, Jonathan W.

    2014-01-01

    We present quantitative estimates of near-surface rock strength relevant to landscape evolution and landslide hazard assessment for 15 geologic map units of the Longmen Shan, China. Strength estimates are derived from a novel method that inverts earthquake peak ground acceleration models and coseismic landslide inventories to obtain material proper- ties and landslide thickness. Aggregate rock strength is determined by prescribing a friction angle of 30° and solving for effective cohesion. Effective cohesion ranges are from 70 kPa to 107 kPa for 15 geologic map units, and are approximately an order of magnitude less than typical laboratory measurements, probably because laboratory tests on hand-sized specimens do not incorporate the effects of heterogeneity and fracturing that likely control near-surface strength at the hillslope scale. We find that strength among the geologic map units studied varies by less than a factor of two. However, increased weakening of units with proximity to the range front, where precipitation and active fault density are the greatest, suggests that cli- matic and tectonic factors overwhelm lithologic differences in rock strength in this high-relief tectonically active setting.

  2. Diversity of Orientia tsutsugamushi clinical isolates in Cambodia reveals active selection and recombination process.

    PubMed

    Duong, Veasna; Blassdell, Kim; May, Thinh Thi Xuan; Sreyrath, Lay; Gavotte, Laurent; Morand, Serge; Frutos, Roger; Buchy, Philippe

    2013-04-01

    Orientia tsutsugamushi, the causative agent of scrub typhus in South East Asia and Pacific, is an obligate intracellular bacterium closely related to the Rickettsia. The pathogen is transmitted to humans through the bites of infected larvae of trombiculid mites of the genus Leptotrombidium in which is maintained trough vertical transmission mechanism. The infection in rodents has been described in over 20 species. Scrub typhus is commonly confused with other tropical fevers and late diagnosis and treatment can lead to severe organ failures and a strain-dependent mortality rate of up to 50%. A MLST scheme associating seven core function genes: adk, lepB, lipA, lipB, secY, sodB and sucA was developed and validated on seven Cambodian strains detected in patients and two complete reference genomes from Korea and Japan. Sequence data were analyzed both with respect to sequence type (ST) diversity and DNA polymorphism. Differing trends were revealed. DNA polymorphism and phylogeny of individual gene loci indicated a significant level of recombination and genetic diversity. However, the ST distribution is clearly clonal and the clinical situation can be summarized by the formula: one patient, one strain, one ST. This contradiction is only apparent and is most likely the consequence of the unique life cycle of O. tsutsugamushi. The quasi exclusive vertical transmission mode in mites generates repeated bottlenecks and small-size populations and strongly limits genetic diversity. O. tsutsugamushi has developed specific mechanisms for generating genetic diversity which include recombination, duplication and conjugation. Recombination and other mechanisms for increasing genetic diversity are likely to occur in rodents which can act as maintenance hosts, although occurrence in mites cannot be excluded. Consequences for the epidemiology of scrub typhus are discussed.

  3. Regulation of the fishing activities in the lagoon of Venice, Italy: Results from a socio-economic study

    NASA Astrophysics Data System (ADS)

    Nunes, Paulo A. L. D.; Silvestri, Silvia; Pellizzato, Michele; Boatto, Vasco

    2008-10-01

    In the last years, the overall fish industry in the lagoon of Venice has shown a gradual decline. In order to better understand this process, we carry out a socio-economic questionnaire next to the fisherman population. Questionnaire contains significant qualitative and quantitative data that allow us to evaluate the social and the cultural profile of the respondents, including information with respect to the different technological fishing characteristics involved, type and amount of the species harvested as well as the overall productivity of the activity. Furthermore, the questionnaire contains an economic valuation exercise so as to assess in monetary terms the preferences of the fishermen with respect to different alternative policy options that may characterize a future regulation of this economic activity. Estimation results show that fishermen welcome any regulation initiative that is characterized by: (1) banning all fishing activities during the night, (2) allocating fishing concessions areas to each fishermen in a way that minimize the distance between the fishing area and the harbor, and (3) by introducing of a labeling mechanism that certifies the origin of the product. Moreover, the underlying economic valuation mechanism reveals to sensitive to respondent's motivational profile, including the overall trust and confidence that fisherman community places on the current institutional bodies. This result reveals to be of particular significance when attempting the design of an efficient, widely supported regulation of the fishing activity in the lagoon of Venice.

  4. Spatio-Temporal Cellular Dynamics of the Arabidopsis Flagellin Receptor Reveal Activation Status-Dependent Endosomal Sorting[C][W

    PubMed Central

    Beck, Martina; Zhou, Ji; Faulkner, Christine; MacLean, Daniel; Robatzek, Silke

    2012-01-01

    The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor FLAGELLIN SENSING2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)–sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b– and ARA6/Rab F1–positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor. PMID:23085733

  5. Tectonic activity revealed by morphostructural analysis: Development of the Sierra de la Candelaria range, northwestern Argentina

    NASA Astrophysics Data System (ADS)

    Barcelona, H.; Peri, G.; Tobal, J.; Sagripanti, L.; Favetto, A.

    2014-12-01

    The tectonically active broken foreland of NW Argentina is a recent analog of the eastern margin of the Puna plateau during Mio-Pliocene times and likely of other broken forelands worldwide. In order to evaluate active tectonism in the broken foreland of the NW Argentine Andes, we examined the complex geomorphology in the vicinity of the basement-cored Sierra de la Candelaria range at ˜26°S and deciphered multiple episodes of crustal deformation spanning the Pliocene to the Quaternary. Digital elevation models, satellite images and geological data within a GIS environment allowed us to analyze the terrain, drainage networks, river dynamics and structure, as well as to obtain detailed geomorphological mapping, active tectonic indices, longitudinal river profiles and structural sections. Three morphostructural segments were defined based on the structural features, the differential vertical dissection pattern over the basement, the faulted Pliocene to recent deposits, the stepwise propagation of anticlines and the distortion over the fluvial system. By combining the several lines of evidence, we concluded that the Sierra de la Candelaria range was subjected to a multi-stage development. The first stage uplifted the central segment concomitant with the formation of the surrounding ranges and with the main partition phase of the foreland. After a significant time lapse, the mountain range was subjected to southward thick-skinned growth and northward growth via stepwise thin-skinned deformation and exerted control over the dynamics of the Río Rosario. Taking into account the surrounding basins and ranges of the Sierra de la Candelaria, the southern Santa Bárbara System is characterized by partially isolated intramontane basins (Choromoro and Rosario) limited by shielded ranges that caused moisture block and shows continuous deformation. These features were related to early stages of a broken foreland evolution model and modern analogs were found at the northern

  6. In Vivo Activation of Azipropofol Prolongs Anesthesia and Reveals Synaptic Targets*

    PubMed Central

    Weiser, Brian P.; Kelz, Max B.; Eckenhoff, Roderic G.

    2013-01-01

    General anesthetic photolabels have been instrumental in discovering and confirming protein binding partners and binding sites of these promiscuous ligands. We report the in vivo photoactivation of meta-azipropofol, a potent analog of propofol, in Xenopus laevis tadpoles. Covalent adduction of meta-azipropofol in vivo prolongs the primary pharmacologic effect of general anesthetics in a behavioral phenotype we termed “optoanesthesia.” Coupling this behavior with a tritiated probe, we performed unbiased, time-resolved gel proteomics to identify neuronal targets of meta-azipropofol in vivo. We have identified synaptic binding partners, such as synaptosomal-associated protein 25, as well as voltage-dependent anion channels as potential facilitators of the general anesthetic state. Pairing behavioral phenotypes elicited by the activation of efficacious photolabels in vivo with time-resolved proteomics provides a novel approach to investigate molecular mechanisms of general anesthetics. PMID:23184948

  7. Unmasking the ancestral activity of integron integrases reveals a smooth evolutionary transition during functional innovation

    PubMed Central

    Escudero, Jose Antonio; Loot, Celine; Parissi, Vincent; Nivina, Aleksandra; Bouchier, Christiane; Mazel, Didier

    2016-01-01

    Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood. Among them, integron integrases are hybrid systems recombining single- and double-stranded DNA partners. These reactions are asymmetric and need a replicative resolution pathway, an exception to the canonical second strand exchange model of Y-recombinases. Integron integrases possess a specific domain for this specialized pathway. Here we show that despite this, integrases are still capable of efficiently operating the ancestral second strand exchange in symmetrical reactions between double-stranded substrates. During these reactions, both strands are reactive and Holliday junction resolution can follow either pathway. A novel deep-sequencing approach allows mapping of the crossover point for the second strand exchange. The persistence of the ancestral activity in integrases illustrates their robustness and shows that innovation towards new recombination substrates and resolution pathways was a smooth evolutionary process. PMID:26961432

  8. Screening bioactives reveals nanchangmycin as a broad spectrum antiviral active against Zika virus

    PubMed Central

    Rausch, Keiko; Hackett, Brent; Weinbren, Nathan; Reeder, Sophia; Sadovsky, Yoel; Hunter, Christopher; Schultz, David C.; Coyne, Carolyn; Cherry, Sara

    2017-01-01

    Zika virus is an emerging arthropod-borne flavivirus for which there are no vaccines or specific therapeutics. We screened a library of 2000 ‘bioactive’ compounds for their ability to block Zika virus infection in three distinct cell-types with two different strains of Zika virus. Using a microscopy-based assay, we validated 38 drugs that inhibited Zika virus infection, including FDA approved nucleoside analogs. Cells expressing high levels of the attachment factor AXL can be protected from infection with receptor tyrosine kinase inhibitors, while placental-derived cells that lack AXL expression are insensitive to this inhibition. Importantly, we identified nanchangmycin as a potent inhibitor of Zika virus entry across all cell types tested including physiologically relevant primary cells. Nanchanmycin was also active against other medically relevant viruses including West Nile, dengue, and chikungunya virus that use a similar route of entry. This study provides a resource of small molecules to study Zika virus pathogenesis. PMID:28099856

  9. Transcription activator structure reveals redox control of a replication initiation reaction†

    PubMed Central

    Sanders, Cyril M.; Sizov, Dmytro; Seavers, Philippa R.; Ortiz-Lombardía, Miguel; Antson, Alfred A.

    2007-01-01

    Redox changes are one of the factors that influence cell-cycle progression and that control the processes of cellular proliferation, differentiation, senescence and apoptosis. Proteins regulated through redox-sensitive cysteines have been characterized but specific ‘sulphydryl switches’ in replication proteins remain to be identified. In bovine papillomavirus type-1, DNA replication begins when the viral transcription factor E2 recruits the viral initiator protein E1 to the origin of DNA replication (ori). Here we show that a novel dimerization interface in the E2 transcription activation domain is stabilized by a disulphide bond. Oxidative cross-linking via Cys57 sequesters the interaction surface between E1 and E2, preventing pre-initiation and replication initiation complex formation. Our data demonstrate that as well as a mechanism for regulating DNA binding, redox reactions can control replication by modulating the tertiary structure of critical protein factors using a specific redox sensor. PMID:17478495

  10. Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity

    PubMed Central

    Soethoudt, Marjolein; Grether, Uwe; Fingerle, Jürgen; Grim, Travis W.; Fezza, Filomena; de Petrocellis, Luciano; Ullmer, Christoph; Rothenhäusler, Benno; Perret, Camille; van Gils, Noortje; Finlay, David; MacDonald, Christa; Chicca, Andrea; Gens, Marianela Dalghi; Stuart, Jordyn; de Vries, Henk; Mastrangelo, Nicolina; Xia, Lizi; Alachouzos, Georgios; Baggelaar, Marc P.; Martella, Andrea; Mock, Elliot D.; Deng, Hui; Heitman, Laura H.; Connor, Mark; Di Marzo, Vincenzo; Gertsch, Jürg; Lichtman, Aron H.; Maccarrone, Mauro; Pacher, Pal; Glass, Michelle; van der Stelt, Mario

    2017-01-01

    The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research. PMID:28045021

  11. Metaproteogenomics reveals the soil microbial communities active in nutrient cycling processes under different tree species

    NASA Astrophysics Data System (ADS)

    Keiblinger, Katharina Maria; Masse, Jacynthe; Zühlke, Daniela; Riedel, Katharina; Zechmeister-Boltenstern, Sophie; Prescott, Cindy E.; Grayston, Sue

    2016-04-01

    Tree species exert strong effects on microbial communities in litter and soil and may alter rates of soil processes fundamental to nutrient cycling and carbon fluxes (Prescott and Grayston 2013). However, the influence of tree species on decomposition processes are still contradictory and poorly understood. An understanding of the mechanisms underlying plant influences on soil processes is important for our ability to predict ecosystem response to altered global/environmental conditions. In order to link microbial community structure and function to forest-floor nutrient cycling processes, we sampled forest floors under western redcedar (Thuja plicata), Douglas-fir (Pseudotsuga menziesii) and Sitka spruce (Picea sitchensis) grown in nutrient-poor sites in common garden experiments on Vancouver island (Canada). We measured forest-floor total N, total C, initial NH4+ and NO3- concentrations, DOC, Cmic and Nmic. Gross rates of ammonification and NH4+ consumption were measured using the 15N pool-dilution method. Organic carbon quality was assessed through FTIR analyses. Microbial community structure was analysed by a metaproteogenomic approach using 16S and ITS amplification and sequencing with MiSeq platform. Proteins were extracted and peptides characterized via LC-MS/MS on a Velos Orbitrap to assess the active microbial community. Different microbial communities were active under the three tree species and variation in process rates were observed and will be discussed. This research provides new insights on microbial processes during organic matter decomposition. The metaproteogenomic approach enables us to investigate these changes with respect to possible effects on soil C-storage at even finer taxonomic resolution.

