The Circadian Clock in Cancer Development and Therapy

PubMed Central

Fu, Loning; Kettner, Nicole M.

2014-01-01

Most aspects of mammalian function display circadian rhythms driven by an endogenous clock. The circadian clock is operated by genes and comprises a central clock in the brain that responds to environmental cues and controls subordinate clocks in peripheral tissues via circadian output pathways. The central and peripheral clocks coordinately generate rhythmic gene expression in a tissue-specific manner in vivo to couple diverse physiological and behavioral processes to periodic changes in the environment. However, as the world industrialized, activities that disrupt endogenous homeostasis with external circadian cues have increased. This change in lifestyle has been linked to increased risk of diseases in all aspects of human health, including cancer. Studies in humans and animal models have revealed that cancer development in vivo is closely associated with the loss of circadian homeostasis in energy balance, immune function and aging that are supported by cellular functions important for tumor suppression including cell proliferation, senescence, metabolism and DNA damage response. The clock controls these cellular functions both locally in cells of peripheral tissues and at the organismal level via extracellular signaling. Thus, the hierarchical mammalian circadian clock provides a unique system to study carcinogenesis as a deregulated physiological process in vivo. The asynchrony between host and malignant tissues in cell proliferation and metabolism also provides new and exciting options for novel anti-cancer therapies. PMID:23899600

CLHM-1 is a functionally conserved and conditionally toxic Ca2+-permeable ion channel in Caenorhabditis elegans.

PubMed

Tanis, Jessica E; Ma, Zhongming; Krajacic, Predrag; He, Liping; Foskett, J Kevin; Lamitina, Todd

2013-07-24

Disruption of neuronal Ca(2+) homeostasis contributes to neurodegenerative diseases through mechanisms that are not fully understood. A polymorphism in CALHM1, a recently described ion channel that regulates intracellular Ca(2+) levels, is a possible risk factor for late-onset Alzheimer's disease. Since there are six potentially redundant CALHM family members in humans, the physiological and pathophysiological consequences of CALHM1 function in vivo remain unclear. The nematode Caenorhabditis elegans expresses a single CALHM1 homolog, CLHM-1. Here we find that CLHM-1 is expressed at the plasma membrane of sensory neurons and muscles. Like human CALHM1, C. elegans CLHM-1 is a Ca(2+)-permeable ion channel regulated by voltage and extracellular Ca(2+). Loss of clhm-1 in the body-wall muscles disrupts locomotory kinematics and biomechanics, demonstrating that CLHM-1 has a physiologically significant role in vivo. The motility defects observed in clhm-1 mutant animals can be rescued by muscle-specific expression of either C. elegans CLHM-1 or human CALHM1, suggesting that the function of these proteins is conserved in vivo. Overexpression of either C. elegans CLHM-1 or human CALHM1 in neurons is toxic, causing degeneration through a necrotic-like mechanism that is partially Ca(2+) dependent. Our data show that CLHM-1 is a functionally conserved ion channel that plays an important but potentially toxic role in excitable cell function.

Free-radical probes for functional in vivo EPR imaging

NASA Astrophysics Data System (ADS)

Subramanian, S.; Krishna, M. C.

2007-02-01

Electron paramagnetic resonance imaging (EPRI) is one of the recent functional imaging modalities that can provide valuable in vivo physiological information on its own merit and aids as a complimentary imaging technique to MRI and PET of tissues especially with respect to in vivo pO II (oxygen partial pressure), redox status and pharmacology. EPR imaging mainly deals with the measurement of distribution and in vivo dynamics and redox changes using special nontoxic paramagnetic spin probes that can be infused into the object of investigation. These spin probes should be characterized by simple EPR spectra, preferably with narrow EPR lines. The line width should be reversibly sensitive to the concentration of in vivo pO II with a linear dependence. Several non-toxic paramagnetic probes, some particulate and insoluble and others water-soluble and infusible (by intravenous or intramuscular injection) have been developed which can be effectively used to quantitatively assess tissue redox status, and tumor hypoxia. Quantitative assessment of the redox status of tissue in vivo is important in investigating oxidative stress, and that of tissue pO II is very important in radiation oncology. Other areas in which EPR imaging and oxymetry may help are in the investigation of tumorangiogenesis, wound healing, oxygenation of tumor tissue by the ingestion of oxygen-rich gases, etc. The correct choice of the spin probe will depend on the modality of measurement (whether by CW or time-domain EPR imaging) and the particular physiology interrogated. Examples of the available spin probes and some EPR imaging applications employing them are presented.

In vivo physiological recording from the lateral line of juvenile zebrafish

PubMed Central

Olt, Jennifer; Allen, Claire E.

2016-01-01

Key points Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells.We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish.The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills.The same approach could be used for in vivo functional studies in other sensory and non‐sensory systems from juvenile and adult zebrafish. Abstract Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l−1 was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l−1 did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K+ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish. PMID:27161862

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo

PubMed Central

Juhas, Mark; Engelmayr, George C.; Fontanella, Andrew N.; Palmer, Gregory M.; Bursac, Nenad

2014-01-01

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration. PMID:24706792

In Vitro Maturation and In Vivo Integration and Function of an Engineered Cell-Seeded Disc-like Angle Ply Structure (DAPS) for Total Disc Arthroplasty.

PubMed

Martin, J T; Gullbrand, S E; Kim, D H; Ikuta, K; Pfeifer, C G; Ashinsky, B G; Smith, L J; Elliott, D M; Smith, H E; Mauck, R L

2017-11-17

Total disc replacement with an engineered substitute is a promising avenue for treating advanced intervertebral disc disease. Toward this goal, we developed cell-seeded disc-like angle ply structures (DAPS) and showed through in vitro studies that these constructs mature to match native disc composition, structure, and function with long-term culture. We then evaluated DAPS performance in an in vivo rat model of total disc replacement; over 5 weeks in vivo, DAPS maintained their structure, prevented intervertebral bony fusion, and matched native disc mechanical function at physiologic loads in situ. However, DAPS rapidly lost proteoglycan post-implantation and did not integrate into adjacent vertebrae. To address this, we modified the design to include polymer endplates to interface the DAPS with adjacent vertebrae, and showed that this modification mitigated in vivo proteoglycan loss while maintaining mechanical function and promoting integration. Together, these data demonstrate that cell-seeded engineered discs can replicate many characteristics of the native disc and are a viable option for total disc arthroplasty.

What can imaging tell us about physiology? Lung growth and regional mechanical strain.

PubMed

Hsia, Connie C W; Tawhai, Merryn H

2012-09-01

The interplay of mechanical forces transduces diverse physico-biochemical processes to influence lung morphogenesis, growth, maturation, remodeling and repair. Because tissue stress is difficult to measure in vivo, mechano-sensitive responses are commonly inferred from global changes in lung volume, shape, or compliance and correlated with structural changes in tissue blocks sampled from postmortem-fixed lungs. Recent advances in noninvasive volumetric imaging technology, nonrigid image registration, and deformation analysis provide valuable tools for the quantitative analysis of in vivo regional anatomy and air and tissue-blood distributions and when combined with transpulmonary pressure measurements, allow characterization of regional mechanical function, e.g., displacement, strain, shear, within and among intact lobes, as well as between the lung and the components of its container-rib cage, diaphragm, and mediastinum-thereby yielding new insights into the inter-related metrics of mechanical stress-strain and growth/remodeling. Here, we review the state-of-the-art imaging applications for mapping asymmetric heterogeneous physical interactions within the thorax and how these interactions permit as well as constrain lung growth, remodeling, and compensation during development and following pneumonectomy to illustrate how advanced imaging could facilitate the understanding of physiology and pathophysiology. Functional imaging promises to facilitate the formulation of realistic computational models of lung growth that integrate mechano-sensitive events over multiple spatial and temporal scales to accurately describe in vivo physiology and pathophysiology. Improved computational models in turn could enhance our ability to predict regional as well as global responses to experimental and therapeutic interventions.

Multi-organ autonomic dysfunction in Parkinson disease

PubMed Central

2010-01-01

Both pathologic and clinical studies of autonomic pathways have expanded the concept of Parkinson disease (PD) from a movement disorder to a multi-level widespread neurodegenerative process with non-motor features spanning several organ systems. This review integrates neuropathologic findings and autonomic physiology in PD as it relates to end organ autonomic function. Symptoms, pathology and physiology of the cardiovascular, skin/sweat gland, urinary, gastrointestinal, pupillary and neuroendocrine systems can be probed by autopsy, biopsy and non-invasive electrophysiological techniques in vivo which assess autonomic anatomy and function. There is mounting evidence that PD affects a chain of neurons in autonomic pathways. Consequently, autonomic physiology may serve as a window into non-motor PD progression and allow the development of mechanistically based treatment strategies for several non-motor features of PD. End-organ physiologic markers may be used to inform a model of PD pathophysiology and non-motor progression. PMID:20851033

Titin Based Viscosity in Ventricular Physiology: An Integrative Investigation of PEVK-Actin Interactions

PubMed Central

Chung, Charles S; Methawasin, Methajit; Nelson, O Lynne; Radke, Michael H; Hidalgo, Carlos G; Gotthardt, Michael; Granzier, Henk L

2011-01-01

Viscosity is proposed to modulate diastolic function, but only limited understanding of the source(s) of viscosity exists. In-vitro experiments have shown that the proline-glutamic acid-valine-lysine (PEVK) rich element of titin interacts with actin, causing a viscous force in the sarcomere. It is unknown whether this mechanism contributes to viscosity in-vivo. We tested the hypothesis that PEVK-actin interaction causes cardiac viscosity and is important in-vivo via an integrative physiological study on a unique PEVK-knockout (KO) model. Both skinned cardiomyocytes and papillary muscle fibers were isolated from wildtype (WT) and PEVK KO mice and passive viscosity was examined using stretch-hold-release and sinusoidal analysis. Viscosity was reduced by ~60% in KO myocytes and ~50% in muscle fibers at room temperature. The PEVK-actin interaction was not modulated by temperature or diastolic calcium, but was increased by lattice compression. Stretch-hold and sinusoidal frequency protocols on intact isolated mouse hearts showed a smaller, 30–40% reduction in viscosity, possibly due to actomyosin interactions, and showed that microtubules did not contribute to viscosity. Transmitral Doppler echocardiography similarly revealed a 40% decrease in LV chamber viscosity in the PEVK KO in-vivo. This integrative study is the first to quantify the influence of a specific molecular (PEVK-actin) viscosity in-vivo and shows that PEVK-actin interactions are an important physiological source of viscosity. PMID:21708170

Long-term in vivo study of vertebrate embryonic development using noninvasive harmonics optical microscopy

NASA Astrophysics Data System (ADS)

Chen, Szu-Yu; Hsieh, C.-S.; Chu, S.-W.; Lin, Cheng-Yung; Ko, C.-Y.; Chen, Y.-C.; Tsai, Huai-Jen; Hu, C.-H.; Sun, Chi-Kuang

2005-03-01

Harmonics optical microscopy (HOM) provides a truly "noninvasive" tool for in vivo and long-term study of vertebrate embryonic development. Based on the nonlinear natures, it provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power (~1μm axial resolution) without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamages. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can be used to do functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Zebrafish embryos now have been used to study many vertebrate physiological systems. We have demonstrated an in vivo HOM study of developmental dynamics of several embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.

In vivo human cardiac shortening and lengthening velocity is region dependent and not coupled with heart rate: 'longitudinal' strain rate markedly underestimates apical contribution.