  12. RNA-Seq Reveals Activation of Both Common and Cytokine-Specific Pathways following Neutrophil Priming

    PubMed Central

    Moots, Robert J.; Edwards, Steven W.

    2013-01-01

    Neutrophils are central to the pathology of inflammatory diseases, where they can damage host tissue through release of reactive oxygen metabolites and proteases, and drive inflammation via secretion of cytokines and chemokines. Many cytokines, such as those generated during inflammation, can induce a similar “primed” phenotype in neutrophils, but it is unknown if different cytokines utilise common or cytokine-specific pathways to induce these functional changes. Here, we describe the transcriptomic changes induced in control human neutrophils during priming in vitro with pro-inflammatory cytokines (TNF-α and GM-CSF) using RNA-seq. Priming led to the rapid expression of a common set of transcripts for cytokines, chemokines and cell surface receptors (CXCL1, CXCL2, IL1A, IL1B, IL1RA, ICAM1). However, 580 genes were differentially regulated by TNF-α and GM-CSF treatment, and of these 58 were directly implicated in the control of apoptosis. While these two cytokines both delayed apoptosis, they induced changes in expression of different pro- and anti-apoptotic genes. Bioinformatics analysis predicted that these genes were regulated via differential activation of transcription factors by TNF-α and GM-CSF and these predictions were confirmed using functional assays: inhibition of NF-κB signalling abrogated the protective effect of TNF-α (but not that of GM-CSF) on neutrophil apoptosis, whereas inhibition of JAK/STAT signalling abrogated the anti-apoptotic effect of GM-CSF, but not that of TNF-α (p<0.05). These data provide the first characterisation of the human neutrophil transcriptome following GM-CSF and TNF-α priming, and demonstrate the utility of this approach to define functional changes in neutrophils following cytokine exposure. This may provide an important, new approach to define the molecular properties of neutrophils after in vivo activation during inflammation. PMID:23554905

  13. The crystal structure of phosphorylated MAPK13 reveals common structural features and differences in p38 MAPK family activation

    PubMed Central

    Yurtsever, Zeynep; Scheaffer, Suzanne M.; Romero, Arthur G.; Holtzman, Michael J.; Brett, Tom J.

    2015-01-01

    The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors. PMID:25849390

  14. The crystal structure of phosphorylated MAPK13 reveals common structural features and differences in p38 MAPK family activation.

    PubMed

    Yurtsever, Zeynep; Scheaffer, Suzanne M; Romero, Arthur G; Holtzman, Michael J; Brett, Tom J

    2015-04-01

    The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors.

  15. Rap1 overexpression reveals that activated RasD induces separable defects during Dictyostelium development.

    PubMed

    Louis, S A; Weeks, G; Spiegelman, G B

    1997-10-15

    One of the Dictyostelium ras genes, rasD, is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymond et al., 1986, Nature, 323, 340-343). The ability of the Dictyostelium rap1 gene to suppress this abnormal developmental phenotype was investigated. The rap1 gene and G12V activated and G10V negative mutant forms of the rap1 gene were independently linked to the rasD promoter and each construct used to transform M1, a Dictyostelium cell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). The rap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louis et al., 1997, Mol. Biol. Cell, 8, 303-312). During the development of ME cells, ecmA mRNA levels were restored to those seen for Ax3, and tagB mRNA levels were also markedly reduced, although not to Ax3 levels. cotC expression in ME cells was enhanced severalfold relative to M1

  16. Representing Microbial Dormancy in Soil Decomposition Models Improves Model Performance and Reveals Key Ecosystem Controls on Microbial Activity

    NASA Astrophysics Data System (ADS)

    He, Y.; Yang, J.; Zhuang, Q.; Wang, G.; Liu, Y.

    2014-12-01

    Climate feedbacks from soils can result from environmental change and subsequent responses of plant and microbial communities and nutrient cycling. Explicit consideration of microbial life history traits and strategy may be necessary to predict climate feedbacks due to microbial physiology and community changes and their associated effect on carbon cycling. In this study, we developed an explicit microbial-enzyme decomposition model and examined model performance with and without representation of dormancy at six temperate forest sites with observed soil efflux ranged from 4 to 10 years across different forest types. We then extrapolated the model to all temperate forests in the Northern Hemisphere (25-50°N) to investigate spatial controls on microbial and soil C dynamics. Both models captured the observed soil heterotrophic respiration (RH), yet no-dormancy model consistently exhibited large seasonal amplitude and overestimation in microbial biomass. Spatially, the total RH from temperate forests based on dormancy model amounts to 6.88PgC/yr, and 7.99PgC/yr based on no-dormancy model. However, no-dormancy model notably overestimated the ratio of microbial biomass to SOC. Spatial correlation analysis revealed key controls of soil C:N ratio on the active proportion of microbial biomass, whereas local dormancy is primarily controlled by soil moisture and temperature, indicating scale-dependent environmental and biotic controls on microbial and SOC dynamics. These developments should provide essential support to modeling future soil carbon dynamics and enhance the avenue for collaboration between empirical soil experiment and modeling in the sense that more microbial physiological measurements are needed to better constrain and evaluate the models.

  17. Revealing Rembrandt

    PubMed Central

    Parker, Andrew J.

    2014-01-01

    The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI). Our results emphasized the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt's portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings. PMID:24795552

  18. Prefrontal activation during two Japanese Stroop tasks revealed with multi-channel near-infrared spectroscopy.

    PubMed

    Watanabe, Yukina; Sumitani, Satsuki; Hosokawa, Mai; Ohmori, Tetsuro

    2015-01-01

    The Stroop task is sometimes used in psychiatric research to elicit prefrontal activity, which presumably reflects cognitive functioning. Although there are two Stroop tasks (Kana script and Kanji script) in Japan, it is unclear whether these tasks elicit the same hemoglobin changes. Moreover, it is unclear whether psychological conditions or characteristics influence hemoglobin changes in the Japanese Stroop task. The aim of this study was to clarify whether hemoglobin changes elicited by the two Japanese Stroop tasks accurately reflected cognitive functioning. Hemoglobin changes were measured with multi-channel near-infrared spectroscopy (NIRS) in 100 healthy Japanese participants performing two Japanese Stroop tasks. The Beck-Depression Inventory (BDI), State-Trait-Anxiety Inventory (STAI), and Maudsley Obsessive Compulsive Inventory (MOCI) were administered to participants to identify psychological conditions or personality characteristics. Compared with the Kanji task, the Kana task produced a greater Stroop effect and a larger increase in oxyhemoglobin (oxy-Hb) concentration. Moreover there were no significant correlations between oxy-Hb concentration and BDI, STAI-trait, STAI-state, or MOCI scores. Therefore we found that a participant's psychological conditions or characteristics did not influence the hemodynamic changes during either task. These data suggest the Kana Stroop task is more useful than the Kanji Stroop task for NIRS studies in psychiatric research.

  19. Amplitude-modulated stimuli reveal auditory-visual interactions in brain activity and brain connectivity

    PubMed Central

    Laing, Mark; Rees, Adrian; Vuong, Quoc C.

    2015-01-01

    The temporal congruence between auditory and visual signals coming from the same source can be a powerful means by which the brain integrates information from different senses. To investigate how the brain uses temporal information to integrate auditory and visual information from continuous yet unfamiliar stimuli, we used amplitude-modulated tones and size-modulated shapes with which we could manipulate the temporal congruence between the sensory signals. These signals were independently modulated at a slow or a fast rate. Participants were presented with auditory-only, visual-only, or auditory-visual (AV) trials in the fMRI scanner. On AV trials, the auditory and visual signal could have the same (AV congruent) or different modulation rates (AV incongruent). Using psychophysiological interaction analyses, we found that auditory regions showed increased functional connectivity predominantly with frontal regions for AV incongruent relative to AV congruent stimuli. We further found that superior temporal regions, shown previously to integrate auditory and visual signals, showed increased connectivity with frontal and parietal regions for the same contrast. Our findings provide evidence that both activity in a network of brain regions and their connectivity are important for AV integration, and help to bridge the gap between transient and familiar AV stimuli used in previous studies. PMID:26483710

  20. Revealing the spin–vibronic coupling mechanism of thermally activated delayed fluorescence

    PubMed Central

    Etherington, Marc K.; Gibson, Jamie; Higginbotham, Heather F.; Penfold, Thomas J.; Monkman, Andrew P.

    2016-01-01

    Knowing the underlying photophysics of thermally activated delayed fluorescence (TADF) allows proper design of high efficiency organic light-emitting diodes. We have proposed a model to describe reverse intersystem crossing (rISC) in donor–acceptor charge transfer molecules, where spin–orbit coupling between singlet and triplet states is mediated by one of the local triplet states of the donor (or acceptor). This second order, vibronically coupled mechanism describes the basic photophysics of TADF. Through a series of measurements, whereby the energy ordering of the charge transfer (CT) excited states and the local triplet are tuned in and out of resonance, we show that TADF reaches a maximum at the resonance point, substantiating our model of rISC. Moreover, using photoinduced absorption, we show how the populations of both singlet and triplet CT states and the local triplet state change in and out of resonance. Our vibronic coupling rISC model is used to predict this behaviour and describes how rISC and TADF are affected by external perturbation. PMID:27901046

  1. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

    PubMed Central

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  2. Revealing the spin-vibronic coupling mechanism of thermally activated delayed fluorescence

    NASA Astrophysics Data System (ADS)

    Etherington, Marc K.; Gibson, Jamie; Higginbotham, Heather F.; Penfold, Thomas J.; Monkman, Andrew P.

    2016-11-01

    Knowing the underlying photophysics of thermally activated delayed fluorescence (TADF) allows proper design of high efficiency organic light-emitting diodes. We have proposed a model to describe reverse intersystem crossing (rISC) in donor-acceptor charge transfer molecules, where spin-orbit coupling between singlet and triplet states is mediated by one of the local triplet states of the donor (or acceptor). This second order, vibronically coupled mechanism describes the basic photophysics of TADF. Through a series of measurements, whereby the energy ordering of the charge transfer (CT) excited states and the local triplet are tuned in and out of resonance, we show that TADF reaches a maximum at the resonance point, substantiating our model of rISC. Moreover, using photoinduced absorption, we show how the populations of both singlet and triplet CT states and the local triplet state change in and out of resonance. Our vibronic coupling rISC model is used to predict this behaviour and describes how rISC and TADF are affected by external perturbation.

  3. Comparative Analysis of Carbohydrate Active Enzymes in Clostridium termitidis CT1112 Reveals Complex Carbohydrate Degradation Ability

    PubMed Central

    Munir, Riffat I.; Schellenberg, John; Henrissat, Bernard; Verbeke, Tobin J.; Sparling, Richard; Levin, David B.

    2014-01-01

    Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly. PMID:25101643

  4. Improved detection reveals active β-papillomavirus infection in skin lesions from kidney transplant recipients.

    PubMed

    Borgogna, Cinzia; Lanfredini, Simone; Peretti, Alberto; De Andrea, Marco; Zavattaro, Elisa; Colombo, Enrico; Quaglia, Marco; Boldorini, Renzo; Miglio, Umberto; Doorbar, John; Bavinck, Jan N Bouwes; Quint, Koen D; de Koning, Maurits N C; Landolfo, Santo; Gariglio, Marisa

    2014-08-01

    The aim of this study was to determine whether detection of β-HPV gene products, as defined in epidermodysplasia verruciformis skin cancer, could also be observed in lesions from kidney transplant recipients alongside the viral DNA. A total of 111 samples, corresponding to 79 skin lesions abscised from 17 kidney transplant recipients, have been analyzed. The initial PCR analysis demonstrated that β-HPV-DNA was highly present in our tumor series (85%). Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize productive infection in 4 out of 19 actinic keratoses, and in the pathological borders of 1 out of 14 squamous cell carcinomas and 1 out of 31 basal cell carcinomas. Increased expression of the cellular proliferation marker minichromosome maintenance protein 7 (MCM7), that extended into the upper epithelial layers, was a common feature of all the E4-positive areas, indicating that cells were driven into the cell cycle in areas of productive viral infections. Although the present study does not directly demonstrate a causal role of these viruses, the detection of E4 and L1 positivity in actinic keratosis and the adjacent pathological epithelium of skin cancer, clearly shows that β-HPV are actively replicating in the intraepidermal precursor lesions of kidney transplant recipients and can therefore cooperate with other carcinogenic agents, such as UVB, favoring skin cancer promotion.

  5. Revealing Physical Activity of GRB Central Engine with Macronova/Kilonova Data

    NASA Astrophysics Data System (ADS)

    Shen, Zhao-Qiang; Jin, Zhi-Ping; Liang, Yun-Feng; Li, Xiang; Fan, Yi-Zhong; Wei, Da-Ming

    2017-02-01

    The modeling of Li-Paczyński macronova/kilonova signals gives a reasonable estimate on the neutron-rich material ejected during the neutron star mergers. Usually the accretion disk is more massive than the macronova ejecta, with which the efficiencies of converting the disk mass into prompt emission of three merger-driven GRBs can hence be directly constrained. Supposing the macronovae/kilonovae associated with GRB 050709, GRB 060614, and GRB 130603B arose from radioactive decay of the r-process material, the upper limit on energy conversion efficiencies are found to be as low as ∼10‑6–10‑4. Moreover, for all three events, neutrino annihilation is likely powerful enough to account for the brief gamma-ray flashes. Neutrino annihilation can also explain the “extended” emission lasting ∼100 s in GRB 050709, but does not work for the one in GRB 060614. These progresses demonstrate that the macronova can serve as a novel probe of the central engine activity.

  6. Synchrotron X-ray imaging reveals a correlation of tumor copper speciation with Clioquinol's anticancer activity

    SciTech Connect

    Barrea, Raul A.; Chen, Di; Irving, Thomas C.; Dou, Q. Ping

    2009-10-21

    Tumor development and metastasis depend on angiogenesis that requires certain growth factors, proteases, and the trace element copper (Cu). Recent studies suggest that Cu could be used as a novel target for cancer therapies. Clioquinol (CQ), an antibiotic that is able to form stable complexes with Cu or zinc (Zn), has shown proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human cancer cells and xenografts. The mechanisms underlying the interaction of CQ with cellular Cu, the alteration of the Cu/Zn ratio and the antitumor role of CQ in vivo have not been fully elucidated. We report here that Cu accumulates in tumor tissue and that the Cu/Zn balances in tumor, but not normal, tissue change significantly after the treatment with CQ. Cu speciation analysis showed that the Cu(I) species is predominant in both normal and tumor tissues and that Cu(II) content was significantly increased in tumor, but not normal tissue after CQ treatment. Our findings indicate that CQ can interact with cellular Cu in vivo, dysregulates the Cu/Zn balance and is able to convert Cu(I) to Cu(II) in tumor tissue. This conversion of Cu(I) to Cu(II) may be associated with CQ-induced proteasome inhibition and growth suppression in the human prostate tumor xenografts.

  7. Interspecific metabolic diversity of root-colonizing endophytic fungi revealed by enzyme activity tests.

    PubMed

    Knapp, Dániel G; Kovács, Gábor M

    2016-12-01

    Although dark septate endophytes (DSE) represent a worldwide dispersed form group of root-colonizing endophytic fungi, our knowledge on their role in ecosystem functioning is far limited. In this study, we aimed to test if functional diversity exists among DSE fungi representing different lineages of root endophytic fungal community of semiarid sandy grasslands. To address this question and to gain general information on function of DSE fungi, we adopted api-ZYM and BioLog FF assays to study those non-sporulating filamentous fungi and characterized the metabolic activity of 15 different DSE species. Although there were striking differences among the species, all of the substrates tested were utilized by the DSE fungi. When endophytes characteristic to grasses and non-grass host plants were separately considered, we found that the whole substrate repertoire was used by both groups. This might illustrate the complementary functional diversity of the communities root endophytic plant-associated fungi. The broad spectra of substrates utilized by these root endophytes illustrate the functional importance of their diversity, which can play role not only in nutrient mobilization and uptake of plants from with nutrient poor soils, but also in general plant performance and ecosystem functioning.

  8. Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process

    PubMed Central

    Valle, Mikel; Sengupta, Jayati; Swami, Neil K.; Grassucci, Robert A.; Burkhardt, Nils; Nierhaus, Knud H.; Agrawal, Rajendra K.; Frank, Joachim

    2002-01-01

    During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon–anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition. PMID:12093756

  9. Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

    PubMed Central

    Barkauskaite, Eva; Brassington, Amy; Tan, Edwin S.; Warwicker, Jim; Dunstan, Mark S.; Banos, Benito; Lafite, Pierre; Ahel, Marijan; Mitchison, Timothy J.; Ahel, Ivan; Leys, David

    2013-01-01

    Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios. PMID:23917065

  10. Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

    PubMed Central

    Lü, Fan; Bize, Ariane; Guillot, Alain; Monnet, Véronique; Madigou, Céline; Chapleur, Olivier; Mazéas, Laurent; He, Pinjing; Bouchez, Théodore

    2014-01-01

    Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially. PMID:23949661

  11. IFNAR signaling directly modulates T lymphocyte activity, resulting in milder experimental autoimmune encephalomyelitis development

    PubMed Central

    Kavrochorianou, Nadia; Evangelidou, Maria; Markogiannaki, Melina; Tovey, Michael; Thyphronitis, George; Haralambous, Sylva

    2016-01-01

    Although interferon-β is used as first-line therapy for multiple sclerosis, the cell type-specific activity of type I interferons in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis, remains obscure. In this study, we have elucidated the in vivo immunomodulatory role of type I interferon signaling in T cells during experimental autoimmune encephalomyelitis by use of a novel transgenic mouse, carrying a cd2–ifnar1 transgene on a interferon-α/β receptor 1 null genetic background, thus allowing expression of the interferon-α/β receptor 1 and hence, a functional type I interferon receptor exclusively on T cells. These transgenic mice exhibited milder experimental autoimmune encephalomyelitis with reduced T cell infiltration, demyelination, and axonal damage in the central nervous system. It is noteworthy that interferon-β administration in transgenic mice generated a more pronounced, protective effect against experimental autoimmune encephalomyelitis compared with untreated littermates. In vivo studies demonstrated that before experimental autoimmune encephalomyelitis onset, endogenous type I interferon receptor signaling in T cells led to impaired T-helper 17 responses, with a reduced fraction of CCR6+ CD4+ T cells in the periphery. At the acute phase, an increased proportion of interleukin-10- and interferon-γ-producing CD4+ T cells was detected in the periphery of the transgenic mice, accompanied by up-regulation of the interferon-γ-induced gene Irgm1 in peripheral T cells. Together, these results reveal a hitherto unknown T cell-associated protective role of type I interferon in experimental autoimmune encephalomyelitis that may provide valuable clues for designing novel therapeutic strategies for multiple sclerosis. PMID:26232452

  12. Results of experiments on iodine dissociation in active medium of oxygen-iodine laser

    NASA Astrophysics Data System (ADS)

    Zagidullin, Marsel V.; Khvatov, Nickolay A.; Malyshev, Mikhail S.

    2017-01-01

    Results of experiments on dissociation of iodine molecules in the presence of singlet oxygen molecules are presented for wide range of oxygen-iodine media composition. Rate constants values have been obtained: 4.3ṡ10-17cm3/s for the reaction O2(1Δ)+O2(1Δ)->O2(1Σ) +O2(3Σ) - (1), 2.8ṡ10-13 cm3/s for the reactionO2(1Δ)+I(2P1/2)->O2(1Σ)+I(2P3/2) - (4) and 8.3ṡ10-11 cm3/s for the reaction O2(1Σ) +I2->O2(3Σ)+2I - (2). Analysis of experiments shows that for the wide range of oxygen-iodine medium composition the dissociation occurs via the chain of reactions (1), (2), O2(1Δ)+I(2P3/2)->O2(3Σ)+I(2P1/2), (4) and via cascade process I2+I(2P1/2)->I2(v)+I(2P3/2), I2(v)+O2(1Δ)→2I+O2(3Σ). Contributions of each mechanism in the dissociation of the iodine are comparable for the typical composition of the active medium of the supersonic chemical oxygen-iodine laser. The experiments did not reveal the contribution of vibrationally excited oxygen molecules in the dissociation of iodine. Thus, the experiments and the following conclusions are fully confirmed iodine dissociation mechanism previously proposed by Heidner et al. (J. Phys. Chem., 87, 2348 (1983)).

  13. Characterization of AhR agonists reveals antagonistic activity in European herring gull (Larus argentatus) eggs.

    PubMed

    Muusse, Martine; Christensen, Guttorm; Gomes, Tânia; Kočan, Anton; Langford, Katherine; Tollefsen, Knut Erik; Vaňková, Lenka; Thomas, Kevin V

    2015-05-01

    European herring gull (Larus argentatus) eggs from two Norwegian islands, Musvær in the south east and Reiaren in Northern Norway, were screened for dioxins, furans, and dioxin-like and selected non-dioxin-like polychlorinated biphenyls (PCBs), and subjected to non-target analysis to try to identify the aryl hydrocarbon receptor (AhR) agonists, responsible for elevated levels measured using the dioxin responsive chemically activated luciferase expression (DR-CALUX) assay. Eggs from Musvær contained chemically calculated toxic equivalent (WHO TEQ) levels of between 109 and 483 pg TEQ/g lw, and between 82 and 337 pg TEQ/g lw was determined in eggs from Reiaren. In particular PCB126 contributed highly to the total TEQ (69-82%). In 19 of the 23 samples the calculated WHO TEQ was higher than the TEQCALUX. Using CALUX specific relative effect potencies (REPs), the levels were lower at between 77 and 292 pg/g lw in eggs from Musvær and between 55 and 223 pg/g lw in eggs from Reiaren, which was higher than the TEQCALUX in 16 of the 23 samples. However, the means of the REP values and the TEQCALUX were not significantly different. This suggests the presence of compounds that can elicit antagonist effects, with a low binding affinity to the AhR. Non-target analysis identified the presence of hexachlorobenzene (HCB) (quantified at 9.6-185 pg/g lw) but neither this compound nor high concentrations of PCB126 and non-dioxin-like PCBs could explain the differences between the calculated TEQ or REP values and the TEQCALUX. Even though, for most AhR agonists, the sensitivity of herring gulls is not known, the reported levels can be considered to represent a risk for biological effects in the developing embryo, compared to LC50 values in chicken embryos. For human consumers of herring gull eggs, these eggs contain TEQ levels up to four times higher than the maximum tolerable weekly intake.

  14. Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.

    PubMed

    Lee, Eunice R; Lamplugh, Lisa; Kluczyk, Beata; Mort, John S; Leblond, Charles Philippe

    2006-09-01

    A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

  15. Early activation of quorum sensing in Pseudomonas aeruginosa reveals the architecture of a complex regulon

    PubMed Central

    Schuster, Martin; Greenberg, E Peter

    2007-01-01

    Background Quorum-sensing regulation of gene expression in Pseudomonas aeruginosa is complex. Two interconnected acyl-homoserine lactone (acyl-HSL) signal-receptor pairs, 3-oxo-dodecanoyl-HSL-LasR and butanoyl-HSL-RhlR, regulate more than 300 genes. The induction of most of the genes is delayed during growth of P. aeruginosa in complex medium, cannot be advanced by addition of exogenous signal, and requires additional regulatory components. Many of these late genes can be induced by addition of signals early by using specific media conditions. While several factors super-regulate the quorum receptors, others may co-regulate target promoters or may affect expression posttranscriptionally. Results To better understand the contributions of super-regulation and co-regulation to quorum-sensing gene expression, and to better understand the general structure of the quorum sensing network, we ectopically expressed the two receptors (in the presence of their cognate signals) and another component that affects quorum sensing, the stationary phase sigma factor RpoS, early in growth. We determined the effect on target gene expression by microarray and real-time PCR analysis. Our results show that many target genes (e.g. lasB and hcnABC) are directly responsive to receptor protein levels. Most genes (e.g. lasA, lecA, and phnAB), however, are not significantly affected, although at least some of these genes are directly regulated by quorum sensing. The majority of promoters advanced by RhlR appeared to be regulated directly, which allowed us to build a RhlR consensus sequence. Conclusion The direct responsiveness of many quorum sensing target genes to receptor protein levels early in growth confirms the role of super-regulation in quorum sensing gene expression. The observation that the induction of most target genes is not affected by signal or receptor protein levels indicates that either target promoters are co-regulated by other transcription factors, or that expression is

  16. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates

  17. Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions.