PubMed

Stöhr, Eric J; Stembridge, Mike; Esformes, Joseph I

2015-04-20

What is the central question of this study? Regulation of cardiac function is typically achieved by changes in heart rate (HR) and cardiac shortening velocity (strain rate; SR), but their interdependence in vivo remains poorly understood. What is the main finding and its importance? Using resistance exercise to increase heart rate and arterial resistance physiologically in humans and measuring regional cardiac SR (at the base and apex), we found that HR and SR were not strictly coupled because SR at the base and apex responded differently, despite the same HR. Importantly, our data show that the region-averaged 'longitudinal' SR, which is currently popular in the clinical setting, markedly underestimates the contribution of the apex. The fundamental importance of cardiac shortening and lengthening velocity (i.e. strain rate; SR) has been demonstrated in vitro. Currently, the interdependence between in vivo SR and HR is poorly understood because studies have typically assessed region-averaged 'longitudinal' strain rate, which is likely to underestimate the apical contribution, and have used non-physiological interventions that may also have been influenced by multicollinearity caused by concomitant reductions in arterial resistance. Resistance exercise acutely raises HR, blood pressure and arterial resistance and transiently disassociates these cardiovascular factors following exercise. Therefore, we measured SR, HR, blood pressure and arterial resistance in nine healthy men (aged 20 ± 1 years) immediately before, during and after double-leg-press exercise at 30 and 60% of maximal strength. Resistance exercise caused a disproportionate SR response at the left ventricular base and apex (interaction effect, P < 0.05). Consequently, associations between HR and regional peak SR were inconsistent and mostly very weak (r(2)  = 0.0004-0.24). Likewise, the areas under the curve for systolic and diastolic SR and their relationship with systolic and diastolic duration were variable and weak. Importantly, region-averaged 'longitudinal' SR was identical to basal SR, thus, markedly underestimating the apical contribution. In conclusion, in vivo HR and SR are not strictly coupled in healthy humans, which is explained by the region-specific responses of SR that are not captured by 'longitudinal SR'. This novel observation emphasizes the independent role of in vivo SR in overall cardiac function during stress and may cause a 'revival' of SR as a marker of regional left ventricular (dys)function. © 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.

Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy.

PubMed

Terrill, Jessica R; Pinniger, Gavin J; Graves, Jamie A; Grounds, Miranda D; Arthur, Peter G

2016-06-01

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation, oxidative stress and myofibre necrosis. Cysteine precursor antioxidants such as N-acetyl cysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) reduce dystropathology in the mdx mouse model for DMD, and we propose this is via increased synthesis of the amino acid taurine. We compared the capacity of OTC and taurine treatment to increase taurine content of mdx muscle, as well as effects on in vivo and ex vivo muscle function, inflammation and oxidative stress. Both treatments increased taurine in muscles, and improved many aspects of muscle function and reduced inflammation. Taurine treatment also reduced protein thiol oxidation and was overall more effective, as OTC treatment reduced body and muscle weight, suggesting some adverse effects of this drug. These data suggest that increasing dietary taurine is a better candidate for a therapeutic intervention for DMD. Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease for which there is no widely available cure. Whilst the mechanism of loss of muscle function in DMD and the mdx mouse model are not fully understood, disruptions in intracellular calcium homeostasis, inflammation and oxidative stress are implicated. We have shown that protein thiol oxidation is increased in mdx muscle, and that the indirect thiol antioxidant l-2-oxothiazolidine-4-carboxylate (OTC), which increases cysteine availability, decreases pathology and increases in vivo strength. We propose that the protective effects of OTC are a consequence of conversion of cysteine to taurine, which has itself been shown to be beneficial to mdx pathology. This study compares the efficacy of taurine with OTC in decreasing dystropathology in mdx mice by measuring in vivo and ex vivo contractile function and measurements of inflammation and protein thiol oxidation. Increasing the taurine content of mdx muscle improved both in vivo and ex vivo muscle strength and function, potentially via anti-inflammatory and antioxidant effects of taurine. OTC treatment increased taurine synthesis in the liver and taurine content of mdx muscle, improved muscle function and decreased inflammation. However, OTC was less effective than taurine treatment, with OTC also decreasing body and EDL muscle weights, suggesting that OTC had some detrimental effects. These data support continued research into the use of taurine as a therapeutic intervention for DMD, and suggest that increasing dietary taurine is the better strategy for increasing taurine content and decreasing severity of dystropathology. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Rad GTPase is essential for the regulation of bone density and bone marrow adipose tissue in mice.

PubMed

Withers, Catherine N; Brown, Drew M; Byiringiro, Innocent; Allen, Matthew R; Condon, Keith W; Satin, Jonathan; Andres, Douglas A

2017-10-01

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca 2+ channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad -/- calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. Copyright © 2017 Elsevier Inc. All rights reserved.

Sensory neurons that detect stretch and nutrients in the digestive system

PubMed Central

Williams, Erika K.; Chang, Rui B.; Strochlic, David E.; Umans, Benjamin D.; Lowell, Bradford B.; Liberles, Stephen D.

2016-01-01

SUMMARY Neural inputs from internal organs are essential for normal autonomic function. The vagus nerve is a key body-brain connection that monitors the digestive, cardiovascular, and respiratory systems. Within the gastrointestinal tract, vagal sensory neurons detect gut hormones and organ distension. Here, we investigate the molecular diversity of vagal sensory neurons and their roles in sensing gastrointestinal inputs. Genetic approaches allowed targeted investigation of gut-to-brain afferents involved in homeostatic responses to ingested nutrients (GPR65 neurons) and mechanical distension of the stomach and intestine (GLP1R neurons). Optogenetics, in vivo ganglion imaging, and genetically guided anatomical mapping provide direct links between neuron identity, peripheral anatomy, central anatomy, conduction velocity, response properties in vitro and in vivo, and physiological function. These studies clarify the roles of vagal afferents in mediating particular gut hormone responses. Moreover, genetic control over gut-to-brain neurons provides a molecular framework for understanding neural control of gastrointestinal physiology. PMID:27238020

Functionalized graphene oxide-based thermosensitive hydrogel for near-infrared chemo-photothermal therapy on tumor.

PubMed

Zhu, Xiali; Zhang, Yingjie; Huang, Heqing; Zhang, Huijuan; Hou, Lin; Zhang, Zhenzhong

2016-03-01

A functionalized graphene oxide-based thermosensitive hydrogel loaded with docetaxel for intratumoral delivery was designed to enhance therapeutic efficacy and alleviate system toxicity. First, graphene oxide was functionalized with chitosan to acquire high stability in physiological solutions. And then docetaxel-graphene oxide/chitosan gel was formed by mixed docetaxel-graphene oxide/chitosan suspension with hydrogel which was made from Poloxamer 407 and Poloxamer 188. Cellular uptake, antitumor effect in vitro and in vivo, cell apoptosis, and biodistribution of docetaxel-graphene oxide/chitosan gel were investigated, compared with the docetaxel solution. Graphene oxide/chitosan was stable in physiological solution, and docetaxel released much slower from docetaxel-graphene oxide/chitosan gel with a pH-responsive feature. Compared with free docetaxel, docetaxel-graphene oxide/chitosan could afford higher antitumor efficacy in Michigan Cancer Foundation-7 (MCF-7) cells in vitro. Furthermore, docetaxel-grapheme oxide/chitosan gel which was injected within tumor could afford higher concentration and longer resident time in tumor tissues of mice in vivo, without obvious toxic effects to normal organs. Meanwhile, the combination of near-infrared laser irradiation at 808 nm significantly enhanced tumor inhibition in vitro and in vivo. Docetaxel-graphene oxide/chitosan gel in combination with 808 nm near-infrared laser irradiation had great potential for cancer chemo-photothermal therapy. © The Author(s) 2016.

Vortex Formation Time is Not an Index of Ventricular Function

PubMed Central

Vlachos, Pavlos P.; Little, William C.

2015-01-01

The diastolic intraventricular ring vortex formation and pinch-off process may provide clinically useful insights into diastolic function in health and disease. The vortex ring formation time (FT) concept, based on hydrodynamic experiments dealing with unconfined (large tank) flow, has attracted considerable attention and popularity. Dynamic conditions evolving within the very confined space of a filling, expansible ventricular chamber with relaxing and rebounding viscoelastic muscular boundaries, diverge from unconfined (large tank) flow and encompass rebounding walls’ suction and myocardial relaxation. Indeed, clinical/physiological findings seeking validation in vivo failed to support the notion that FT is an index of normal/abnormal diastolic ventricular function. Therefore, FT as originally proposed cannot and should not be utilized as such an index. Evidently, physiologically accurate models accounting for coupled hydrodynamic and (patho)physiological myocardial wall interactions with the intraventricular flow are still needed to enhance our understanding and yield diastolic function indices useful and reliable in the clinical setting. PMID:25609509

Regulation of receptor-type protein tyrosine phosphatases by their C-terminal tail domains.

PubMed

Barnea, Maayan; Olender, Tsviya; Bedford, Mark T; Elson, Ari

2016-10-15

Protein tyrosine phosphatases (PTPs) perform specific functions in vivo, despite being vastly outnumbered by their substrates. Because of this and due to the central roles PTPs play in regulating cellular function, PTP activity is regulated by a large variety of molecular mechanisms. We review evidence that indicates that the divergent C-terminal tail sequences (C-terminal domains, CTDs) of receptor-type PTPs (RPTPs) help regulate RPTP function by controlling intermolecular associations in a way that is itself subject to physiological regulation. We propose that the CTD of each RPTP defines an 'interaction code' that helps determine molecules it will interact with under various physiological conditions, thus helping to regulate and diversify PTP function. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Multimodal optical imaging system for in vivo investigation of cerebral oxygen delivery and energy metabolism

PubMed Central

Yaseen, Mohammad A.; Srinivasan, Vivek J.; Gorczynska, Iwona; Fujimoto, James G.; Boas, David A.; Sakadžić, Sava

2015-01-01

Improving our understanding of brain function requires novel tools to observe multiple physiological parameters with high resolution in vivo. We have developed a multimodal imaging system for investigating multiple facets of cerebral blood flow and metabolism in small animals. The system was custom designed and features multiple optical imaging capabilities, including 2-photon and confocal lifetime microscopy, optical coherence tomography, laser speckle imaging, and optical intrinsic signal imaging. Here, we provide details of the system’s design and present in vivo observations of multiple metrics of cerebral oxygen delivery and energy metabolism, including oxygen partial pressure, microvascular blood flow, and NADH autofluorescence. PMID:26713212

Novel ex vivo culture method for human monocytes uses shear flow to prevent total loss of transendothelial diapedesis function.

PubMed

Tsubota, Yoshiaki; Frey, Jeremy M; Raines, Elaine W

2014-01-01

Monocyte recruitment to inflammatory sites and their transendothelial migration into tissues are critical to homeostasis and pathogenesis of chronic inflammatory diseases. However, even short-term suspension culture of primary human monocytes leads to phenotypic changes. In this study, we characterize the functional effects of ex vivo monocyte culture on the steps involved in monocyte transendothelial migration. Our data demonstrate that monocyte diapedesis is impaired by as little as 4 h culture, and the locomotion step is subsequently compromised. After 16 h in culture, monocyte diapedesis is irreversibly reduced by ∼90%. However, maintenance of monocytes under conditions mimicking physiological flow (5-7.5 dyn/cm²) is sufficient to reduce diapedesis impairment significantly. Thus, through the application of shear during ex vivo culture of monocytes, our study establishes a novel protocol, allowing functional analyses of monocytes not currently possible under static culture conditions. These data further suggest that monocyte-based therapeutic applications may be measurably improved by alteration of ex vivo conditions before their use in patients.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo.

PubMed

Stenzel, Denise; Franco, Claudio A; Estrach, Soline; Mettouchi, Amel; Sauvaget, Dominique; Rosewell, Ian; Schertel, Andreas; Armer, Hannah; Domogatskaya, Anna; Rodin, Sergey; Tryggvason, Karl; Collinson, Lucy; Sorokin, Lydia; Gerhardt, Holger

2011-10-28

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

PubMed Central

Stenzel, Denise; Franco, Claudio A; Estrach, Soline; Mettouchi, Amel; Sauvaget, Dominique; Rosewell, Ian; Schertel, Andreas; Armer, Hannah; Domogatskaya, Anna; Rodin, Sergey; Tryggvason, Karl; Collinson, Lucy; Sorokin, Lydia; Gerhardt, Holger

2011-01-01

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo. PMID:21979816

What can imaging tell us about physiology? Lung growth and regional mechanical strain

PubMed Central

Tawhai, Merryn H.