    PubMed

    Harmer, Jenny E; Hiscox, Martyn J; Dinis, Pedro C; Fox, Stephen J; Iliopoulos, Andreas; Hussey, James E; Sandy, James; Van Beek, Florian T; Essex, Jonathan W; Roach, Peter L

    2014-11-15

    Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized lipoyl synthase (LipA) complex contains 5'-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modelling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.

  18. Active microrheology reveals molecular-level variations in the viscoelastic properties of Chaetopterus mucus

    NASA Astrophysics Data System (ADS)

    Weigand, William; Messmore, Ashley; Anderson, Rae

    The sea annelid, Chaetopterus Variopedatus, secretes a bioluminescent mucus that also exhibits complex viscoelastic properties. The constituents of the mucus are relatively unknown but it does play an important role in the development of the worms' parchment-like housing tubes. In order to determine how and why this mucus can exhibit material properties ranging from fluidity to rigidity we perform microrheology experiments. We determine the microscale viscoelastic properties by using optical tweezers to produce small oscillations in the mucus which allow us to determine both the linear storage and loss moduli (G',G'') along with the viscosity of the fluid. By varying the size of the microspheres (2-10 µm) and oscillation amplitude (.5-10 µm) we are able to determine the dominant intrinsic length scales of the molecular mesh comprising the mucus. By varying the oscillation frequency (1-15Hz) we determine the crossover frequency at which G' surpasses G'', to quantify the longest relaxation time of the mesh network. Initial results show a strong dependence on bead size which indicate that the dominant entanglement lengthscale of the mucus mesh is ~5 um. Microspheres of this size exhibit a wide variety of stress responses in different regions of the mucus demonstrating the substantial microscale heterogeneity of the mucus. We carry out measurements on a population of worms of varying size and age to determine mucus variability between worms.

  19. Low dose cadmium poisoning results in sustained ERK phosphorylation and caspase activation

    SciTech Connect

    Martin, Patrick . E-mail: pmartin@unice.fr; Poggi, Marie Christine . E-mail: poggi@unice.fr; Chambard, Jean Claude . E-mail: chambard@unice.fr; Boulukos, Kim E. . E-mail: boulukos@unice.fr; Pognonec, Philippe . E-mail: pognonec@unice.fr

    2006-11-24

    Cadmium poisoning has been known to result in a wide variety of cellular responses, including oxidative stress and kinase activation. It has been reported that ERK is activated following acute cadmium exposure, and this response is commonly seen as a classical ERK survival mechanism. Here, we analyzed different cell types for their responses to low concentrations of cadmium poisoning. We found that there is an association between cell susceptibility to cadmium toxicity and ERK activation. This activation is atypical, since it consists of a sustained ERK phosphorylation, that lasts up to 6 days post stimulation. This activation is associated with the appearance of cleaved caspases 8 and 3, processed PARP, and irreversible damage. Pharmacological inhibition of ERK phosphorylation results in the ability of cells to resist cadmium poisoning. Our data indicate that low cadmium concentrations result in an unconventional ERK sustained phosphorylation, which in turn leads to death signaling.

  20. Glycoproteomics Reveals Decorin Peptides with Anti-Myostatin Activity in Human Atrial Fibrillation

    PubMed Central

    Barallobre-Barreiro, Javier; Gupta, Shashi K.; Zoccarato, Anna; Kitazume-Taneike, Rika; Fava, Marika; Yin, Xiaoke; Werner, Tessa; Hirt, Marc N; Zampetaki, Anna; Viviano, Alessandro; Chong, Mei; Bern, Marshall; Kourliouros, Antonios; Domenech, Nieves; Willeit, Peter; Shah, Ajay M; Jahangiri, Marjan; Schaefer, Liliana; Fischer, Jens W.; Iozzo, Renato V.; Viner, Rosa; Thum, Thomas; Heineke, Joerg; Kichler, Antoine; Otsu, Kinya; Mayr, Manuel

    2016-01-01

    Background Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). Methods Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. Results ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. Upon enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were consistently identified in the flow through. Out of all ECM proteins identified, decorin was found to be most fragmented. Within its protein core, eighteen different cleavage sites were identified. In contrast, no cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was predominantly detected in ventricles compared to atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had higher levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. Conclusions This proteomics study is the first to analyse the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages can regulate

  1. Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

    PubMed

    Kittas, Aristotelis; Delobelle, Aurélien; Schmitt, Sabrina; Breuhahn, Kai; Guziolowski, Carito; Grabe, Niels

    2016-01-01

    An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg.

  2. Active drumlin field revealed at the margin of Múlajökull, Iceland: a surge-type glacier

    NASA Astrophysics Data System (ADS)

    Schomacker, A.; Johnson, M. D.; Benediktsson, I.; Ingolfsson, O.; Geiger, A. J.; Ferguson, A.

    2010-12-01

    Recent marginal retreat of Múlajökull, a surge-type, outlet glacier of the Hofsjökull ice cap, central Iceland, has revealed a drumlin field consisting of over 50 drumlins. The drumlins are 90-320 m long, 30-105 m wide, 5-10 m in relief, and composed of multiple beds of till deposited by lodgement and bed deformation. The youngest till layer truncates the older units with an erosion surface that parallels the drumlin form. Thus, the drumlins are built up and formed by a combination of subglacial depositional and erosional processes. Field evidence suggests each till bed to be associated with individual, recent surges. We consider the drumlin field to be active in the sense that the drumlins are shaped by the current glacial regime. The Múlajökull field is the only known active drumlin field and is, therefore, a unique analogue to Pleistocene drumlin fields.

  3. Does Pedometer Goal Setting Improve Physical Activity among Native Elders? Results from a Randomized Pilot Study

    ERIC Educational Resources Information Center

    Sawchuk, Craig N.; Russo, Joan E.; Charles, Steve; Goldberg, Jack; Forquera, Ralph; Roy-Byrne, Peter; Buchwald, Dedra

    2011-01-01

    We examined if step-count goal setting resulted in increases in physical activity and walking compared to only monitoring step counts with pedometers among American Indian/Alaska Native elders. Outcomes included step counts, self-reported physical activity and well-being, and performance on the 6-minute walk test. Although no significant…

  4. A Study Of The Radionuclidic Content Of The Environmental Samples Resulting From NIPNE Activities

    SciTech Connect

    Stochioiu, Ana I.; Bercea, Sorin I.; Ivan, Constantin G.; Dumitru, Octavian R.; Pantelica, Ana I.

    2007-04-23

    The results of alpha, beta, gamma global activity measurements and gamma spectrometric measurements are presented. For more detailed characterization of samples, gamma-spectrometric measurement, implying identification of radionuclides and their activities are carried-out on significant ambiental annual samples.

  5. Do Media Use and Physical Activity Compete in Adolescents? Results of the MoMo Study

    PubMed Central

    Spengler, Sarah; Mess, Filip; Woll, Alexander

    2015-01-01

    Purpose The displacement hypothesis predicts that physical activity and media use compete in adolescents; however, findings are inconsistent. A more differentiated approach at determining the co-occurrence of physical activity and media use behaviors within subjects may be warranted. The aim of this study was to determine the co-occurrence of physical activity and media use by identifying clusters of adolescents with specific behavior patterns including physical activity in various settings (school, sports club, leisure time) and different types of media use (watching TV, playing console games, using PC / Internet). Methods Cross-sectional data of 2,083 adolescents (11–17 years) from all over Germany were collected between 2009 and 2012 in the Motorik-Modul Study. Physical activity and media use were self-reported. Cluster analyses (Ward’s method and K-means analysis) were used to identify behavior patterns of boys and girls separately. Results Eight clusters were identified for boys and seven for girls. The clusters demonstrated that a high proportion of boys (33%) as well as girls (42%) show low engagement in both physical activity and media use, irrespective of setting or type of media. Other adolescents are engaged in both behaviors, but either physical activity (35% of boys, 27% of girls) or media use (31% of boys and girls) predominates. These adolescents belong to different clusters, whereat in most clusters either one specific setting of physical activity or a specific combination of different types of media predominates. Conclusion The results of this study support to some extent the hypothesis that media use and physical activity compete: Very high media use occurred with low physical activity behavior, but very high activity levels co-occurred with considerable amounts of time using any media. There was no evidence that type of used media was related to physical activity levels, neither setting of physical activity was related to amount of media use

  6. Reducing contralateral SI activity reveals hindlimb receptive fields in the SI forelimb-stump representation of neonatally amputated rats.

    PubMed

    Pluto, Charles P; Chiaia, Nicolas L; Rhoades, Robert W; Lane, Richard D

    2005-09-01

    In adult rats that sustained forelimb amputation on the day of birth, >30% of multiunit recording sites in the forelimb-stump representation of primary somatosensory cortex (SI) also respond to cutaneous hindlimb stimulation when cortical GABA(A+B) receptors are blocked (GRB). This study examined whether hindlimb receptive fields could also be revealed in forelimb-stump sites by reducing one known source of excitatory input to SI GABAergic neurons, the contralateral SI cortex. Corpus callosum projection neurons connect homotopic SI regions, making excitatory contacts onto pyramidal cells and interneurons. Thus in addition to providing monosynaptic excitation in SI, callosal fibers can produce disynaptic inhibition through excitatory synapses with inhibitory interneurons. Based on the latter of these connections, we hypothesized that inactivating the contralateral (intact) SI forelimb region would "unmask" normally suppressed hindlimb responses by reducing the activity of SI GABAergic neurons. The SI forelimb-stump representation was first mapped under normal conditions and then during GRB to identify stump/hindlimb responsive sites. After GRB had dissipated, the contralateral (intact) SI forelimb region was mapped and reversibly inactivated with injections of 4% lidocaine, and selected forelimb-stump sites were retested. Contralateral SI inactivation revealed hindlimb responses in approximately 60% of sites that were stump/hindlimb responsive during GRB. These findings indicate that activity in the contralateral SI contributes to the suppression of reorganized hindlimb receptive fields in neonatally amputated rats.

  7. In silico Analysis of Combinatorial microRNA Activity Reveals Target Genes and Pathways Associated with Breast Cancer Metastasis

    PubMed Central

    Dombkowski, Alan A.; Sultana, Zakia; Craig, Douglas B.; Jamil, Hasan

    2011-01-01

    This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited. Aberrant microRNA activity has been reported in many diseases, and studies often find numerous microRNAs concurrently dysregulated. Most target genes have binding sites for multiple microRNAs, and mounting evidence indicates that it is important to consider their combinatorial effect on target gene repression. A recent study associated the coincident loss of expression of six microRNAs with metastatic potential in breast cancer. Here, we used a new computational method, miR-AT!, to investigate combinatorial activity among this group of microRNAs. We found that the set of transcripts having multiple target sites for these microRNAs was significantly enriched with genes involved in cellular processes commonly perturbed in metastatic tumors: cell cycle regulation, cytoskeleton organization, and cell adhesion. Network analysis revealed numerous target genes upstream of cyclin D1 and c-Myc, indicating that the collective loss of the six microRNAs may have a focal effect on these two key regulatory nodes. A number of genes previously implicated in cancer metastasis are among the predicted combinatorial targets, including TGFB1, ARPC3, and RANKL. In summary, our analysis reveals extensive combinatorial interactions that have notable implications for their potential role in breast cancer metastasis and in therapeutic development. PMID:21552493

  8. Artemisinins, New Miconazole Potentiators Resulting in Increased Activity against Candida albicans Biofilms

    PubMed Central

    De Cremer, Kaat; Lanckacker, Ellen; Cools, Tanne L.; Bax, Marijke; De Brucker, Katrijn; Cos, Paul; Thevissen, Karin

    2014-01-01

    Mucosal biofilm-related fungal infections are very common, and the incidence of recurrent oral and vulvovaginal candidiasis is significant. As resistance to azoles (the preferred treatment) is occurring, we aimed at identifying compounds that increase the activity of miconazole against Candida albicans biofilms. We screened 1,600 compounds of a drug-repositioning library in combination with a subinhibitory concentration of miconazole. Synergy between the best identified potentiators and miconazole was characterized by checkerboard analyses and fractional inhibitory concentration indices. Hexachlorophene, pyrvinium pamoate, and artesunate act synergistically with miconazole in affecting C. albicans biofilms. Synergy was most pronounced for artesunate and structural homologues thereof. No synergistic effect could be observed between artesunate and fluconazole, caspofungin, or amphotericin B. Our data reveal enhancement of the antibiofilm activity of miconazole by artesunate, pointing to potential combination therapy consisting of miconazole and artesunate to treat C. albicans biofilm-related infections. PMID:25367916

  9. Incomplete suppression of distractor-related activity in the frontal eye field results in curved saccades.

    PubMed

    McPeek, Robert M

    2006-11-01

    Saccades in the presence of distractors show significant trajectory curvature. Based on previous work in the superior colliculus (SC), we speculated that curvature arises when a movement is initiated before competition between the target and distractor goals has been fully resolved. To test this hypothesis, we recorded frontal eye field (FEF) activity for curved and straight saccades in search. In contrast to the SC, activity in FEF is normally poorly correlated with saccade dynamics. However, the FEF, like the SC, is involved in target selection. Thus if curvature is caused by incomplete target selection, we expect to see its neural correlates in the FEF. We found that saccades that curve toward a distractor are accompanied by an increase in perisaccadic activity of FEF neurons coding the distractor location, and saccades that curve away are accompanied by a decrease in activity. In contrast, for FEF neurons coding the target location, there is no significant difference in activity between curved and straight saccades. To establish that the distractor-related activity is causally related to saccade curvature, we applied microstimulation to sites in the FEF before saccades to targets presented without distractors. The stimulation was subthreshold for evoking saccades and the temporal structure of the stimulation train resembled the activity recorded for curved saccades. The resulting movements curved toward the location coded by the stimulation site. These results support the idea that saccade curvature results from incomplete suppression of distractor-related activity during target selection.

  10. Simulating natural light and temperature cycles in the laboratory reveals differential effects on activity/rest rhythm of four Drosophilids.

    PubMed

    Prabhakaran, Priya M; Sheeba, Vasu

    2014-10-01

    Recent studies under semi-natural conditions have revealed various unique features of activity/rest rhythms in Drosophilids that differ from those under standard laboratory conditions. An additional afternoon peak (A-peak) has been reported for Drosophila melanogaster and another species D. malerkotliana while D. ananassae exhibited mostly unimodal diurnal activity. To tease apart the role of light and temperature in mediating these species-specific behaviours of four Drosophilid species D. melanogaster, D. malerkotliana, D. ananassae, and Zaprionus indianus we simulated gradual natural light and/or temperature cycles conditions in laboratory. The pattern observed under semi-natural conditions could be reproduced in the laboratory for all the species under a variety of simulated conditions. D. melanogaster and D. malerkotliana showed similar patterns where as D. ananassae consistently exhibited predominant morning activity under almost all regimes. Z. indianus showed clearly rhythmic activity mostly when temperature cycles were provided. We find that gradually changing light intensities reaching a sufficiently high peak value can elicit A-peak in D. melanogaster, D. malerkotliana, and D. ananassae even at mild ambient temperature. Furthermore, we show that high mid-day temperature could induce A-peak in all species even under constant light conditions suggesting that this A-peak is likely to be a stress response.

  11. Integration of conventional quantitative and phospho-proteomics reveals new elements in activated Jurkat T-cell receptor pathway maintenance.

    PubMed

    Jouy, Florent; Müller, Stephan A; Wagner, Juliane; Otto, Wolfgang; von Bergen, Martin; Tomm, Janina M

    2015-01-01

    Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T-cell receptor (TCR) pathway in a T-cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T-cells and phosphopeptides enriched via a TiO2-based chromatographic step. Both phosphopeptide-enriched and flow-through fractions were analyzed by LC-MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow-through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR-related function were detected. A kinase-substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR-related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T-cells.

  12. Structural Analysis of Glycine Sarcosine N-methyltransferase from Methanohalophilus portucalensis Reveals Mechanistic Insights into the Regulation of Methyltransferase Activity

    PubMed Central

    Lee, Yi-Ru; Lin, Te-Sheng; Lai, Shu-Jung; Liu, Mu-Sen; Lai, Mei-Chin; Chan, Nei-Li

    2016-01-01

    Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways’ end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. Here, we report the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT), which represents the first structural elucidation of the GSMT methyltransferase family. As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. This work implicates a structural basis by which feedback inhibition of biosynthetic methyltransferases may be achieved. PMID:27934872

  13. Distribution of glass transition temperatures Tg in polystyrene thin films as revealed by low-energy muon spin relaxation: A comparison with neutron reflectivity results.

    PubMed

    Kanaya, Toshiji; Ogawa, Hiroki; Kishimoto, Mizuki; Inoue, Rintaro; Suter, Andreas; Prokscha, Thomas

    2015-08-01

    In a previous paper [Phys. Rev. E 83, 021801 (2011)] we performed neutron reflectivity (NR) measurements on a five-layer polystyrene (PS) thin film consisting of alternatively stacked deuterated polystyrene (dPS) and hydrogenated polystyrene (hPS) layers (dPS/hPS/dPS/hPS/dPS, ∼100 nm thick) on a Si substrate to reveal the distribution of Tg along the depth direction. Information on the Tg distribution is very useful to understand the interesting but unusual properties of polymer thin films. However, one problem that we have to clarify is if there are effects of deuterium labeling on Tg or not. To tackle the problem we performed low-energy muon spin relaxation (μSR) measurements on the above-mentioned deuterium-labeled five-layer PS thin film as well as dPS and hPS single-layer thin films ∼100 nm thick as a function of muon implantation energy. It was found that the deuterium labeling had no significant effects on the Tg distribution, guaranteeing that we can safely discuss the unusual thin film properties based on the Tg distribution revealed by NR on the deuterium-labeled thin films. In addition, the μSR result suggested that the higher Tg near the Si substrate is due to the strong orientation of phenyl rings.

  14. Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition.

    PubMed

    Leight, Jennifer L; Tokuda, Emi Y; Jones, Caitlin E; Lin, Austin J; Anseth, Kristi S

    2015-04-28

    Matrix metalloproteinases (MMPs) are important for many different types of cancer-related processes, including metastasis. Understanding the functional impact of changes in MMP activity during cancer treatment is an important facet not typically evaluated as part of preclinical research. With MMP activity being a critical component of the metastatic cascade, we designed a 3D hydrogel system to probe whether pharmacological inhibition affected human melanoma cell proteolytic activity; metastatic melanoma is a highly aggressive and drug-resistant form of skin cancer. The relationship between MMP activity and drug treatment is unknown, and therefore we used an in situ fluorogenic MMP sensor peptide to determine how drug treatment affects melanoma cell MMP activity in three dimensions. We encapsulated melanoma cells from varying stages of progression within PEG-based hydrogels to examine the relationship between drug treatment and MMP activity. From these results, a metastatic melanoma cell line (A375) and two inhibitors that inhibit RAF (PLX4032 and sorafenib) were studied further to determine whether changes in MMP activity led to a functional change in cell behavior. A375 cells exhibited increased MMP activity despite an overall decrease in metabolic activity with PLX4032 treatment. The changes in proteolytic activity correlated with increased cell elongation and increased single-cell migration. In contrast, sorafenib did not alter MMP activity or cell motility, showing that the changes induced by PLX4032 were not a universal response to small-molecule inhibition. Therefore, we argue the importance of studying MMP activity with drug treatment and its possible implications for unwanted side effects.

  15. Influence of mesophase activation conditions on the specific capacitance of the resulting carbons

    NASA Astrophysics Data System (ADS)

    Mora, E.; Ruiz, V.; Santamaría, R.; Blanco, C.; Granda, M.; Menéndez, R.; Juarez-Galán, J. M.; Rodríguez-Reinoso, F.

    Mesophase pitch AR24 was directly activated with KOH using different proportions of the activating agent and activation temperatures, to study the effect on the textural characteristics of the resultant activated carbons and how these characteristics influence their behaviour as electrodes in supercapacitors. The textural properties of the activated carbons were studied by gas adsorption and immersion calorimetry. The results indicate that all the carbons produced were mainly microporous, with pore size around 1 nm. The behaviour of these carbons as electrodes in supercapacitors was studied from galvanostatic charge-discharge cycles. The specific capacitance values obtained were very high, reaching 400 and 200 F g -1 at low and high current densities respectively, for the sample activated with (5:1) KOH to mesophase ratio. Nevertheless, the reasons for this high capacitance values cannot be explained only on the basis of the textural characteristics of the activated carbons, as the results indicated that other factors might be also playing a significant role in their electrochemical behaviour.

  16. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    SciTech Connect

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  17. Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

    PubMed Central

    Frelinger, Andrew L.; Gerrits, Anja J.; Garner, Allen L.; Torres, Andrew S.; Caiafa, Antonio; Morton, Christine A.; Berny-Lang, Michelle A.; Carmichael, Sabrina L.; Neculaes, V. Bogdan; Michelson, Alan D.

    2016-01-01

    Background Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. Aims To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. Methods PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. Results PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the

  18. Charge Profile Analysis Reveals That Activation of Pro-apoptotic Regulators Bax and Bak Relies on Charge Transfer Mediated Allosteric Regulation

    PubMed Central

    Ionescu, Crina-Maria; Svobodová Vařeková, Radka; Prehn, Jochen H. M.; Huber, Heinrich J.; Koča, Jaroslav

    2012-01-01

    The pro-apoptotic proteins Bax and Bak are essential for executing programmed cell death (apoptosis), yet the mechanism of their activation is not properly understood at the structural level. For the first time in cell death research, we calculated intra-protein charge transfer in order to study the structural alterations and their functional consequences during Bax activation. Using an electronegativity equalization model, we investigated the changes in the Bax charge profile upon activation by a functional peptide of its natural activator protein, Bim. We found that charge reorganizations upon activator binding mediate the exposure of the functional sites of Bax, rendering Bax active. The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction. We further identified a network of charge reorganizations that confirms previous speculations of allosteric sensing, whereby the activation information is conveyed from the activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. The network was mediated by a hub of three residues on helix 5 of the hydrophobic core of Bax. Sequence and structural alignment revealed that this hub was conserved in the Bak amino acid sequence, and in the 3D structure of folded Bak. Our results suggest that allostery mediated by charge transfer is responsible for the activation of both Bax and Bak, and that this might be a prototypical mechanism for a fast activation of proteins during signal transduction. Our method can be applied to any protein or protein complex in order to map the progress of allosteric changes through the proteins' structure. PMID:22719244

  19. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    PubMed

    Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

    2010-10-14

    A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  20. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    PubMed Central

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo. PMID:28045057

  1. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    NASA Astrophysics Data System (ADS)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

  2. Post-Knowledge of Results Delay: Effects of Interpolated Activity on Learning and Performance.

    ERIC Educational Resources Information Center

    Benedetti, Carol; McCullagh, Penny

    1987-01-01

    Experimental evidence strongly supports the assumption that knowledge of results (KR) is necessary for learning to occur. This study compares the effects of KR or no-KR with the effects of an interpolated verbal activity on learning and performance of a timed motor task. Results are presented. (Author/MT)

  3. Real-time optical recording of β1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol

    PubMed Central

    Rochais, Francesca; Vilardaga, Jean-Pierre; Nikolaev, Viacheslav O.; Bünemann, Moritz; Lohse, Martin J.; Engelhardt, Stefan

    2007-01-01

    Antagonists of β-adrenergic receptors (β-ARs) have become a main therapeutic regimen for the treatment of heart failure even though the mechanisms of their beneficial effects are still poorly understood. Here, we used fluorescent resonance energy transfer–based (FRET-based) approaches to directly monitor activation of the β1-AR and downstream signaling. While the commonly used β-AR antagonists metoprolol, bisoprolol, and carvedilol displayed varying degrees of inverse agonism on the Gly389 variant of the receptor (i.e., actively switching off the β1-AR), surprisingly, only carvedilol showed very specific and marked inverse agonist effects on the more frequent Arg389 variant. These specific effects of carvedilol on the Arg389 variant of the β1-AR were also seen for control of beating frequency in rat cardiac myocytes expressing the 2 receptor variants. This FRET sensor permitted direct observation of activation of the β1-AR in living cells in real time. It revealed that β1-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use. PMID:17200720

  4. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

  5. Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells

    PubMed Central

    Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

    2016-01-01

    L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells. PMID:26983598

  6. Activated FcgammaRII and signalling molecules revealed in rafts by ultra-structural observations of plasma-membrane sheets.