2012-01-01

The interplay of mechanical forces transduces diverse physico-biochemical processes to influence lung morphogenesis, growth, maturation, remodeling and repair. Because tissue stress is difficult to measure in vivo, mechano-sensitive responses are commonly inferred from global changes in lung volume, shape, or compliance and correlated with structural changes in tissue blocks sampled from postmortem-fixed lungs. Recent advances in noninvasive volumetric imaging technology, nonrigid image registration, and deformation analysis provide valuable tools for the quantitative analysis of in vivo regional anatomy and air and tissue-blood distributions and when combined with transpulmonary pressure measurements, allow characterization of regional mechanical function, e.g., displacement, strain, shear, within and among intact lobes, as well as between the lung and the components of its container—rib cage, diaphragm, and mediastinum—thereby yielding new insights into the inter-related metrics of mechanical stress-strain and growth/remodeling. Here, we review the state-of-the-art imaging applications for mapping asymmetric heterogeneous physical interactions within the thorax and how these interactions permit as well as constrain lung growth, remodeling, and compensation during development and following pneumonectomy to illustrate how advanced imaging could facilitate the understanding of physiology and pathophysiology. Functional imaging promises to facilitate the formulation of realistic computational models of lung growth that integrate mechano-sensitive events over multiple spatial and temporal scales to accurately describe in vivo physiology and pathophysiology. Improved computational models in turn could enhance our ability to predict regional as well as global responses to experimental and therapeutic interventions. PMID:22582216

Correlative Ultratructural Investigations of Airway Epithelium Following Experimental Exposure to Defined Air Pollutants and Lifestyle Exposure to Tobacco Smoke

EPA Science Inventory

Context: Investigations of cell/molecular level effects of in vivo exposure of airway mucosa of experimental animals to common irritant gases have demonstrated structural and physiological changes reflective of breaches in epithelial barrier function, presence of inflammatory cel...

Dopamine receptors – IUPHAR Review 13

PubMed Central

Beaulieu, Jean-Martin; Espinoza, Stefano; Gainetdinov, Raul R

2015-01-01

The variety of physiological functions controlled by dopamine in the brain and periphery is mediated by the D1, D2, D3, D4 and D5 dopamine GPCRs. Drugs acting on dopamine receptors are significant tools for the management of several neuropsychiatric disorders including schizophrenia, bipolar disorder, depression and Parkinson's disease. Recent investigations of dopamine receptor signalling have shown that dopamine receptors, apart from their canonical action on cAMP-mediated signalling, can regulate a myriad of cellular responses to fine-tune the expression of dopamine-associated behaviours and functions. Such signalling mechanisms may involve alternate G protein coupling or non-G protein mechanisms involving ion channels, receptor tyrosine kinases or proteins such as β-arrestins that are classically involved in GPCR desensitization. Another level of complexity is the growing appreciation of the physiological roles played by dopamine receptor heteromers. Applications of new in vivo techniques have significantly furthered the understanding of the physiological functions played by dopamine receptors. Here we provide an update of the current knowledge regarding the complex biology, signalling, physiology and pharmacology of dopamine receptors. PMID:25671228

Long-term in vivo harmonics imaging of zebrafish embryonic development based on a femtosecond Cr:forsterite laser

NASA Astrophysics Data System (ADS)

Chen, S.-Y.; Tsai, T.-H.; Hsieh, C.-S.; Tai, S.-P.; Lin, C.-Y.; Ko, C.-Y.; Chen, Y.-C.; Tsai, H.-J.; Hu, C.-H.; Sun, C.-K.

2005-03-01

Based on a femtosecond Cr:forsterite laser, harmonics optical microscopy (HOM) provides a truly "noninvasive" tool for in vivo and long-term study of vertebrate embryonic development. Based on optical nonlinearity, HOM provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamage. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can perform functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Here we demonstrate in vivo HOM studies of developmental dynamics of several important embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.

MSX2 in ameloblast cell fate and activity

PubMed Central

Babajko, Sylvie; de La Dure-Molla, Muriel; Jedeon, Katia; Berdal, Ariane

2015-01-01

While many effectors have been identified in enamel matrix and cells via genetic studies, physiological networks underlying their expression levels and thus the natural spectrum of enamel thickness and degree of mineralization are now just emerging. Several transcription factors are candidates for enamel gene expression regulation and thus the control of enamel quality. Some of these factors, such as MSX2, are mainly confined to the dental epithelium. MSX2 homeoprotein controls several stages of the ameloblast life cycle. This chapter introduces MSX2 and its target genes in the ameloblast and provides an overview of knowledge regarding its effects in vivo in transgenic mouse models. Currently available in vitro data on the role of MSX2 as a transcription factor and its links to other players in ameloblast gene regulation are considered. MSX2 modulations are relevant to the interplay between developmental, hormonal and environmental pathways and in vivo investigations, notably in the rodent incisor, have provided insight into dental physiology. Indeed, in vivo models are particularly promising for investigating enamel formation and MSX2 function in ameloblast cell fate. MSX2 may be central to the temporal-spatial restriction of enamel protein production by the dental epithelium and thus regulation of enamel quality (thickness and mineralization level) under physiological and pathological conditions. Studies on MSX2 show that amelogenesis is not an isolated process but is part of the more general physiology of coordinated dental-bone complex growth. PMID:25601840

The physiology of endothelial xanthine oxidase: from urate catabolism to reperfusion injury to inflammatory signal transduction.

PubMed

Meneshian, Avedis; Bulkley, Gregory B

2002-07-01

Xanthine oxidoreductase (XOR) is a ubiquitous metalloflavoprotein that appears in two interconvertible yet functionally distinct forms: xanthine dehydrogenase (XD), which is constitutively expressed in vivo; and xanthine oxidase (XO), which is generated by the posttranslational modification of XD, either through the reversible, incremental thiol oxidation of sulfhydryl residues on XD or the irreversible proteolytic cleavage of a segment of XD, which occurs at low oxygen tension and in the presence of several proinflammatory mediators. Functionally, both XD and XO catalyze the oxidation of purines to urate. However, whereas XD requires NAD+ as an electron acceptor for these redox reactions, thereby generating the stable product NADH, XO is unable to use NAD+ as an electron acceptor, requiring instead the reduction of molecular oxygen for this purine oxidation and generating the highly reactive superoxide free radical. Nearly 100 years of study has documented the physiologic role of XD in urate catabolism. However, the rapid, posttranslational conversion of XD to the oxidant-generating form XO provides a possible physiologic mechanism for rapid, posttranslational, oxidant-mediated signaling. XO-generated reactive oxygen species (ROS) have been implicated in various clinicopathologic entities, including ischemia/reperfusion injury and multisystem organ failure. More recently, the concept of physiologic signal transduction mediated by ROS has been proposed, and the possibility of XD to XO conversion, with subsequent ROS generation, serving as the trigger of the microvascular inflammatory response in vivo has been hypothesized. This review presents the evidence and basis for this hypothesis.

Emulating Host-Microbiome Ecosystem of Human Gastrointestinal Tract in Vitro.

PubMed

Park, Gun-Seok; Park, Min Hee; Shin, Woojung; Zhao, Connie; Sheikh, Sameer; Oh, So Jung; Kim, Hyun Jung

2017-06-01

The human gut microbiome performs prodigious physiological functions such as production of microbial metabolites, modulation of nutrient digestion and drug metabolism, control of immune system, and prevention of infection. Paradoxically, gut microbiome can also negatively orchestrate the host responses in diseases or chronic disorders, suggesting that the regulated and balanced host-gut microbiome crosstalk is a salient prerequisite in gastrointestinal physiology. To understand the pathophysiological role of host-microbiome crosstalk, it is critical to recreate in vivo relevant models of the host-gut microbiome ecosystem in human. However, controlling the multi-species microbial communities and their uncontrolled growth has remained a notable technical challenge. Furthermore, conventional two-dimensional (2D) or 3D culture systems do not recapitulate multicellular microarchitectures, mechanical dynamics, and tissue-specific functions. Here, we review recent advances and current pitfalls of in vitro and ex vivo models that display human GI functions. We also discuss how the disruptive technologies such as 3D organoids or a human organ-on-a-chip microphysiological system can contribute to better emulate host-gut microbiome crosstalks in health and disease. Finally, the medical and pharmaceutical significance of the gut microbiome-based personalized interventions is underlined as a future perspective.

REGENERATIVE MEDICINE AS APPLIED TO GENERAL SURGERY

PubMed Central

Orlando, Giuseppe; Wood, Kathryn J; De Coppi, Paolo; Baptista, Pedro M; Binder, Kyle W; Bitar, Khalil N; Breuer, Christopher; Burnett, Luke; Christ, George; Farney, Alan; Figliuzzi, Marina; Holmes, James H; Koch, Kenneth; Macchiarini, Paolo; Sani, Sayed-Hadi Mirmalek; Opara, Emmanuel; Remuzzi, Andrea; Rogers, Jeffrey; Saul, Justin M; Seliktar, Dror; Shapira-Schweitzer, Keren; Smith, Tom; Solomon, Daniel; Van Dyke, Mark; Yoo, James J; Zhang, Yuanyuan; Atala, Anthony; Stratta, Robert J; Soker, Shay

2012-01-01

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues or organs in order to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate. PMID:22330032

Intravital imaging of cardiac function at the single-cell level.

PubMed

Aguirre, Aaron D; Vinegoni, Claudio; Sebas, Matt; Weissleder, Ralph

2014-08-05

Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

PubMed

Zhdanov, A V; Golubeva, A V; Okkelman, I A; Cryan, J F; Papkovsky, D B

2015-10-01

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-μm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 μM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 μM across the cell, or ∼0.6 μM/μm. Deeply deoxygenated (5-15 μM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues. Copyright © 2015 the American Physiological Society.

A protocol to study ex vivo mouse working heart at human-like heart rate.

PubMed

Feng, Han-Zhong; Jin, Jian-Ping

2018-01-01

Genetically modified mice are widely used as experimental models to study human heart function and diseases. However, the fast rate of normal mouse heart at 400-600bpm limits its capacity of assessing kinetic parameters that are important for the physiology and pathophysiology of human heart that beats at a much slower rate (75-180bpm). To extend the value of mouse models, we established a protocol to study ex vivo mouse working hearts at a human-like heart rate. In the presence of 300μM lidocaine to lower pacemaker and conductive activities and prevent arrhythmia, a stable rate of 120-130bpm at 37°C is achieved for ex vivo mouse working hearts. The negative effects of decreased heart rate on force-frequency dependence and lidocaine as a myocardial depressant on intracellular calcium can be compensated by using a higher but still physiological level of calcium (2.75mM) in the perfusion media. Multiple parameters were studied to compare the function at the human-like heart rate with that of ex vivo mouse working hearts at the standard rate of 480bpm. The results showed that the conditions for slower heart rate in the presence of 300μM lidocaine did not have depressing effect on left ventricular pressure development, systolic and diastolic velocities and stroke volume with maintained positive inotropic and lusitropic responses to β-adrenergic stimulation. Compared with that at 480bpm, the human-like heart rate increased ventricular filling and end diastolic volume with enhanced Frank-Starling responses. Coronary perfusion was increased from longer relaxation time and interval between beats whereas cardiac efficiency was significantly improved. Although the intrinsic differences between mouse and human heart remain, this methodology for ex vivo mouse hearts to work at human-like heart rate extends the value of using genetically modified mouse models to study cardiac function and human heart diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression of Translationally Controlled Tumor Protein in Human Kidney and in Renal Cell Carcinoma.

PubMed

Ambrosio, Maria R; Rocca, Bruno J; Barone, Aurora; Onorati, Monica; Mundo, Lucia; Crivelli, Filippo; Di Nuovo, Franca; De Falco, Giulia; del Vecchio, Maria T; Tripodi, Sergio A; Tosi, Piero

2015-01-01

Translationally controlled tumor protein is a multifaceted protein involved in several physiological and biological functions. Its expression in normal kidney and in renal carcinomas, once corroborated by functional data, may add elements to elucidate renal physiology and carcinogenesis. In this study, translationally controlled tumor protein expression was evaluated by quantitative real time polymerase chain reaction and western blotting, and its localization was examined by immunohistochemistry on 84 nephrectomies for cancer. In normal kidney protein expression was found in the cytoplasm of proximal and distal tubular cells, in cells of the thick segment of the loop of Henle, and in urothelial cells of the pelvis. It was also detectable in cells of renal carcinoma with different pattern of localization (membranous and cytoplasmic) depending on tumor histotype. Our data may suggest an involvement of translationally controlled tumor protein in normal physiology and carcinogenesis. However, functional in vitro and in vivo studies are needed to verify this hypothesis.