    PubMed

    Strzelecka-Kiliszek, Agnieszka; Korzeniowski, Marek; Kwiatkowska, Katarzyna; Mrozińska, Kazimiera; Sobota, Andrzej

    2004-01-01

    To reveal topography of FcgammaRII components of the receptor-signalling complex, large plasma-membrane sheets were obtained by cell cleavage and analysed by immuno-electron microscopy. Non-activated FcgammaRII was dispersed in the plane of the plasma membrane and only rarely was localized in the proximity of Lyn, an Src family tyrosine kinase, and CD55, a glycosylphosphatidylinositol-anchored protein. After FcgammaRII activation by cross-linking with antibodies, clusters of an electron-dense material acquiring about 86% of FcgammaRII and reaching up to 300 nm in diameter were formed within 5 min. These structures also accommodated about 85% of Lyn and 63% of CD55 labels that were located in close vicinity of gold particles attributed to the cross-linked FcgammaRII . The electron-dense structures were also abundant in tyrosine phosphorylated proteins. At their margins PIP2 was preferentially located. Based on a concentration of Lyn, CD55 and activated FcgammaRII , the electron-dense structures seem to reflect coalescent membrane rafts.

  7. Functional ultrasound imaging reveals different odor-evoked patterns of vascular activity in the main olfactory bulb and the anterior piriform cortex.

    PubMed

    Osmanski, B F; Martin, C; Montaldo, G; Lanièce, P; Pain, F; Tanter, M; Gurden, H

    2014-07-15

    Topographic representation of the outside world is a key feature of sensory systems, but so far it has been difficult to define how the activity pattern of the olfactory information is distributed at successive stages in the olfactory system. We studied odor-evoked activation patterns in the main olfactory bulb and the anterior piriform cortex of rats using functional ultrasound (fUS) imaging. fUS imaging is based on the use of ultrafast ultrasound scanners and detects variations in the local blood volume during brain activation. It makes deep brain imaging of ventral structures, such as the piriform cortex, possible. Stimulation with two different odors (hexanal and pentylacetate) induced the activation of odor-specific zones that were spatially segregated in the main olfactory bulb. Interestingly, the same odorants triggered the activation of the entire anterior piriform cortex, in all layers, with no distinguishable odor-specific areas detected in the power Doppler images. These fUS imaging results confirm the spatial distribution of odor-evoked activity in the main olfactory bulb, and furthermore, they reveal the absence of such a distribution in the anterior piriform cortex at the macroscopic scale in vivo.

  8. Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

    PubMed Central

    Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

    2017-01-01

    Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

  9. Electrocardiograhic findings resulting in inappropriate cardiac catheterization laboratory activation for ST-segment elevation myocardial infarction

    PubMed Central

    Shamim, Shariq; McCrary, Justin; Wayne, Lori; Gratton, Matthew

    2014-01-01

    Background Prompt reperfusion has been shown to improve outcomes in patients with acute ST-segment elevation myocardial infarction (STEMI) with a goal of culprit vessel patency in <90 minutes. This requires a coordinated approach between the emergency medical services (EMS), emergency department (ED) and interventional cardiology. The urgency of this process can contribute to inappropriate cardiac catheterization laboratory (CCL) activations. Objectives One of the major determinants of inappropriate activations has been misinterpretation of the electrocardiogram (ECG) in the patient with acute chest pain. Methods We report the ECG findings for all CCL activations over an 18-month period after the inception of a STEMI program at our institution. Results There were a total of 139 activations with 77 having a STEMI diagnosis confirmed and 62 activations where there was no STEMI. The inappropriate activations resulted from a combination of atypical symptoms and misinterpretation of the ECG (45% due to anterior ST-segment elevation) on patient presentation. The electrocardiographic abnormalities were particularly problematic in African-Americans with left ventricular hypertrophy. Conclusions In this single-center, prospective observational study, nearly half of the inappropriate STEMI activations were due to the misinterpretation of anterior ST-segment elevation and this finding was commonly seen in African-Americans with left ventricular hypertrophy. PMID:25009790

  10. The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode†

    PubMed Central

    Caner, Sami; Nguyen, Nham; Aguda, Adeleke; Zhang, Ran; Pan, Yuan T; Withers, Stephen G; Brayer, Gary D

    2013-01-01

    Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation. PMID:23735230

  11. The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode.

    PubMed

    Caner, Sami; Nguyen, Nham; Aguda, Adeleke; Zhang, Ran; Pan, Yuan T; Withers, Stephen G; Brayer, Gary D

    2013-09-01

    Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation.

  12. Crystal structure of Plasmodium vivax FK506-binding protein 25 reveals conformational changes responsible for its noncanonical activity.

    PubMed

    Rajan, Sreekanth; Austin, David; Harikishore, Amaravadhi; Nguyen, Quoc Toan; Baek, Kwanghee; Yoon, Ho Sup

    2014-07-01

    The malarial parasites currently remain one of the most dreadful parasites, which show increasing trend of drug resistance to the currently available antimalarial drugs. Thus, the need to identify and characterize new protein targets in these parasites can aid to design novel therapeutic strategies to combat malaria. Recently, the conserved FK506-binding protein family members with molecular weight of 35 kDa from Plasmodium falciparum and Plasmodium vivax (referred to as PfFKBP35 and PvFKBP35, respectively) were identified for drug targeting. Further data mining revealed a 25-kDa FKBP (FKBP25) family member present in the parasites. FKBP25 belongs to a unique class of FKBP, because it is a nuclear FKBP with multiple protein-binding partners. Apart from immune regulation, it is also known for its chaperoning role in various cellular processes such as transcription regulation and trafficking. Here, we present the biochemical characterization and 1.9-Å crystal structure of an N-terminal truncated FKBP25 from P. vivax (PvFKBP25(72-209)). The protein reveals the noncanonical nature with unique structural changes observed in the loops flanking the active site, concealing the binding pocket. Further, a potential calmodulin-binding domain, which is absent in human FKBP25, is observed in this protein. Although the functional implication of Plasmodium FKBP25 in malaria still remains elusive, we speculate that the notable conformational changes in its structure might serve as an overture in understanding its molecular mechanism.

  13. Mitochondrial proteome analysis reveals depression of the Ndufs3 subunit and activity of complex I in diabetic rat brain.

    PubMed

    Taurino, Federica; Stanca, Eleonora; Siculella, Luisa; Trentadue, Raffaella; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2012-04-18

    Type-1 diabetes resulting from defective insulin secretion and consequent hyperglycemia, is associated with "diabetic encephalopathy." This is characterized by brain neurophysiological and structural changes resulting in impairment of cognitive function. The present proteomic analysis of brain mitochondrial proteins from streptozotocin-induced type-1 diabetic rats, shows a large decrement of the Ndufs3 protein subunit of complex I, decreased level of the mRNA and impaired catalytic activity of the complex in the diabetic rats as compared to controls. The severe depression of the expression and enzymatic activity of complex I can represent a critical contributing factor to the onset of the diabetic encephalopathy in type-1 diabetes.

  14. Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design

    PubMed Central

    Hilbert, Brendan J.; Grossman, Steven R.; Schiffer, Celia A.; Royer, William E.

    2014-01-01

    The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD+ revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD+ phosphate. Neither feature is present in other D2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors. PMID:24657618

  15. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.

    PubMed

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.

  16. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    PubMed Central

    Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A. PMID:28052131

  17. Efficacy of physical activity in the adjunctive treatment of major depressive disorders: preliminary results

    PubMed Central

    2007-01-01

    Background No controlled trials have evaluated the long term efficacy of exercise activity to improve the treatment of patients with Major Depressive Disorders. The aim of the present study was to confirm the efficacy of the adjunctive physical activity in the treatment of major depressive disorders, with a long term follow up (8 months). Methods Trial with randomized naturalistic control. Patients selected from the clinical activity registries of the Psychiatric Unit of the University of Cagliari, Italy. Inclusion criteria: female, between 40 and 60 years, diagnosis of Major Depressive Disorders (DSM-IV TR) resistant to the ongoing treatment. Exclusion criteria: diagnosis of psychotic disorders; any contraindications to physical activity. 30 patients (71.4% of the eligible) participated to the study. Cases: 10 randomized patients undergoing pharmacological treatment plus physical activity. Controls: 20 patients undergoing only pharmacological therapy. The following tools were collected from each patient by two different psychiatric physicians at baseline and 8 month after the beginning of exercise program: SCID-I, HAM-D, CGI (Clinical Global Impression), GAF. Results The patients that made physical activity had their HAM-D, GAF and CGI score improved from T0 to T8, all differences were statistically significant. In the control group HAM-D, GAF and CGI scores do not show any statistically significant differences between T0 and T8. Limits Small sample size limited to female in adult age; control group was not subject to any structured rehabilitation activity or placebo so it was impossible to evaluate if the improvement was due to a non specific therapeutic effect associated with taking part in a social activity. Conclusion Physical activity seems a good adjunctive treatment in the long term management of patients with MDD. Randomized placebo controlled trials are needed to confirm the results. PMID:17620123

  18. High-resolution, terrestrial radar velocity observations and model results reveal a strong bed at stable, tidewater Rink Isbræ, West Greenland

    NASA Astrophysics Data System (ADS)

    Bartholomaus, T. C.; Walker, R. T.; Stearns, L. A.; Fahnestock, M. A.; Cassotto, R.; Catania, G. A.; Felikson, D.; Fried, M.; Sutherland, D.; Nash, J. D.; Shroyer, E.

    2015-12-01

    At tidewater Rink Isbræ, on the central west coast of Greenland, satellite observations reveal that glacier velocities and terminus positions have remained stable, while the lowest 25 km have thinned 30 m since 1985. Over this same time period, other tidewater glaciers in central west Greenland have retreated, thinned and accelerated. Here we present field observations and model results to show that the flow of Rink Isbræ is resisted by unusually high basal shear stresses. Terrestrial radar interferometry (TRI) observations over 9 days in summer 2014 demonstrate weak velocity response to 4 km wide, full thickness calving events. Velocities at the terminus change by +/- 10% in response to rising and falling tides within a partial-width, 2.5-km-long floating ice tongue; however these tidal perturbations damp out within 2 km of the grounding line. Inversions for basal shear stress and force balance analyses together show that basal shear stresses in excess of 300 kPa support the majority of the driving stress at thick, steep Rink Isbræ. These observational and modeling results tell a consistent story in which a strong bed may limit the unstable tidewater glacier retreats observed elsewhere. Rink Isbræ has an erosion resistant quartzite bed with low fracture density. We hypothesize that this geology may play a major role in the bed strength.

  19. aPKC Phosphorylation of HDAC6 Results in Increased Deacetylation Activity

    PubMed Central

    Du, Yifeng; Seibenhener, Michael L.; Yan, Jin; Jiang, Jianxiong; Wooten, Michael C.

    2015-01-01

    The Class II histone deacetylase, HDAC6, has been shown to be involved in cell motility, aggresome formation and mitochondria transport. HDAC6 deacetylase activity regulates α-tubulin acetylation levels and thus plays a critical role in these processes. In turn, HDAC6 activity can be regulated by interaction with various proteins including multiple kinases. Kinase mediated phosphorylation of HDAC6 can lead to either increased or reduced activity. Our previous research has shown that sequestosome1/p62 (SQSTM1/p62) interacts with HDAC6 and regulates its activity. As SQSTM1/p62 is a scaffolding protein known to interact directly with the zeta isoform of Protein Kinase C (PKCζ), we sought to examine if HDAC6 could be a substrate for PKCζ phosphorylation and if so, how its activity might be regulated. Our data demonstrate that HDAC6 is not only present in a protein complex with PKCζ but can also be phosphorylated by PKCζ. We also show that specific phosphorylation of HDAC6 by PKCζ increases HDAC6 deacetylase activity resulting in reduced acetylated tubulin levels. Our findings provide novel insight into the molecular mechanism by which HDAC6, PKCζ and SQSTM1/p62 function together in protein aggregate clearance. These results also highlight a new research direction which may prove fruitful for understanding the underlying cause of several neurodegenerative diseases. PMID:25860570

  20. aPKC phosphorylation of HDAC6 results in increased deacetylation activity.

    PubMed

    Du, Yifeng; Seibenhener, Michael L; Yan, Jin; Jiang, Jianxiong; Wooten, Michael C

    2015-01-01

    The Class II histone deacetylase, HDAC6, has been shown to be involved in cell motility, aggresome formation and mitochondria transport. HDAC6 deacetylase activity regulates α-tubulin acetylation levels and thus plays a critical role in these processes. In turn, HDAC6 activity can be regulated by interaction with various proteins including multiple kinases. Kinase mediated phosphorylation of HDAC6 can lead to either increased or reduced activity. Our previous research has shown that sequestosome1/p62 (SQSTM1/p62) interacts with HDAC6 and regulates its activity. As SQSTM1/p62 is a scaffolding protein known to interact directly with the zeta isoform of Protein Kinase C (PKCζ), we sought to examine if HDAC6 could be a substrate for PKCζ phosphorylation and if so, how its activity might be regulated. Our data demonstrate that HDAC6 is not only present in a protein complex with PKCζ but can also be phosphorylated by PKCζ. We also show that specific phosphorylation of HDAC6 by PKCζ increases HDAC6 deacetylase activity resulting in reduced acetylated tubulin levels. Our findings provide novel insight into the molecular mechanism by which HDAC6, PKCζ and SQSTM1/p62 function together in protein aggregate clearance. These results also highlight a new research direction which may prove fruitful for understanding the underlying cause of several neurodegenerative diseases.

  1. Return to sporting activity after Birmingham hip resurfacing arthroplasty: Mid term results

    PubMed Central

    Sandiford, Nemandra; Muirhead-Allwood, SK; Skinner, JA

    2015-01-01

    Background: Hip resurfacing arthroplasty (HRA) is primarily indicated for young, active patients with disabling coxarthrosis who wish to remain active and return to sports after surgery. Relatively few prospective studies have assessed return to sporting activity and impact of gender and age on this. Materials and Methods: Seventy-nine consecutive patients treated with HRA were included. Patients were reviewed clinically and radiologically. Function was assessed using the modified University of California Los Angeles (UCLA) activity score. The Oxford, Harris and WOMAC hip scores were calculated. Results: Average age at the time of surgery was 54.9 years (range 34.5–73.6 years). Average preoperative and postoperative UCLA scores were 4 and 7.6 respectively. Patients were involved in 2 (0–4) sporting activities preoperatively and 2 (0–5) postoperatively. Preoperative and postoperative Oxford Hip Scores, Harris Hip Score and WOMAC scores were 40, 46 and 51 and 16, 94 and 3 respectively (P < 0.0001). Patients returned to sports at an average of 3 months postoperatively. Conclusion: Patients were able to return to sports by 3 months and perform the same number of activities at preoperative intensity. Activity levels are maintained up to the medium term with few complications. PMID:26806965

  2. Status of data, major results, and plans for geophysical activities, Yucca Mountain Project

    SciTech Connect

    Oliver, H.W.; Hardin, E.L.; Nelson, P.H.

    1990-07-01

    This report describes past and planned geophysical activities associated with the Yucca Mountain Project and is intended to serve as a starting point for integration of geophysical activities. This report relates past results to site characterization plans, as presented in the Yucca Mountain Site Characterization Plan (SCP). This report discusses seismic exploration, potential field methods, geoelectrical methods, teleseismic data collection and velocity structural modeling, and remote sensing. This report discusses surface-based, airborne, borehole, surface-to-borehole, crosshole, and Exploratory Shaft Facility-related activities. The data described in this paper, and the publications discussed, have been selected based on several considerations; location with respect to Yucca Mountain, whether the success or failure of geophysical data is important to future activities, elucidation of features of interest, and judgment as to the likelihood that the method will produce information that is important for site characterization. 65 refs., 19 figs., 12 tabs.

  3. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    SciTech Connect

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W.

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  4. Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling.

    PubMed

    Yeh, Yuh-Ying; Ozer, Hatice Gulcin; Lehman, Amy M; Maddocks, Kami; Yu, Lianbo; Johnson, Amy J; Byrd, John C

    2015-05-21

    Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile.

  5. Long-Term Spatiotemporal Reconfiguration of Neuronal Activity Revealed by Voltage-Sensitive Dye Imaging in the Cerebellar Granular Layer

    PubMed Central

    Gandolfi, Daniela; Mapelli, Jonathan; D'Angelo, Egidio

    2015-01-01

    Understanding the spatiotemporal organization of long-term synaptic plasticity in neuronal networks demands techniques capable of monitoring changes in synaptic responsiveness over extended multineuronal structures. Among these techniques, voltage-sensitive dye imaging (VSD imaging) is of particular interest due to its good spatial resolution. However, improvements of the technique are needed in order to overcome limits imposed by its low signal-to-noise ratio. Here, we show that VSD imaging can detect long-term potentiation (LTP) and long-term depression (LTD) in acute cerebellar slices. Combined VSD imaging and patch-clamp recordings revealed that the most excited regions were predominantly associated with granule cells (GrCs) generating EPSP-spike complexes, while poorly responding regions were associated with GrCs generating EPSPs only. The correspondence with cellular changes occurring during LTP and LTD was highlighted by a vector representation obtained by combining amplitude with time-to-peak of VSD signals. This showed that LTP occurred in the most excited regions lying in the core of activated areas and increased the number of EPSP-spike complexes, while LTD occurred in the less excited regions lying in the surround. VSD imaging appears to be an efficient tool for investigating how synaptic plasticity contributes to the reorganization of multineuronal activity in neuronal circuits. PMID:26294979

  6. Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling

    PubMed Central

    Yeh, Yuh-Ying; Ozer, Hatice Gulcin; Lehman, Amy M.; Maddocks, Kami; Yu, Lianbo; Byrd, John C.

    2015-01-01

    Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile. PMID:25833959

  7. Transcriptomic Analysis of Tail Regeneration in the Lizard Anolis carolinensis Reveals Activation of Conserved Vertebrate Developmental and Repair Mechanisms

    PubMed Central

    Hutchins, Elizabeth D.; Markov, Glenn J.; Eckalbar, Walter L.; George, Rajani M.; King, Jesse M.; Tokuyama, Minami A.; Geiger, Lauren A.; Emmert, Nataliya; Ammar, Michael J.; Allen, April N.; Siniard, Ashley L.; Corneveaux, Jason J.; Fisher, Rebecca E.; Wade, Juli; DeNardo, Dale F.; Rawls, J. Alan; Huentelman, Matthew J.; Wilson-Rawls, Jeanne; Kusumi, Kenro

    2014-01-01

    Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies. PMID:25140675

  8. Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

    PubMed

    Hutchins, Elizabeth D; Markov, Glenn J; Eckalbar, Walter L; George, Rajani M; King, Jesse M; Tokuyama, Minami A; Geiger, Lauren A; Emmert, Nataliya; Ammar, Michael J; Allen, April N; Siniard, Ashley L; Corneveaux, Jason J; Fisher, Rebecca E; Wade, Juli; DeNardo, Dale F; Rawls, J Alan; Huentelman, Matthew J; Wilson-Rawls, Jeanne; Kusumi, Kenro

    2014-01-01

    Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

  9. Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon induced antiviral activity

    PubMed Central

    Huang, Hao; Zeqiraj, Elton; Dong, Beihua; Jha, Babal Kant; Duffy, Nicole; Orlicky, Stephen; Thevakumaran, Neroshan; Talukdar, Manisha; Pillon, Monica C.; Ceccarelli, Derek F.; Wan, Leo; Juang, Yu-Chi; Mao, Daniel Y.L.; Gaughan, Christina; Brinton, Margo A.; Perelygin, Andrey A.; Kourinov, Igor; Guarné, Alba; Silverman, Robert H.; Sicheri, Frank

    2014-01-01

    Summary RNase L is an ankyrin repeat domain containing dual endoribonuclease-pseudokinase that is activated by unusual 2′,5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here, we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small angle x-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the non-hydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain together with nucleotide binding, impose a rigid intertwined dimer configuration that is essential for RNase catalytic and anti-viral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold. PMID:24462203

  10. Changes in breath trihalomethane levels resulting from household water-use activities.

    PubMed

    Gordon, Sydney M; Brinkman, Marielle C; Ashley, David L; Blount, Benjamin C; Lyu, Christopher; Masters, John; Singer, Philip C

    2006-04-01

    Common household water-use activities such as showering, bathing, drinking, and washing clothes or dishes are potentially important contributors to individual exposure to trihalomethanes (THMs), the major class of disinfection by-products of water treated with chlorine. Previous studies have focused on showering or bathing activities. In this study, we selected 12 common water-use activities and determined which may lead to the greatest THM exposures and result in the greatest increase in the internal dose. Seven subjects performed the various water-use activities in two residences served by water utilities with relatively high and moderate total THM levels. To maintain a consistent exposure environment, the activities, exposure times, air exchange rates, water flows, water temperatures, and extraneous THM emissions to the indoor air were carefully controlled. Water, indoor air, blood, and exhaled-breath samples were collected during each exposure session for each activity, in accordance with a strict, well-defined protocol. Although showering (for 10 min) and bathing (for 14 min), as well as machine washing of clothes and opening mechanical dishwashers at the end of the cycle, resulted in substantial increases in indoor air chloroform concentrations, only showering and bathing caused significant increases in the breath chloroform levels. In the case of bromodichloromethane (BDCM), only bathing yielded a significantly higher air level in relation to the preexposure concentration. For chloroform from showering, strong correlations were observed for indoor air and exhaled breath, blood and exhaled breath, indoor air and blood, and tap water and blood. Only water and breath, and blood and breath were significantly associated for chloroform from bathing. For BDCM, significant correlations were obtained for blood and air, and blood and water from showering. Neither dibromochloromethane nor bromoform gave measurable breath concentrations for any of the activities

  11. Analysis of cytotoxic activity at short incubation times reveals profound differences among Annonaceus acetogenins, inhibitors of mitochondrial Complex I.

    PubMed

    de Pedro, Nuria; Cautain, Bastien; Melguizo, Angeles; Cortes, Diego; Vicente, Francisca; Genilloud, Olga; Tormo, Jose R; Peláez, Fernando

    2013-02-01

    Annonaceous acetogenins are potent cytotoxic agents against tumor cell lines as well as potent inhibitors of mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116-120, 1994; Degli Esposti et al. Biochem J 301(Pt 1):161-167, 1994; Tormo et al. Arch Biochem Biophys 369:119-126, 1999). Eighteen different ACGs belonging to seven structural sub-families were tested against six tumor and two non tumor cell lines in a MTT cytotoxicity assay to evaluate the correlation between mitochondrial Complex I inhibition and cytotoxic activity potency and selectivity. The results showed a substantial heterogeneity in potency and selectivity among the different compounds tested, although no clear overall structure-activity relationships could be established. To further characterize the biological activity of these compounds, four ACGs were selected based on their inhibition binding sites to Complex I, to evaluate their cytotoxic activity over a 15-minute to 48-hour period using a more sensitive time-course LDH cytotoxicity assay. Our results indicate that, although all of the ACGs were highly cytotoxic in HepG2 cell lines at 24 h, each sub-class behaves rather differently at shorter times. Perhaps other aspects related to how these compounds reach or bind to their target sites, or differences in their ability to cross the cell and/or the mitochondrial membranes, could help explain their different activities. This different behavior between ACGs may provide new clues for a better understanding of their potential antitumor properties.

  12. The secret life of ground squirrels: accelerometry reveals sex-dependent plasticity in above-ground activity.

    PubMed

    Williams, Cory T; Wilsterman, Kathryn; Zhang, Victor; Moore, Jeanette; Barnes, Brian M; Buck, C Loren

    2016-09-01

    The sexes differ in how and when they allocate energy towards reproduction, but how this influences phenotypic plasticity in daily activity patterns is unclear. Here, we use collar-mounted light loggers and triaxial accelerometers to examine factors that affect time spent above ground and overall dynamic body acceleration (ODBA), an index of activity-specific energy expenditure, across the active season of free-living, semi-fossorial arctic ground squirrels (Urocitellus parryii). We found high day-to-day variability in time spent above ground and ODBA with most of the variance explained by environmental conditions known to affect thermal exchange. In both years, females spent more time below ground compared with males during parturition and early lactation; however, this difference was fourfold larger in the second year, possibly, because females were in better body condition. Daily ODBA positively correlated with time spent above ground in both sexes, but females were more active per unit time above ground. Consequently, daily ODBA did not differ between the sexes when females were early in lactation, even though females were above ground three to six fewer hours each day. Further, on top of having the additional burden of milk production, ODBA data indicate females also had fragmented rest patterns and were more active during late lactation. Our results indicate that sex differences in reproductive requirements can have a substantial influence on activity patterns, but the size of this effect may be dependent on capital resources accrued during gestation.