Expression of Translationally Controlled Tumor Protein in Human Kidney and in Renal Cell Carcinoma

PubMed Central

Ambrosio, Maria R.; Rocca, Bruno J.; Barone, Aurora; Onorati, Monica; Mundo, Lucia; Crivelli, Filippo; Di Nuovo, Franca; De Falco, Giulia; del Vecchio, Maria T.; Tripodi, Sergio A.; Tosi, Piero

2015-01-01

Translationally controlled tumor protein is a multifaceted protein involved in several physiological and biological functions. Its expression in normal kidney and in renal carcinomas, once corroborated by functional data, may add elements to elucidate renal physiology and carcinogenesis. In this study, translationally controlled tumor protein expression was evaluated by quantitative real time polymerase chain reaction and western blotting, and its localization was examined by immunohistochemistry on 84 nephrectomies for cancer. In normal kidney protein expression was found in the cytoplasm of proximal and distal tubular cells, in cells of the thick segment of the loop of Henle, and in urothelial cells of the pelvis. It was also detectable in cells of renal carcinoma with different pattern of localization (membranous and cytoplasmic) depending on tumor histotype. Our data may suggest an involvement of translationally controlled tumor protein in normal physiology and carcinogenesis. However, functional in vitro and in vivo studies are needed to verify this hypothesis. PMID:26425551

Cyclic reconstruction of 4D retinal blood flow with pulse synchronization

NASA Astrophysics Data System (ADS)

Schmoll, Tilman; Lasser, Theo; Leitgeb, Rainer A.

2009-02-01

Doppler OCT systems allow nowadays to visualize quantitative and qualitative angiographic maps of retinal tissue. We equipped the instrument with a pulse oximeter and recorded the pulse synchronously with the resonant Doppler flow data. Recombination of tomograms according to the heart beat cycles yields full volumes for each cycle instant. We believe such multi-dimensional functional information and the ability to monitor dynamic processes over time to open exciting perspectives that ultimately contribute to a better understanding of retinal physiology and patho-physiology in-vivo.

Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro.

PubMed

Bardy, Cedric; van den Hurk, Mark; Eames, Tameji; Marchand, Cynthia; Hernandez, Ruben V; Kellogg, Mariko; Gorris, Mark; Galet, Ben; Palomares, Vanessa; Brown, Joshua; Bang, Anne G; Mertens, Jerome; Böhnke, Lena; Boyer, Leah; Simon, Suzanne; Gage, Fred H

2015-05-19

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.

Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

PubMed Central

Bardy, Cedric; van den Hurk, Mark; Eames, Tameji; Marchand, Cynthia; Hernandez, Ruben V.; Kellogg, Mariko; Gorris, Mark; Galet, Ben; Palomares, Vanessa; Brown, Joshua; Bang, Anne G.; Mertens, Jerome; Böhnke, Lena; Boyer, Leah; Simon, Suzanne; Gage, Fred H.

2015-01-01

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro. PMID:25870293

Evidence for biochemical and physiological differences between enzyme genotypes in Drosophila melanogaster.

PubMed Central

Cavener, D R; Clegg, M T

1981-01-01

The in vivo flux of carbon through the pentose shunt is investigated as a function of different 6-phosphogluconate dehydrogenase (6Pgd) and glucose-6-phosphate dehydrogenase (G6pd) genotypes by using differential radioactive labeling of the C-1 and C-6 positions of glucose. Alternative 6Pgd-G6pd genotypes are shown to differ in relative in vivo carbon flux through the pentose shunt. The relative in vitro specific activity differences between the 6PgdSS and 6PgdFF genotypes appear to be primarily responsible for these differences. In addition, the pentose-shunt activity is correlated with the rate of lipid synthesis. This correlation is consistent with the major metabolic function of the pathway, which is to produce NADPH for lipid synthesis. Taken together, the results of these experiments show that different genotypes of 6Pgd are associated with measurable biochemical and physiological differences. Higher order phenotypic differences of this kind must be demonstrated to support the hypothesis that natural selection can discriminate among allozymes of a given genetic locus. PMID:6794031

Imaging light responses of foveal ganglion cells in the living macaque eye.

PubMed

Yin, Lu; Masella, Benjamin; Dalkara, Deniz; Zhang, Jie; Flannery, John G; Schaffer, David V; Williams, David R; Merigan, William H

2014-05-07

The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses.

A conserved tryptophan within the WRDPLVDID domain of yeast Pah1 phosphatidate phosphatase is required for its in vivo function in lipid metabolism.

PubMed

Park, Yeonhee; Han, Gil-Soo; Carman, George M

2017-12-01

PAH1 -encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions ( e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Physiological Environment Induces Quick Response – Slow Exhaustion Reactions

PubMed Central

Hiroi, Noriko; Lu, James; Iba, Keisuke; Tabira, Akito; Yamashita, Shuji; Okada, Yasunori; Flamm, Christoph; Oka, Kotaro; Köhler, Gottfried; Funahashi, Akira

2011-01-01

In vivo environments are highly crowded and inhomogeneous, which may affect reaction processes in cells. In this study we examined the effects of intracellular crowding and an inhomogeneity on the behavior of in vivo reactions by calculating the spectral dimension (ds), which can be translated into the reaction rate function. We compared estimates of anomaly parameters obtained from fluorescence correlation spectroscopy (FCS) data with fractal dimensions derived from transmission electron microscopy (TEM) image analysis. FCS analysis indicated that the anomalous property was linked to physiological structure. Subsequent TEM analysis provided an in vivo illustration; soluble molecules likely percolate between intracellular clusters, which are constructed in a self-organizing manner. We estimated a cytoplasmic spectral dimension ds to be 1.39 ± 0.084. This result suggests that in vivo reactions initially run faster than the same reactions in a homogeneous space; this conclusion is consistent with the anomalous character indicated by FCS analysis. We further showed that these results were compatible with our Monte-Carlo simulation in which the anomalous behavior of mobile molecules correlates with the intracellular environment, leading to description as a percolation cluster, as demonstrated using TEM analysis. We confirmed by the simulation that the above-mentioned in vivo like properties are different from those of homogeneously concentrated environments. Additionally, simulation results indicated that crowding level of an environment might affect diffusion rate of reactant. Such knowledge of the spatial information enables us to construct realistic models for in vivo diffusion and reaction systems. PMID:21960972

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip.

PubMed

Dauth, Stephanie; Maoz, Ben M; Sheehy, Sean P; Hemphill, Matthew A; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M; Budnik, Bogdan; Parker, Kevin Kit

2017-03-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. Copyright © 2017 the American Physiological Society.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.

PubMed

Hartwell, Kimberly A; Miller, Peter G; Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2013-12-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

PubMed Central

Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2014-01-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMGCoA reductase. These results illustrate the power of merging physiologically-relevant models with high-throughput screening. PMID:24161946

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc{sub 1} function in vivo

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

Ekiert, Robert; Czapla, Monika; Sarewicz, Marcin

2014-08-22

Highlights: • We used hybrid fusion bc{sub 1} complex to test inter-monomer electron transfer in vivo. • Cross-inactivated complexes were able to sustain photoheterotrophic growth. • Inter-monomer electron transfer supports catalytic cycle in vivo. • bc{sub 1} dimer is functional even when cytochrome b subunits come from different species. - Abstract: Electronic connection between Q{sub o} and Q{sub i} quinone catalytic sites of dimeric cytochrome bc{sub 1} is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the lattermore » remains unclear. Here, using a series of mutated hybrid cytochrome bc{sub 1}-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.« less

A reversible fluorescent probe based on C[double bond, length as m-dash]N isomerization for the selective detection of formaldehyde in living cells and in vivo.

PubMed

Song, Xinyu; Han, Xiaoyue; Yu, Fabiao; Zhang, Jinjin; Chen, Lingxin; Lv, Changjun

2018-01-15

Formaldehyde (FA) is an endogenously produced reactive carbonyl species (RCS) through biological metabolic processes whose concentration is closely related to human health and disease. Noninvasive and real-time detection of FA concentration in organisms is very important for revealing the physiological and pathological functions of FA. Herein, we design and synthesize a reversible fluorescent probe BOD-NH 2 for the detection of FA in living cells and in vivo. The probe is composed of two moieties: the BODIPY fluorophore and the primary amino group response unit. The probe undergoes an intracellular aldimine condensation reaction with FA and forms imine (C[double bond, length as m-dash]N) which will result in C[double bond, length as m-dash]N isomerization and rotation to turn-off the fluorescence of the probe. It is important that the probe can show a reversible response to FA. The probe BOD-NH 2 has been successfully applied for detecting and imaging FA in the cytoplasm of living cells. BOD-NH 2 is capable of detecting fluctuations in the levels of endogenous and exogenous FA in different types of living cells. The probe can be used to visualize the FA concentration in fresh hippocampus and the probe can further qualitatively evaluate the FA concentrations in ex vivo-dissected organs. Moreover, BOD-NH 2 can also be used for imaging in mice. The above applications make our new probe a potential chemical tool for the study of physiological and pathological functions of FA in cells and in vivo.

Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo.

PubMed

Yang, Joon-Mo; Favazza, Christopher; Chen, Ruimin; Yao, Junjie; Cai, Xin; Maslov, Konstantin; Zhou, Qifa; Shung, K Kirk; Wang, Lihong V

2012-08-01

At present, clinicians routinely apply ultrasound endoscopy in a variety of interventional procedures that provide treatment solutions for diseased organs. Ultrasound endoscopy not only produces high-resolution images, but also is safe for clinical use and broadly applicable. However, for soft tissue imaging, its mechanical wave-based image contrast fundamentally limits its ability to provide physiologically specific functional information. By contrast, photoacoustic endoscopy possesses a unique combination of functional optical contrast and high spatial resolution at clinically relevant depths, ideal for imaging soft tissues. With these attributes, photoacoustic endoscopy can overcome the current limitations of ultrasound endoscopy. Moreover, the benefits of photoacoustic imaging do not come at the expense of existing ultrasound functions; photoacoustic endoscopy systems are inherently compatible with ultrasound imaging, thereby enabling multimodality imaging with complementary contrast. Here we present simultaneous photoacoustic and ultrasonic dual-mode endoscopy and show its ability to image internal organs in vivo, thus illustrating its potential clinical application.

Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo

PubMed Central

Yang, Joon-Mo; Favazza, Christopher; Chen, Ruimin; Yao, Junjie; Cai, Xin; Maslov, Konstantin; Zhou, Qifa; Shung, K. Kirk; Wang, Lihong V.

2013-01-01

Presently, clinicians routinely apply ultrasound endoscopy in a variety of interventional procedures which provide treatment solutions for diseased organs. Ultrasound endoscopy not only produces high resolution images, it is also safe for clinical use and broadly applicable. However, for soft tissue imaging, its mechanical wave-based image contrast fundamentally limits its ability to provide physiologically-specific functional information. By contrast, photoacoustic endoscopy possesses a unique combination of functional optical contrast and high spatial resolution at clinically-relevant depths, ideal for soft tissue imaging. With these attributes, photoacoustic endoscopy can overcome the current limitations of ultrasound endoscopy. Moreover, the benefits of photoacoustic imaging do not come at the expense of existing ultrasound functions; photoacoustic endoscopy systems are inherently compatible with ultrasound imaging, enabling multi-modality imaging with complementary contrast. Here, we present simultaneous photoacoustic and ultrasonic dual-mode endoscopy and demonstrate its ability to image internal organs in vivo, illustrating its potential clinical application. PMID:22797808

Development of a Bioreactor to Culture Tissue Engineered Ureters Based on the Application of Tubular OPTIMAIX 3D Scaffolds.

PubMed

Seifarth, Volker; Gossmann, Matthias; Janke, Heinz Peter; Grosse, Joachim O; Becker, Christoph; Heschel, Ingo; Artmann, Gerhard M; Temiz Artmann, Aysegül

2015-01-01

Regenerative medicine, tissue engineering and biomedical research give hope to many patients who need bio-implants. Tissue engineering applications have already been developed based on bioreactors. Physiological ureter implants, however, do not still function sufficiently, as they represent tubular hollow structures with very specific cellular structures and alignments consisting of several cell types. The aim of this study was to a develop a new bioreactor system based on seamless, collagenous, tubular OPTIMAIX 3D prototype sponge as scaffold material for ex-vivo culturing of a tissue engineered ureter replacement for future urological applications. Particular emphasis was given to a great extent to mimic the physiological environment similar to the in vivo situation of a ureter. NIH-3T3 fibroblasts, C2C12, Urotsa and primary genitourinary tract cells were applied as co-cultures on the scaffold and the penetration of cells into the collagenous material was followed. By the end of this study, the bioreactor was functioning, physiological parameter as temperature and pH and the newly developed BIOREACTOR system is applicable to tubular scaffold materials with different lengths and diameters. The automatized incubation system worked reliably. The tubular OPTIMAIX 3D sponge was a suitable scaffold material for tissue engineering purposes and co-cultivation procedures. © 2015 S. Karger AG, Basel.

What have we learned about GPER function in physiology and disease from knockout mice?

PubMed Central

Prossnitz, Eric R.; Hathaway, Helen J.

2015-01-01

Estrogens, predominantly 17β-estradiol, exert diverse effects throughout the body in both normal and patho-physiology, during development and in reproductive, metabolic, endocrine, cardiovascular, nervous, musculoskeletal and immune systems. Estrogen and its receptors also play important roles in carcinogenesis and therapy, particularly for breast cancer. In addition to the classical nuclear estrogen receptors (ERα and ERβ) that traditionally mediate predominantly genomic signaling, the G protein-coupled estrogen receptor GPER has become recognized as a critical mediator of rapid signaling in response to estrogen. Mouse models, and in particular knockout (KO) mice, represent an important approach to understand the functions of receptors in normal physiology and disease. Whereas ERα KO mice display multiple significant defects in reproduction and mammary gland development, ERβ KO phenotypes are more limited, and GPER KO exhibit no reproductive deficits. However, the study of GPER KO mice over the last six years has revealed that GPER deficiency results in multiple physiological alterations including obesity, cardiovascular dysfunction, insulin resistance and glucose intolerance. In addition, the lack of estrogen-mediated effects in numerous tissues of GPER KO mice, studied in vivo or ex vivo, including those of the cardiovascular, endocrine, nervous and immune systems, reveals GPER as a genuine mediator of estrogen action. Importantly, GPER KO mice have also revealed roles for GPER in breast carcinogenesis and metastasis. In combination with the supporting effects of GPER-selective ligands and GPER knockdown approaches, GPER KO mice demonstrate the therapeutic potential of targeting GPER activity in diseases as diverse as obesity, diabetes, multiple sclerosis, hypertension, atherosclerosis, myocardial infarction, stroke and cancer. PMID:26189910

The endothelial glycocalyx

PubMed Central

Yang, Yimu; Schmidt, Eric P.

2013-01-01

Once thought to be a structure of small size and uncertain significance, the endothelial glycocalyx is now known to be an important regulator of endothelial function. Studies of the systemic vasculature have demonstrated that the glycocalyx forms a substantial in vivo endothelial surface layer (ESL) critical to inflammation, barrier function and mechanotransduction. The pulmonary ESL is significantly thicker than the systemic ESL, suggesting unique physiologic function. We have recently demonstrated that the pulmonary ESL regulates exposure of endothelial surface adhesion molecules, thereby serving as a barrier to neutrophil adhesion and extravasation. While the pulmonary ESL is not a critical structural component of the endothelial barrier to fluid and protein, it serves a major role in the mechanotransduction of vascular pressure, with impact on the active regulation of endothelial permeability. It is likely that the ESL serves numerous additional functions in vascular physiology, representing a fertile area for future investigation. PMID:24073386

Fyn kinase genetic ablation causes structural abnormalities in mature retina and defective Müller cell function.

PubMed

Chavez-Solano, Marbella; Ibarra-Sanchez, Alfredo; Treviño, Mario; Gonzalez-Espinosa, Claudia; Lamas, Monica

2016-04-01

Fyn kinase is widely expressed in neuronal and glial cells of the brain, where it exerts multiple functional roles that affect fundamental physiological processes. The aim of our study was to investigate the, so far unknown, functional role of Fyn in the retina. We report that Fyn is expressed, in vivo, in a subpopulation of Müller glia. We used a mouse model of Fyn genetic ablation and Müller-enriched primary cultures to demonstrate that Fyn deficiency induces morphological alterations in the mature retina, a reduction in the thickness of the outer and inner nuclear layers and alterations in postnatal Müller cell physiology. These include shortening of Müller cell processes, a decrease in cell proliferation, inactivation of the Akt signal transduction pathway, a reduced number of focal adhesions points and decreased adhesion of these cells to the ECM. As abnormalities in Müller cell physiology have been previously associated to a compromised retinal function we evaluated behavioral responses to visual stimulation. Our results associate Fyn deficiency with impaired visual optokinetic responses under scotopic and photopic light conditions. Our study reveals novel roles for Fyn kinase in retinal morphology and Müller cell physiology and suggests that Fyn is required for optimal visual processing. Copyright © 2016 Elsevier Inc. All rights reserved.

A telehealth architecture for networked embedded systems: a case study in in vivo health monitoring.

PubMed

Dabiri, Foad; Massey, Tammara; Noshadi, Hyduke; Hagopian, Hagop; Lin, C K; Tan, Robert; Schmidt, Jacob; Sarrafzadeh, Majid

2009-05-01

The improvement in processor performance through continuous breakthroughs in transistor technology has resulted in the proliferation of lightweight embedded systems. Advances in wireless technology and embedded systems have enabled remote healthcare and telemedicine. While medical examinations could previously extract only localized symptoms through snapshots, now continuous monitoring can discretely analyze how a patient's lifestyle affects his/her physiological conditions and if additional symptoms occur under various stimuli. We demonstrate how medical applications in particular benefit from a hierarchical networking scheme that will improve the quantity and quality of ubiquitous data collection. Our Telehealth networking infrastructure provides flexibility in terms of functionality and the type of applications that it supports. We specifically present a case study that demonstrates the effectiveness of our networked embedded infrastructure in an in vivo pressure application. Experimental results of the in vivo system demonstrate how it can wirelessly transmit pressure readings measuring from 0 to 1.5 lbf/in (2) with an accuracy of 0.02 lbf/in (2). The challenges in biocompatible packaging, transducer drift, power management, and in vivo signal transmission are also discussed. This research brings researchers a step closer to continuous, real-time systemic monitoring that will allow one to analyze the dynamic human physiology.

Brain Basis of Early Parent-Infant Interactions: Psychology, Physiology, and "in vivo" Functional Neuroimaging Studies

ERIC Educational Resources Information Center

Swain, James E.; Lorberbaum, Jeffrey P.; Kose, Samet; Strathearn, Lane

2007-01-01

Parenting behavior critically shapes human infants' current and future behavior. The parent-infant relationship provides infants with their first social experiences, forming templates of what they can expect from others and how to best meet others' expectations. In this review, we focus on the neurobiology of parenting behavior, including our own…

An in vivo multiplexed small molecule screening platform

PubMed Central

Yang, Dian; Ogasawara, Daisuke; Dix, Melissa M.; Rogers, Zoë N.; Chuang, Chen-Hua; McFarland, Christopher D.; Chiou, Shin-Heng; Brown, J. Mark; Cravatt, Benjamin F.; Bogyo, Matthew; Winslow, Monte M.

2016-01-01

Phenotype-based small molecule screening is a powerful method to identify regulators of cellular function. However, such screens are generally performed in vitro using conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of libraries of small molecules. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed towards hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo. PMID:27617390

Passive Ventricular Mechanics Modelling Using MRI of Structure and Function

PubMed Central

Wang, V.Y.; Lam, H.I.; Ennis, D.B.; Young, A.A.; Nash, M.P.

2009-01-01

Patients suffering from dilated cardiomyopathy or myocardial infarction can develop left ventricular (LV) diastolic impairment. The LV remodels its structure and function to adapt to pathophysiological changes in geometry and loading conditions and this remodeling process can alter the passive ventricular mechanics. In order to better understand passive ventricular mechanics, a LV finite element model was developed to incorporate physiological and mechanical information derived from in vivo magnetic resonance imaging (MRI) tissue tagging, in vivo LV cavity pressure recording and ex vivo diffusion tensor MRI (DTMRI) of a canine heart. MRI tissue tagging enables quantitative evaluation of cardiac mechanical function with high spatial and temporal resolution, whilst the direction of maximum water diffusion (the primary eigenvector) in each voxel of a DTMRI directly correlates with the myocardial fibre orientation. This model was customized to the geometry of the canine LV during diastasis by fitting the segmented epicardial and endocardial surface data from tagged MRI using nonlinear finite element fitting techniques. Myofibre orientations, extracted from DTMRI of the same heart, were incorporated into this geometric model using a free form deformation methodology. Pressure recordings, temporally synchronized to the tissue tagging MRI data, were used to simulate the LV deformation during diastole. Simulation of the diastolic LV mechanics allowed us to estimate the stiffness of the passive LV myocardium based on kinematic data obtained from tagged MRI. This integrated physiological model will allow more insight into the regional passive diastolic mechanics of the LV on an individualized basis, thereby improving our understanding of the underlying structural basis of mechanical dysfunction in pathological conditions. PMID:18982680

Passive ventricular mechanics modelling using MRI of structure and function.

PubMed

Wang, V Y; Lam, H I; Ennis, D B; Young, A A; Nash, M P

2008-01-01

Patients suffering from dilated cardiomyopathy or myocardial infarction can develop left ventricular (LV) diastolic impairment. The LV remodels its structure and function to adapt to pathophysiological changes in geometry and loading conditions and this remodeling process can alter the passive ventricular mechanics. In order to better understand passive ventricular mechanics, a LV finite element model was developed to incorporate physiological and mechanical information derived from in vivo magnetic resonance imaging (MRI) tissue tagging, in vivo LV cavity pressure recording and ex vivo diffusion tensor MRI (DTMRI) of a canine heart. MRI tissue tagging enables quantitative evaluation of cardiac mechanical function with high spatial and temporal resolution, whilst the direction of maximum water diffusion (the primary eigenvector) in each voxel of a DTMRI directly correlates with the myocardial fibre orientation. This model was customized to the geometry of the canine LV during diastasis by fitting the segmented epicardial and endocardial surface data from tagged MRI using nonlinear finite element fitting techniques. Myofibre orientations, extracted from DTMRI of the same heart, were incorporated into this geometric model using a free form deformation methodology. Pressure recordings, temporally synchronized to the tissue tagging MRI data, were used to simulate the LV deformation during diastole. Simulation of the diastolic LV mechanics allowed us to estimate the stiffness of the passive LV myocardium based on kinematic data obtained from tagged MRI. This integrated physiological model will allow more insight into the regional passive diastolic mechanics of the LV on an individualized basis, thereby improving our understanding of the underlying structural basis of mechanical dysfunction in pathological conditions.

In vivo recruitment patterns in the anterior oblique and dorsoradial ligaments of the first carpometacarpal joint.

PubMed

Halilaj, Eni; Rainbow, Michael J; Moore, Douglas C; Laidlaw, David H; Weiss, Arnold-Peter C; Ladd, Amy L; Crisco, Joseph J

2015-07-16

The anterior oblique ligament (AOL) and the dorsoradial ligament (DRL) are both regarded as mechanical stabilizers of the thumb carpometacarpal (CMC) joint, which in older women is often affected by osteoarthritis. Inferences on the potential relationship of these ligaments to joint pathomechanics are based on clinical experience and studies of cadaveric tissue, but their functions has been studied sparsely in vivo. The purpose of this study was to gain insight into the functions of the AOL and DRL using in vivo joint kinematics data. The thumbs of 44 healthy subjects were imaged with a clinical computed tomography scanner in functional-task and thumb range-of-motion positions. The origins and insertion sites of the AOL and the DRL were identified on the three-dimensional bone models and each ligament was modeled as a set of three fibers whose lengths were the minimum distances between insertion sites. Ligament recruitment, which represented ligament length as a percentage of the maximum length across the scanned positions, was computed for each position and related to joint posture. Mean AOL recruitment was lower than 91% across the CMC range of motion, whereas mean DRL recruitment was generally higher than 91% in abduction and flexion. Under the assumption that ligaments do not strain by more than 10% physiologically, our findings of mean ligament recruitments across the CMC range of motion indicate that the AOL is likely slack during most physiological positions, whereas the DRL may be taut and therefore support the joint in positions of CMC joint abduction and flexion. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vivo recruitment patterns in the anterior oblique and dorsoradial ligaments of the first carpometacarpal joint

PubMed Central

Halilaj, Eni; Rainbow, Michael J.; Moore, Douglas C.; Laidlaw, David H.; Weiss, Arnold-Peter C.; Ladd, Amy L.; Crisco, Joseph J.