  13. The secret life of ground squirrels: accelerometry reveals sex-dependent plasticity in above-ground activity

    PubMed Central

    Wilsterman, Kathryn; Zhang, Victor; Moore, Jeanette; Barnes, Brian M.; Buck, C. Loren

    2016-01-01

    The sexes differ in how and when they allocate energy towards reproduction, but how this influences phenotypic plasticity in daily activity patterns is unclear. Here, we use collar-mounted light loggers and triaxial accelerometers to examine factors that affect time spent above ground and overall dynamic body acceleration (ODBA), an index of activity-specific energy expenditure, across the active season of free-living, semi-fossorial arctic ground squirrels (Urocitellus parryii). We found high day-to-day variability in time spent above ground and ODBA with most of the variance explained by environmental conditions known to affect thermal exchange. In both years, females spent more time below ground compared with males during parturition and early lactation; however, this difference was fourfold larger in the second year, possibly, because females were in better body condition. Daily ODBA positively correlated with time spent above ground in both sexes, but females were more active per unit time above ground. Consequently, daily ODBA did not differ between the sexes when females were early in lactation, even though females were above ground three to six fewer hours each day. Further, on top of having the additional burden of milk production, ODBA data indicate females also had fragmented rest patterns and were more active during late lactation. Our results indicate that sex differences in reproductive requirements can have a substantial influence on activity patterns, but the size of this effect may be dependent on capital resources accrued during gestation. PMID:27703706

  14. Activity-dependent isolation of the presenilin– γ-secretase complex reveals nicastrin and a γ substrate

    PubMed Central

    Esler, William P.; Kimberly, W. Taylor; Ostaszewski, Beth L.; Ye, Wenjuan; Diehl, Thekla S.; Selkoe, Dennis J.; Wolfe, Michael S.

    2002-01-01

    Presenilin heterodimers apparently contain the active site of γ-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-β precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimer's disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional γ-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (β-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated γ-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-β precursor protein substrate of γ-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active γ-secretase complex, indicate that β-catenin, calsenilin, and presenilin-associated protein are not required for γ activity, and suggest an unprecedented mechanism of substrate–protease interaction. PMID:11867728

  15. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity.

    PubMed

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K; Sunil, Sujatha

    2016-11-30

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models.

  16. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity

    PubMed Central

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K.; Sunil, Sujatha

    2016-01-01

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models. PMID:27901124

  17. Control of a Shunt Active Power Filter with Neural Networks—Theory and Practical Results

    NASA Astrophysics Data System (ADS)

    Villalva, Marcelo G.; Filho, Ernesto Ruppert

    This paper presents theoretical studies and practical results obtained with a four-wire shunt active power filter fully controlled with neural networks. The paper is focused on a current compensation method based on adaptive linear elements (adalines), which are powerful and easy-to-use neural networks. The reader will find here an introduction about these networks, an explanatory section about the achievement of Fourier series with adalines, and the full description of an adaline-based selective current compensator. The paper also brings a quick discussion about the use of a feedforward neural network in the current controller of the active filter, as well as simulation and experimental results obtained with the prototype of an active power filter.

  18. Smith-Magenis syndrome results in disruption of CLOCK gene transcription and reveals an integral role for RAI1 in the maintenance of circadian rhythmicity.

    PubMed

    Williams, Stephen R; Zies, Deborah; Mullegama, Sureni V; Grotewiel, Michael S; Elsea, Sarah H

    2012-06-08

    Haploinsufficiency of RAI1 results in Smith-Magenis syndrome (SMS), a disorder characterized by intellectual disability, multiple congenital anomalies, obesity, neurobehavioral abnormalities, and a disrupted circadian sleep-wake pattern. An inverted melatonin rhythm (i.e., melatonin peaks during the day instead of at night) and associated sleep-phase disturbances in individuals with SMS, as well as a short-period circadian rhythm in mice with a chromosomal deletion of Rai1, support SMS as a circadian-rhythm-dysfunction disorder. However, the molecular cause of the circadian defect in SMS has not been described. The circadian oscillator temporally orchestrates metabolism, physiology, and behavior largely through transcriptional modulation. Data support RAI1 as a transcriptional regulator, but the genes it might regulate are largely unknown. Investigation into the role that RAI1 plays in the regulation of gene transcription and circadian maintenance revealed that RAI1 regulates the transcription of circadian locomotor output cycles kaput (CLOCK), a key component of the mammalian circadian oscillator that transcriptionally regulates many critical circadian genes. Data further show that haploinsufficiency of RAI1 and Rai1 in SMS fibroblasts and the mouse hypothalamus, respectively, results in the transcriptional dysregulation of the circadian clock and causes altered expression and regulation of multiple circadian genes, including PER2, PER3, CRY1, BMAL1, and others. These data suggest that heterozygous mutation of RAI1 and Rai1 leads to a disrupted circadian rhythm and thus results in an abnormal sleep-wake cycle, which can contribute to an abnormal feeding pattern and dependent cognitive performance. Finally, we conclude that RAI1 is a positive transcriptional regulator of CLOCK, pinpointing a novel and important role for this gene in the circadian oscillator.

  19. [The results of the introduction of a protocol of preventive activities in a primary care center].

    PubMed

    Tamayo Marco, B; Deniel Rosanas, J; López Plana, A; Trallero Fort, J C; Lafuente Navarro, A; Lafuente Navarro, C

    1991-04-01

    A sample of 360 people was analyzed. They had received the protocol of preventive activities of the "Unitat Docent de Medicina Familiar i Comunitària de Barcelona" during 1989. The results of those activities directed to the prevention of cardiovascular risk and to the detection of excessive alcohol intake are reported, as they are those with the easiest and widest implementation. Overall results include hypercholesterolemia in 43.9%, abnormal blood pressure in 10.5%, a rate of smokers of 31.7%, 7.7% with an excessive alcohol intake, and 51.9% of obese individuals. The results are also shown by age and sex groups. In the discussion, the results are compared with those expected for our population. It is concluded that preventive measures should be encouraged by primary health are teams.

  20. Factor IX Amagasaki: A new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

    SciTech Connect

    Miyata, Toshiyuki; Iwanaga, Sadaaki ); Sakai, Toshiyuki; Sugimoto, Mitsuhiko; Naka, Hiroyuki; Yamamoto, Kazukuni; Yoshioka, Akira; Fukui, Hiromu ); Mitsui, Kotoko; Kamiya, Kensyu; Umeyama, Hideaki )

    1991-11-26

    Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. The authors identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate for factor X in the presence of factor VIII, phospholipids, and Ca{sup 2+}, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311 {yields} Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

  1. Structural characterization of native autoinducing peptides and abiotic analogues reveals key features essential for activation and inhibition of an AgrC quorum sensing receptor in Staphylococcus aureus.

    PubMed

    Tal-Gan, Yftah; Ivancic, Monika; Cornilescu, Gabriel; Cornilescu, Claudia C; Blackwell, Helen E

    2013-12-11

    Staphylococcus aureus is a major human pathogen that uses quorum sensing (QS) to control virulence. Its QS system is regulated by macrocyclic peptide signals (or autoinducing peptides (AIPs)) and their cognate transmembrane receptors (AgrCs). Four different specificity groups of S. aureus have been identified to date (groups I-IV), each of which uses a different AIP:AgrC pair. Non-native ligands capable of intercepting AIP:AgrC binding, and thereby QS, in S. aureus have attracted considerable interest as chemical tools to study QS pathways and as possible antivirulence strategies for the treatment of infection. We recently reported a set of analogues of the group-III AIP that are capable of strongly modulating the activity of all four AgrC receptors. Critical to the further development of such ligands is a detailed understanding of the structural features of both native AIPs and non-native analogues that are essential for activity. Herein, we report the first three-dimensional structural analysis of the known native AIP signals (AIPs-I-IV) and several AIP-III analogues with varied biological activities using NMR spectroscopy. Integration of these NMR studies with the known agonism and antagonism profiles of these peptides in AgrC-III revealed two key structural elements that control AIP-III (and non-native peptide) activity: (1) a tri-residue hydrophobic "knob" essential for both activation and inhibition and (2) a fourth anchor point on the exocyclic tail needed for receptor activation. These results provide strong structural support for a mechanism of AIP-mediated AgrC activation and inhibition in S. aureus , and should facilitate the design of new AgrC ligands with enhanced activities (as agonists or antagonists) and simplified chemical structures.

  2. "Exercise Dependence"--A Problem or Natural Result of High Activity?

    ERIC Educational Resources Information Center

    Phelan, Suzanne; Bond, Dale S.; Lang, Wei; Jordan, Dustin; Wing, Rena R.

    2011-01-01

    Objectives: To compare physical activity (PA) and exercise dependence (ED) in 267 weight-loss maintainers (WLM) and 213 normal-weight (NW) controls. Methods: PA and ED assessed via accelerometery and the Exercise Dependence Questionnaire. Results: WLM had higher PA levels and ED scores than those of NW (P less than 0.0001). WLM status (P = 0.006)…

  3. Dynamic Docking Test System (DDTS) active table frequency response test results. [Apollo Soyuz Test Project

    NASA Technical Reports Server (NTRS)

    Gates, R. M.

    1974-01-01

    Results are presented of the frequency response test performed on the dynamic docking test system (DDTS) active table. Sinusoidal displacement commands were applied to the table and the dynamic response determined from measured actuator responses and accelerometers mounted to the table and one actuator.

  4. Social Work Roles and Activities Regarding Psychiatric Medication: Results of a National Survey

    ERIC Educational Resources Information Center

    Bentley, Kia J.; Walsh, Joseph; Farmer, Rosemary L.

    2005-01-01

    This article reports the findings of a 2001 national survey of social workers regarding their everyday practice roles and activities regarding psychiatric medication. The results of this quantitative study indicate variability in the types of roles carried out by social workers with regard to psychiatric medication, but that perceptions of…

  5. Half-width plots, a simple tool to predict peak shape, reveal column kinetics and characterise chromatographic columns in liquid chromatography: state of the art and new results.

    PubMed

    Baeza-Baeza, J J; Ruiz-Ángel, M J; García-Álvarez-Coque, M C; Carda-Broch, S

    2013-11-01

    Peak profiles in chromatography are characterised by their height, position, width and asymmetry; the two latter depend on the values of the left and right peak half-widths. Simple correlations have been found between the peak half-widths and the retention times. The representation of such correlations has been called half-width plots. For isocratic elution, the plots are parabolic, although often, the parabolas can be approximated to straight-lines. The plots can be obtained with the half-widths/retention time data for a set of solutes experiencing the same kinetics, eluted with a mobile phase at fixed or varying composition. When the analysed solutes experience different resistance to mass transfer, the plots will be solute dependent, and should be obtained with the data for each solute eluted with mobile phases at varying composition. The half-width plots approach is a simple tool that facilitates the prediction of peak shape (width and asymmetry) with optimisation purposes, reveal the interaction kinetics of solutes in different columns, and characterise chromatographic columns. This work shows half-width plots for different situations in isocratic elution, including the use of different flows, the effect of temperature, the modification of the stationary phase surface by an additive, the existence of specific interactions within the column, and the comparison of columns. The adaptation to gradient elution is also described. Previous knowledge on half-width plots is structured and analysed, to which new results are added.

  6. Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

    PubMed

    Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

    2015-09-01

    Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes

  7. Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes

    PubMed Central

    Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

    2015-01-01

    Abstract The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling. Key points Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The

  8. Parallel Functional Activity Profiling Reveals Valvulopathogens Are Potent 5-Hydroxytryptamine2B Receptor Agonists: Implications for Drug Safety Assessment

    PubMed Central

    Huang, Xi-Ping; Setola, Vincent; Yadav, Prem N.; Allen, John A.; Rogan, Sarah C.; Hanson, Bonnie J.; Revankar, Chetana; Robers, Matt; Doucette, Chris

    2009-01-01

    Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine2B (5-HT2B) receptor agonists. We have shown that activation of 5-HT2B receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT2B receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT2B receptor agonists (hits); 14 of these had previously been identified as 5-HT2B receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then “functionally profiled” (i.e., assayed in parallel for 5-HT2B receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC50 data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT2B receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease. PMID:19570945

  9. Perceptual distortions in pitch and time reveal active prediction and support for an auditory pitch-motion hypothesis.

    PubMed

    Henry, Molly J; McAuley, J Devin

    2013-01-01

    A number of accounts of human auditory perception assume that listeners use prior stimulus context to generate predictions about future stimulation. Here, we tested an auditory pitch-motion hypothesis that was developed from this perspective. Listeners judged either the time change (i.e., duration) or pitch change of a comparison frequency glide relative to a standard (referent) glide. Under a constant-velocity assumption, listeners were hypothesized to use the pi