2015-01-01

The anterior oblique ligament (AOL) and the dorsoradial ligament (DRL) are both regarded as mechanical stabilizers of the thumb carpometacarpal (CMC) joint, which in older women is often affected by osteoarthritis. Inferences on the potential relationship of these ligaments to joint pathomechanics are based on clinical experience and studies of cadaveric tissue, but their function has been studied sparsely in vivo. The purpose of this study was to gain insight into the functions of the AOL and DRL using in vivo joint kinematic data. The thumbs of 44 healthy subjects were imaged with a clinical computed tomography scanner in functional-task and thumb range-of-motion positions. The origins and insertion sites of the AOL and the DRL were identified on the 3D bone models and each ligament was modeled as a set of three fibers whose lengths were the minimum distances between insertion sites. Ligament recruitment, which represented ligament length as a percentage of the maximum length across the scanned positions, was computed for each position and related to joint posture. Mean AOL recruitment was lower than 91% across the CMC range of motion, whereas mean DRL recruitment was generally higher than 91% in abduction and flexion. Under the assumption that ligaments do not strain by more than 10% physiologically, our findings of mean ligament recruitments across the CMC range of motion indicate that the AOL is likely slack during most physiological positions, whereas the DRL may be taut and therefore support the joint in positions of CMC joint abduction and flexion. PMID:25964211

Fibre Bragg grating manometry catheters for in vivo monitoring of peristalsis

NASA Astrophysics Data System (ADS)

Arkwright, John W.; Underhill, Ian

2017-02-01

The human gastrointestinal tract or `gut' is one of the body's largest functional systems spanning up to 8 metres in length from beginning to end. It is formed of a series of physiologically different sections that perform the various functions required for the digestion of food, absorption of nutrients and water, and the removal of waste products. To enable the gut to perform correctly it must be able to transport digesta through each section at the appropriate rate, and any breakdown or malfunction of this transport mechanism can have severe consequences to on-going good health. Monitoring motor function deep within the gut is challenging due to the need to monitor over extended lengths with high spatial resolution. Fiber Bragg grating (FBG) manometry catheters provide a near ideal method of monitoring physiologically significant lengths of the gut in a minimally invasive fashion. Following the development by our group of the first viable FBG based manometry catheter we have undertaken a series of clinical investigations in the human esophagus, colon, stomach and small bowel. Each region presents its own technological challenge and has required a range of modifications to the basic catheter design. We present the design of these catheters and clinical results from over 100 in-vivo studies.

New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells.

PubMed

Xiao, Wenjin; Perry, Guillaume; Komori, Kikuo; Sakai, Yasuyuki

2015-11-01

To develop an in vitro liver tissue equivalent, hepatocytes should be cocultured with liver non-parenchymal cells to mimic the in vivo physiological microenvironments. In this work, we describe a physiologically-relevant liver tissue model by hierarchically organizing layers of primary rat hepatocytes and human liver sinusoidal endothelial cells (TMNK-1) on an oxygen-permeable polydimethylsiloxane (PDMS) membrane, which facilitates direct oxygenation by diffusion through the membrane. This in vivo-mimicking hierarchical coculture was obtained by simply proceeding the overlay of TMNK-1 cells on the hepatocyte layer re-formed on the collagen immobilized PDMS membranes. The comparison of hepatic functionalities was achieved between coculture and sandwich culture with Matrigel, in the presence and absence of direct oxygenation. A complete double-layered structure of functional liver cells with vertical contact between hepatocytes and TMNK-1 was successfully constructed in the coculture with direct oxygen supply and was well-maintained for 14 days. The hepatocytes in this hierarchical culture exhibited improved survival, functional bile canaliculi formation, cellular level polarization and maintenance of metabolic activities including Cyp1A1/2 activity and albumin production. By contrast, the two cell populations formed discontinuous monolayers on the same surfaces in the non-oxygen-permeable cultures. These results demonstrate that (i) the direct oxygenation through the PDMS membranes enables very simple formation of a hierarchical structure consisting of a hepatocyte layer and a layer of TMNK-1 and (ii) we may include other non-parenchymal cells in this format easily, which can be widely applicable to other epithelial organs.

Improving the physiological realism of experimental models.

PubMed

Vinnakota, Kalyan C; Cha, Chae Y; Rorsman, Patrik; Balaban, Robert S; La Gerche, Andre; Wade-Martins, Richard; Beard, Daniel A; Jeneson, Jeroen A L

2016-04-06

The Virtual Physiological Human (VPH) project aims to develop integrative, explanatory and predictive computational models (C-Models) as numerical investigational tools to study disease, identify and design effective therapies and provide an in silico platform for drug screening. Ultimately, these models rely on the analysis and integration of experimental data. As such, the success of VPH depends on the availability of physiologically realistic experimental models (E-Models) of human organ function that can be parametrized to test the numerical models. Here, the current state of suitable E-models, ranging from in vitro non-human cell organelles to in vivo human organ systems, is discussed. Specifically, challenges and recent progress in improving the physiological realism of E-models that may benefit the VPH project are highlighted and discussed using examples from the field of research on cardiovascular disease, musculoskeletal disorders, diabetes and Parkinson's disease.

Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

PubMed

Coppola, Daniela; Giordano, Daniela; Milazzo, Lisa; Howes, Barry D; Ascenzi, Paolo; di Prisco, Guido; Smulevich, Giulietta; Poole, Robert K; Verde, Cinzia

2018-02-28

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O 2 -rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O 2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O 2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217. Copyright © 2017. Published by Elsevier Inc.

Microfluidic 3D cell culture: potential application for tissue-based bioassays

PubMed Central

Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

2014-01-01

Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

NASA Technical Reports Server (NTRS)

Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

2017-01-01

Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

PubMed Central

Di Buduo, Christian A.; Wray, Lindsay S.; Tozzi, Lorenzo; Malara, Alessandro; Chen, Ying; Ghezzi, Chiara E.; Smoot, Daniel; Sfara, Carla; Antonelli, Antonella; Spedden, Elise; Bruni, Giovanna; Staii, Cristian; De Marco, Luigi; Magnani, Mauro; Kaplan, David L.

2015-01-01

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production. PMID:25575540

Remote in vivo stress assessment of aquatic animals with microencapsulated biomarkers for environmental monitoring

NASA Astrophysics Data System (ADS)

Gurkov, Anton; Shchapova, Ekaterina; Bedulina, Daria; Baduev, Boris; Borvinskaya, Ekaterina; Meglinski, Igor; Timofeyev, Maxim

2016-11-01

Remote in vivo scanning of physiological parameters is a major trend in the development of new tools for the fields of medicine and animal physiology. For this purpose, a variety of implantable optical micro- and nanosensors have been designed for potential medical applications. At the same time, the important area of environmental sciences has been neglected in the development of techniques for remote physiological measurements. In the field of environmental monitoring and related research, there is a constant demand for new effective and quick techniques for the stress assessment of aquatic animals, and the development of proper methods for remote physiological measurements in vivo may significantly increase the precision and throughput of analyses in this field. In the present study, we apply pH-sensitive microencapsulated biomarkers to remotely monitor the pH of haemolymph in vivo in endemic amphipods from Lake Baikal, and we compare the suitability of this technique for stress assessment with that of common biochemical methods. For the first time, we demonstrate the possibility of remotely detecting a change in a physiological parameter in an aquatic organism under ecologically relevant stressful conditions and show the applicability of techniques using microencapsulated biomarkers for remote physiological measurements in environmental monitoring.

Mechanical characterization and numerical simulation of a subcutaneous implantable 3D printed cell encapsulation system.

PubMed

Adamo, Federica; Farina, Marco; Thekkedath, Usha R; Grattoni, Alessandro; Sesana, Raffaella

2018-06-01

Cell transplantation in bioengineered scaffolds and encapsulation systems has shown great promise in regenerative medicine. Depending on the site of implantation, type of cells and their expected function, these systems are designed to provide cells with a physiological-like environment while providing mechanical support and promoting long-term viability and function of the graft. A minimally invasive 3D printed system termed neovascularized implantable cell homing and encapsulation (NICHE) was developed in polylactic acid for subcutaneous transplantation of endocrine cells, including pancreatic islets. The suitability of the NICHE for long term in vivo deployment is investigated by assessing mechanical behavior of both fresh devices under simulated subcutaneous conditions and NICHE retrieved from subcutaneous implantation in pigs. Both experimental and numerical studies were performed with a focus on validating the constitutive material model used in the numerical analysis for accuracy and reliability. Notably, homogeneous isotropic constitutive material model calibrated by means of uniaxial testing well suited experimental results. The results highlight the long term durability for in vivo applications and the potential applicability of the model to predict the mechanical behavior of similar devices in various physiological settings. Copyright © 2018 Elsevier Ltd. All rights reserved.

Precision cut lung slices as an efficient tool for in vitro lung physio-pharmacotoxicology studies.

PubMed

Morin, Jean-Paul; Baste, Jean-Marc; Gay, Arnaud; Crochemore, Clément; Corbière, Cécile; Monteil, Christelle

2013-01-01

1.We review the specific approaches for lung tissue slices preparation and incubation systems and the research application fields in which lung slices proved to be a very efficient alternative to animal experimentation for biomechanical, physiological, pharmacological and toxicological approaches. 2.Focus is made on air-liquid interface dynamic organ culture systems that allow direct tissue exposure to complex aerosol and that best mimic in vivo lung tissue physiology. 3.A compilation of research applications in the fields of vascular and airway reactivity, mucociliary transport, polyamine transport, xenobiotic biotransformation, chemicals toxicology and complex aerosols supports the concept that precision cut lung slices are a very efficient tool maintaining highly differentiated functions similar to in vivo lung organ when kept under dynamic organ culture. They also have been successfully used for lung gene transfer efficiency assessment, for lung viral infection efficiency assessment, for studies of tissue preservation media and tissue post-conditioning to optimize lung tissue viability before grafting. 4.Taken all together, the reviewed studies point to a great interest for precision cut lung slices as an efficient and valuable alternative to in vivo lung organ experimentation.

GPER/GPR30 knockout mice: effects of GPER on metabolism

PubMed Central

Sharma, Geetanjali; Prossnitz, Eric R.

2015-01-01

i. Summary Endogenous estrogens, predominantly 17β-estradiol (E2), mediate various very diverse effects throughout the body in both normal physiology and disease. Actions include development (including puberty) and reproduction as well as additional effects throughout life in the metabolic, endocrine, musculoskeletal, nervous, cardiovascular and immune systems. The actions of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERα and ERβ) that largely mediate transcriptional/genomic activities. However, more recently the G protein-coupled estrogen receptor GPER/GPR30 has become recognized as an essential mediator of certain, and particularly rapid, signaling events in response to E2. Murine genetic knockout (KO) models represent an important approach to understand the mechanisms of E2 action in physiology and disease. Studies of GPER KO mice over the last years have revealed functions for GPER in the regulation of obesity, insulin resistance and glucose intolerance, among other areas of (patho)physiology. This chapter focuses on methods for the evaluation of metabolic parameters in vivo and ex vivo with an emphasis on glucose homeostasis and metabolism through the use of glucose and insulin tolerance tests, pancreatic islet and adipocyte isolation and characterization. PMID:26585159

GPER/GPR30 Knockout Mice: Effects of GPER on Metabolism.

PubMed

Sharma, Geetanjali; Prossnitz, Eric R

2016-01-01

Endogenous estrogens, predominantly 17β-estradiol (E2), mediate various diverse effects throughout the body in both normal physiology and disease. Actions include development (including puberty) and reproduction as well as additional effects throughout life in the metabolic, endocrine, musculoskeletal, nervous, cardiovascular, and immune systems. The actions of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERα and ERβ) that largely mediate transcriptional/genomic activities. However, more recently the G protein-coupled estrogen receptor GPER/GPR30 has become recognized as an essential mediator of certain, and particularly rapid, signaling events in response to E2. Murine genetic knockout (KO) models represent an important approach to understand the mechanisms of E2 action in physiology and disease. Studies of GPER KO mice over the last years have revealed functions for GPER in the regulation of obesity, insulin resistance and glucose intolerance, among other areas of (patho)physiology. This chapter focuses on methods for the evaluation of metabolic parameters in vivo and ex vivo with an emphasis on glucose homeostasis and metabolism through the use of glucose and insulin tolerance tests, pancreatic islet and adipocyte isolation and characterization.

Visualization and contractile activity of cochlear pericytes in the capillaries of the spiral ligament.

PubMed

Dai, Min; Nuttall, Alfred; Yang, Yue; Shi, Xiaorui

2009-08-01

Pericytes, mural cells located on microvessels, are considered to play an important role in the formation of the vasculature and the regulation of local blood flow in some organs. Little is known about the physiology of cochlear pericytes. In order to investigate the function of cochlear pericytes, we developed a method to visualize cochlear pericytes using diaminofluorescein-2 diacetate (DAF-2DA) and intravital fluorescence microscopy. This method can permit the study of the effect of vasoactive agents on pericytes under the in vivo and normal physiological condition. The specificity of the labeling method was verified by the immunofluorescence labeling of pericyte maker proteins such as desmin, neural proteoglycan (NG2), and thymocyte differentiation antigen 1 (Thy-1). Superfused K(+) and Ca(2+) to the cochlear lateral wall resulted in localized constriction of capillaries at pericyte locations both in vivo and in vitro, while there was no obvious change in cochlear capillary diameters with application of the adrenergic neurotransmitter noradrenaline. The method could be an effective way to visualize cochlear pericytes and microvessels and study lateral wall vascular physiology. Moreover, we demonstrate for the first time that cochlear pericytes have contractility, which may be important for regulation of cochlear blood flow.

Programming and Isolation of Highly Pure Physiologically and Pharmacologically Functional Sinus-Nodal Bodies from Pluripotent Stem Cells

PubMed Central

Jung, Julia Jeannine; Husse, Britta; Rimmbach, Christian; Krebs, Stefan; Stieber, Juliane; Steinhoff, Gustav; Dendorfer, Andreas; Franz, Wolfgang-Michael; David, Robert

2014-01-01

Summary Therapeutic approaches for “sick sinus syndrome” rely on electrical pacemakers, which lack hormone responsiveness and bear hazards such as infection and battery failure. These issues may be overcome via “biological pacemakers” derived from pluripotent stem cells (PSCs). Here, we show that forward programming of PSCs with the nodal cell inducer TBX3 plus an additional Myh6-promoter-based antibiotic selection leads to cardiomyocyte aggregates consisting of >80% physiologically and pharmacologically functional pacemaker cells. These induced sinoatrial bodies (iSABs) exhibited highly increased beating rates (300–400 bpm), coming close to those found in mouse hearts, and were able to robustly pace myocardium ex vivo. Our study introduces iSABs as highly pure, functional nodal tissue that is derived from PSCs and may be important for future cell therapies and drug testing in vitro. PMID:24936448

International Union of Basic and Clinical Pharmacology. LXXVI. Current Progress in the Mammalian TRP Ion Channel Family

PubMed Central

Wu, Long-Jun; Sweet, Tara-Beth

2010-01-01

Transient receptor potential (TRP) channels are a large family of ion channel proteins, surpassed in number in mammals only by voltage-gated potassium channels. TRP channels are activated and regulated through strikingly diverse mechanisms, making them suitable candidates for cellular sensors. They respond to environmental stimuli such as temperature, pH, osmolarity, pheromones, taste, and plant compounds, and intracellular stimuli such as Ca2+ and phosphatidylinositol signal transduction pathways. However, it is still largely unknown how TRP channels are activated in vivo. Despite the uncertainties, emerging evidence using TRP channel knockout mice indicates that these channels have broad function in physiology. Here we review the recent progress on the physiology, pharmacology and pathophysiological function of mammalian TRP channels. PMID:20716668

Implementation of physiological fluids to provide insight into the characterization, fate, and biological interactions of silver nanoparticles.

PubMed

Breitner, Emily K; Burns, Katherine E; Hussain, Saber M; Comfort, Kristen K

2018-06-22

Silver nanoparticles (AgNPs) are being increasingly utilized in consumer and medical applications. However, there remains conflicting reports on their safety, which are evaluated through a combination of in vitro and in vivo exposure models. These discrepancies may arise, in part, due to the inherent differences between cell-based and animal systems. It is well established that nanotoxicological effects are highly dependent on the unique physicochemical properties and behavior of the particle set, including size, surface chemistry, agglomeration, and ionic dissolution. However, recent studies have identified that these properties vary as a function of exposure environment; providing a rationale for the contradictory results between in vitro and in vivo assessments. Artificial physiological fluids are emerging as a powerful tool as they allow for the characterization of NPs in an environment which they would likely encounter in vivo, in addition to having the experimental advantages of flexibility and consistency. Here, we demonstrated that the utilization of artificial fluids provided a mechanism to assess AgNP behavior and induced bioresponses in environments that they would likely encounter in vivo. AgNPs were introduced within an alveolar-based exposure model, which included alveolar epithelial (A549) cells incubated within artificial alveolar fluid (AF). Additionally, the particles underwent extensive characterization within both AF and lysosomal fluid, which the AgNPs would encounter following cellular internalization. Following incubation in physiological environments AgNP properties were significantly modified versus a traditional media environment, including alterations to both extent of agglomeration and rate of ionic dissolution. Moreover, when A549s were exposed to AgNPs in AF, the cells displayed lower cytotoxicity and stress rates, corresponding to a fluid-dependent drop in silver ion production. This work highlights the need for enhanced in vitro models that more closely mimic in vivo exposure environments in order to capture true NP behaviors and cellular interactions.

Implementation of physiological fluids to provide insight into the characterization, fate, and biological interactions of silver nanoparticles

NASA Astrophysics Data System (ADS)

Breitner, Emily K.; Burns, Katherine E.; Hussain, Saber M.; Comfort, Kristen K.

2018-06-01

Silver nanoparticles (AgNPs) are being increasingly utilized in consumer and medical applications. However, there remains conflicting reports on their safety, which are evaluated through a combination of in vitro and in vivo exposure models. These discrepancies may arise, in part, due to the inherent differences between cell-based and animal systems. It is well established that nanotoxicological effects are highly dependent on the unique physicochemical properties and behavior of the particle set, including size, surface chemistry, agglomeration, and ionic dissolution. However, recent studies have identified that these properties vary as a function of exposure environment; providing a rationale for the contradictory results between in vitro and in vivo assessments. Artificial physiological fluids are emerging as a powerful tool as they allow for the characterization of NPs in an environment which they would likely encounter in vivo, in addition to having the experimental advantages of flexibility and consistency. Here, we demonstrated that the utilization of artificial fluids provided a mechanism to assess AgNP behavior and induced bioresponses in environments that they would likely encounter in vivo. AgNPs were introduced within an alveolar-based exposure model, which included alveolar epithelial (A549) cells incubated within artificial alveolar fluid (AF). Additionally, the particles underwent extensive characterization within both AF and lysosomal fluid, which the AgNPs would encounter following cellular internalization. Following incubation in physiological environments AgNP properties were significantly modified versus a traditional media environment, including alterations to both extent of agglomeration and rate of ionic dissolution. Moreover, when A549s were exposed to AgNPs in AF, the cells displayed lower cytotoxicity and stress rates, corresponding to a fluid-dependent drop in silver ion production. This work highlights the need for enhanced in vitro models that more closely mimic in vivo exposure environments in order to capture true NP behaviors and cellular interactions.

In vivo demonstration of surgical task assistance using miniature robots.

PubMed

Hawks, Jeff A; Kunowski, Jacob; Platt, Stephen R

2012-10-01

Laparoscopy is beneficial to patients as measured by less painful recovery and an earlier return to functional health compared to conventional open surgery. However, laparoscopy requires the manipulation of long, slender tools from outside the patient's body. As a result, laparoscopy generally benefits only patients undergoing relatively simple procedures. An innovative approach to laparoscopy uses miniature in vivo robots that fit entirely inside the abdominal cavity. Our previous work demonstrated that a mobile, wireless robot platform can be successfully operated inside the abdominal cavity with different payloads (biopsy, camera, and physiological sensors). We hope that these robots are a step toward reducing the invasiveness of laparoscopy. The current study presents design details and results of laboratory and in vivo demonstrations of several new payload designs (clamping, cautery, and liquid delivery). Laboratory and in vivo cooperation demonstrations between multiple robots are also presented.

In vivo imaging of kidney glomeruli transplanted into the anterior chamber of the mouse eye

PubMed Central

Kistler, Andreas D.; Caicedo, Alejandro; Abdulreda, Midhat H.; Faul, Christian; Kerjaschki, Dontscho; Berggren, Per-Olof; Reiser, Jochen; Fornoni, Alessia

2014-01-01

Multiphoton microscopy enables live imaging of the renal glomerulus. However, repeated in vivo imaging of the same glomerulus over extended periods of time and the study of glomerular function independent of parietal epithelial and proximal tubular cell effects has not been possible so far. Here, we report a novel approach for non-invasive imaging of acapsular glomeruli transplanted into the anterior chamber of the mouse eye. After microinjection, glomeruli were capable of engrafting on the highly vascularized iris. Glomerular structure was preserved, as demonstrated by podocyte specific expression of cyan fluorescent protein and by electron microscopy. Injection of fluorescence-labeled dextrans of various molecular weights allowed visualization of glomerular filtration and revealed leakage of 70 kDa dextran in an inducible model of proteinuria. Our findings demonstrate functionality and long-term survival of glomeruli devoid of Bowman's capsule and provide a novel approach for non-invasive longitudinal in vivo study of glomerular physiology and pathophysiology. PMID:24464028

In Vivo Observation of Structural Changes in Neocortical Catecholaminergic Projections in Response to Drugs of Abuse.

PubMed

Morimoto, Mai M; Tanaka, Shinji; Mizutani, Shunsuke; Urata, Shinji; Kobayashi, Kazuto; Okabe, Shigeo

2018-01-01

Catecholaminergic (dopamine and norepinephrine) projections to the cortex play an important role in cognitive functions and dysfunctions including learning, addiction, and mental disorders. While dynamics of glutamatergic synapses have been well studied in such contexts, little is known regarding catecholaminergic projections, owing to lack of robust methods. Here we report a system to monitor catecholaminergic projections in vivo over the timeframes that such events occur. Green fluorescent protein (GFP) expression driven by tyrosine hydroxylase promoter in a transgenic mouse line enabled us to perform two-photon imaging of cortical catecholaminergic projections through a cranial window. Repetitive imaging of the same axons over 24 h revealed the highly dynamic nature of catecholaminergic boutons. Surprisingly, administration of single high dose methamphetamine (MAP) induced a transient increase in bouton volumes. This new method opens avenues for longitudinal in vivo evaluation of structural changes at single release sites of catecholamines in association with physiology and pathology of cortical functions.

In Vivo Observation of Structural Changes in Neocortical Catecholaminergic Projections in Response to Drugs of Abuse

PubMed Central

Morimoto, Mai M.; Tanaka, Shinji; Mizutani, Shunsuke; Urata, Shinji; Kobayashi, Kazuto

2018-01-01

Catecholaminergic (dopamine and norepinephrine) projections to the cortex play an important role in cognitive functions and dysfunctions including learning, addiction, and mental disorders. While dynamics of glutamatergic synapses have been well studied in such contexts, little is known regarding catecholaminergic projections, owing to lack of robust methods. Here we report a system to monitor catecholaminergic projections in vivo over the timeframes that such events occur. Green fluorescent protein (GFP) expression driven by tyrosine hydroxylase promoter in a transgenic mouse line enabled us to perform two-photon imaging of cortical catecholaminergic projections through a cranial window. Repetitive imaging of the same axons over 24 h revealed the highly dynamic nature of catecholaminergic boutons. Surprisingly, administration of single high dose methamphetamine (MAP) induced a transient increase in bouton volumes. This new method opens avenues for longitudinal in vivo evaluation of structural changes at single release sites of catecholamines in association with physiology and pathology of cortical functions. PMID:29445765

Improved Tumor Targeting and Longer Retention Time of NIR Fluorescent Probes Using Bioorthogonal Chemistry.

PubMed

Zhang, Xianghan; Wang, Bo; Zhao, Na; Tian, Zuhong; Dai, Yunpeng; Nie, Yongzhan; Tian, Jie; Wang, Zhongliang; Chen, Xiaoyuan

2017-01-01

The traditional labeling method for targeted NIR fluorescence probes requires directly covalent-bonded conjugation of targeting domains and fluorophores in vitro . Although this strategy works well, it is not sufficient for detecting or treating cancers in vivo , due to steric hindrance effects that relatively large fluorophore molecules exert on the configurations and physiological functions of specific targeting domains. The copper-free, "click-chemistry"-assisted assembly of small molecules in living systems may enhance tumor accumulation of fluorescence probes by improving the binding affinities of the targeting factors. Here, we employed a vascular homing peptide, GEBP11, as a targeting factor for gastric tumors, and we demonstrate its effectiveness for in vivo imaging via click-chemistry-mediated conjugation with fluorescence molecules in tumor xenograft mouse models. This strategy showed higher binding affinities than those of the traditional conjugation method, and our results showed that the tumor accumulation of click-chemistry-mediated probes are 11-fold higher than that of directly labeled probes. The tracking life was prolonged by 12-fold, and uptake of the probes into the kidney was reduced by 6.5-fold. For lesion tumors of different sizes, click-chemistry-mediated probes can achieve sufficient signal-to-background ratios (3.5-5) for in vivo detection, and with diagnostic sensitivity approximately 3.5 times that of traditional labeling probes. The click-chemistry-assisted detection strategy utilizes the advantages of "small molecule" probes while not perturbing their physiological functions; this enables tumor detection with high sensitivity and specific selectivity.

In Vivo Confocal Intrinsic Optical Signal Identification of Localized Retinal Dysfunction

PubMed Central

Zhang, Qiu-Xiang; Lu, Rong-Wen; Curcio, Christine A.; Yao, Xin-Cheng

2012-01-01

Purpose. The purposes of this study were to investigate the physiological mechanism of stimulus-evoked fast intrinsic optical signals (IOSs) recorded in dynamic confocal imaging of the retina, and to demonstrate the feasibility of in vivo confocal IOS mapping of localized retinal dysfunctions. Methods. A rapid line-scan confocal ophthalmoscope was constructed to achieve in vivo confocal IOS imaging of frog (Rana pipiens) retinas at cellular resolution. In order to investigate the physiological mechanism of confocal IOS, comparative IOS and electroretinography (ERG) measurements were made using normal frog eyes activated by variable-intensity stimuli. A dynamic spatiotemporal filtering algorithm was developed to reject the contamination of hemodynamic changes on fast IOS recording. Laser-injured frog eyes were employed to test the potential of confocal IOS mapping of localized retinal dysfunctions. Results. Comparative IOS and ERG experiments revealed a close correlation between the confocal IOS and retinal ERG, particularly the ERG a-wave, which has been widely used to evaluate photoreceptor function. IOS imaging of laser-injured frog eyes indicated that the confocal IOS could unambiguously detect localized (30 μm) functional lesions in the retina before a morphological abnormality is detectable. Conclusions. The confocal IOS predominantly results from retinal photoreceptors, and can be used to map localized photoreceptor lesion in laser-injured frog eyes. We anticipate that confocal IOS imaging can provide applications in early detection of age-related macular degeneration, retinitis pigmentosa, and other retinal diseases that can cause pathological changes in the photoreceptors. PMID:23150616

Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo

PubMed Central

Yang, Yang; Poe, Jonathan C.; Yang, Lisong; Fedoriw, Andrew; Desai, Siddhi; Magnuson, Terry; Li, Zhiguo; Fedoriw, Yuri; Araki, Kimi; Gao, Yanzhe; Tateishi, Satoshi; Sarantopoulos, Stefanie; Vaziri, Cyrus

2016-01-01

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18−/− mice. Moreover, primary Rad18−/− mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18−/− HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18−/− mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting. PMID:26883629

Improving the physiological realism of experimental models

PubMed Central

Vinnakota, Kalyan C.; Cha, Chae Y.; Rorsman, Patrik; Balaban, Robert S.; La Gerche, Andre; Wade-Martins, Richard; Beard, Daniel A.

2016-01-01

The Virtual Physiological Human (VPH) project aims to develop integrative, explanatory and predictive computational models (C-Models) as numerical investigational tools to study disease, identify and design effective therapies and provide an in silico platform for drug screening. Ultimately, these models rely on the analysis and integration of experimental data. As such, the success of VPH depends on the availability of physiologically realistic experimental models (E-Models) of human organ function that can be parametrized to test the numerical models. Here, the current state of suitable E-models, ranging from in vitro non-human cell organelles to in vivo human organ systems, is discussed. Specifically, challenges and recent progress in improving the physiological realism of E-models that may benefit the VPH project are highlighted and discussed using examples from the field of research on cardiovascular disease, musculoskeletal disorders, diabetes and Parkinson's disease. PMID:27051507

Effect of oxygen levels on the physiology of dendritic cells: implications for adoptive cell therapy.

PubMed

Futalan, Diahnn; Huang, Chien-Tze; Schmidt-Wolf, Ingo G H; Larsson, Marie; Messmer, Davorka

2011-01-01

Dendritic cell (DC)-based adoptive tumor immunotherapy approaches have shown promising results, but the incidence of tumor regression is low and there is an evident call for identifying culture conditions that produce DCs with a more potent Th1 potential. Routinely, DCs are differentiated in CO(2) incubators under atmospheric oxygen conditions (21% O(2)), which differ from physiological oxygen levels of only 3-5% in tissue, where most DCs reside. We investigated whether differentiation and maturation of DCs under physiological oxygen levels could produce more potent T-cell stimulatory DCs for use in adoptive immunotherapy. We found that immature DCs differentiated under physiological oxygen levels showed a small but significant reduction in their endocytic capacity. The different oxygen levels did not influence their stimuli-induced upregulation of cluster of differentiation 54 (CD54), CD40, CD83, CD86, C-C chemokine receptor type 7 (CCR7), C-X-C chemokine receptor type 4 (CXCR4) and human leukocyte antigen (HLA)-DR or the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 in response to lipopolysaccharide (LPS) or a cytokine cocktail. However, DCs differentiated under physiological oxygen level secreted higher levels of IL-12(p70) after exposure to LPS or CD40 ligand. Immature DCs differentiated at physiological oxygen levels caused increased T-cell proliferation, but no differences were observed for mature DCs with regard to T-cell activation. In conclusion, we show that although DCs generated under atmospheric or physiological oxygen conditions are mostly similar in function and phenotype, DCs differentiated under physiological oxygen secrete larger amounts of IL-12(p70). This result could have implications for the use of ex vivo-generated DCs for clinical studies, since DCs differentiated at physiological oxygen could induce increased Th1 responses in vivo.

Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells

PubMed Central

Fu, Xing; Du, Min

2018-01-01

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health. PMID:29643147

Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

PubMed Central

Wei, Yiyong; Zhou, Bin; Bernhard, Jonathan; Robinson, Samuel; Burapachaisri, Aonnicha; Guo, X. Edward

2017-01-01

Standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development. Cartilage grown from human mesenchymal stem cells (hMSCs) is poorly organized and unstable in vivo. We report that human cartilage with physiologic organization and in vivo stability can be grown in vitro from self-assembling hMSCs by implementing spatiotemporal regulation during induction. Self-assembling hMSCs formed cartilage discs in Transwell inserts following isotropic chondrogenic induction with transforming growth factor β to set up a dual-compartment culture. Following a switch in the basal compartment to a hypertrophic regimen with thyroxine, the cartilage discs underwent progressive deep-zone hypertrophy and mineralization. Concurrent chondrogenic induction in the apical compartment enabled the maintenance of functional and hyaline cartilage. Cartilage homeostasis, chondrocyte maturation, and terminal differentiation markers were all up-regulated versus isotropic control groups. We assessed the in vivo stability of the cartilage formed under different induction regimens. Cartilage formed under spatiotemporal regulation in vitro resisted endochondral ossification, retained the expression of cartilage markers, and remained organized following s.c. implantation in immunocompromised mice. In contrast, the isotropic control groups underwent endochondral ossification. Cartilage formed from hMSCs remained stable and organized in vivo. Spatiotemporal regulation during induction in vitro recapitulated some aspects of native cartilage development, and potentiated the maturation of self-assembling hMSCs into stable and organized cartilage resembling the native articular cartilage. PMID:28228529

KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine

PubMed Central

Goodwill, Adam G.; Fu, Lijuan; Noblet, Jillian N.; Casalini, Eli D.; Berwick, Zachary C.; Kassab, Ghassan S.; Tune, Johnathan D.

2016-01-01

Hydrogen peroxide (H2O2) and voltage-dependent K+ (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. PMID:26825518

In Vivo Neuromechanics: Decoding Causal Motor Neuron Behavior with Resulting Musculoskeletal Function.

PubMed

Sartori, Massimo; Yavuz, Utku Ş; Farina, Dario

2017-10-18

Human motor function emerges from the interaction between the neuromuscular and the musculoskeletal systems. Despite the knowledge of the mechanisms underlying neural and mechanical functions, there is no relevant understanding of the neuro-mechanical interplay in the neuro-musculo-skeletal system. This currently represents the major challenge to the understanding of human movement. We address this challenge by proposing a paradigm for investigating spinal motor neuron contribution to skeletal joint mechanical function in the intact human in vivo. We employ multi-muscle spatial sampling and deconvolution of high-density fiber electrical activity to decode accurate α-motor neuron discharges across five lumbosacral segments in the human spinal cord. We use complete α-motor neuron discharge series to drive forward subject-specific models of the musculoskeletal system in open-loop with no corrective feedback. We perform validation tests where mechanical moments are estimated with no knowledge of reference data over unseen conditions. This enables accurate blinded estimation of ankle function purely from motor neuron information. Remarkably, this enables observing causal associations between spinal motor neuron activity and joint moment control. We provide a new class of neural data-driven musculoskeletal modeling formulations for bridging between movement neural and mechanical levels in vivo with implications for understanding motor physiology, pathology, and recovery.

Dissection of K+ currents in Caenorhabditis elegans muscle cells by genetics and RNA interference

PubMed Central

Santi, C. M.; Yuan, A.; Fawcett, G.; Wang, Z.-W.; Butler, A.; Nonet, M. L.; Wei, A.; Rojas, P.; Salkoff, L.

2003-01-01

GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K+ currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo. In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca2+ and Cl- were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances. PMID:14612577

Uriniferous tubule: structural and functional organization.

PubMed

Christensen, Erik Ilsø; Wagner, Carsten A; Kaissling, Brigitte

2012-04-01

The uriniferous tubule is divided into the proximal tubule, the intermediate (thin) tubule, the distal tubule and the collecting duct. The present chapter is based on the chapters by Maunsbach and Christensen on the proximal tubule, and by Kaissling and Kriz on the distal tubule and collecting duct in the 1992 edition of the Handbook of Physiology, Renal Physiology. It describes the fine structure (light and electron microscopy) of the entire mammalian uriniferous tubule, mainly in rats, mice, and rabbits. The structural data are complemented by recent data on the location of the major transport- and transport-regulating proteins, revealed by morphological means(immunohistochemistry, immunofluorescence, and/or mRNA in situ hybridization). The structural differences along the uriniferous tubule strictly coincide with the distribution of the major luminal and basolateral transport proteins and receptors and both together provide the basis for the subdivision of the uriniferous tubule into functional subunits. Data on structural adaptation to defined functional changes in vivo and to genetical alterations of specified proteins involved in transepithelial transport importantly deepen our comprehension of the correlation of structure and function in the kidney, of the role of each segment or cell type in the overall renal function,and our understanding of renal pathophysiology. © 2012 American Physiological Society. Compr Physiol 2:933-996, 2012.

Spontaneous Release Regulates Synaptic Scaling in the Embryonic Spinal Network In Vivo

PubMed Central

Garcia-Bereguiain, Miguel Angel; Gonzalez-Islas, Carlos; Lindsly, Casie

2016-01-01

Homeostatic plasticity mechanisms maintain cellular or network spiking activity within a physiologically functional range through compensatory changes in synaptic strength or intrinsic cellular excitability. Synaptic scaling is one form of homeostatic plasticity that is triggered after blockade of spiking or neurotransmission in which the strengths of all synaptic inputs to a cell are multiplicatively scaled upward or downward in a compensatory fashion. We have shown previously that synaptic upscaling could be triggered in chick embryo spinal motoneurons by complete blockade of spiking or GABAA receptor (GABAAR) activation for 2 d in vivo. Here, we alter GABAAR activation in a more physiologically relevant manner by chronically adjusting presynaptic GABA release in vivo using nicotinic modulators or an mGluR2 agonist. Manipulating GABAAR activation in this way triggered scaling in a mechanistically similar manner to scaling induced by complete blockade of GABAARs. Remarkably, we find that altering action-potential (AP)-independent spontaneous release was able to fully account for the observed bidirectional scaling, whereas dramatic changes in spiking activity associated with spontaneous network activity had little effect on quantal amplitude. The reliance of scaling on an AP-independent process challenges the plasticity's relatedness to spiking in the living embryonic spinal network. Our findings have implications for the trigger and function of synaptic scaling and suggest that spontaneous release functions to regulate synaptic strength homeostatically in vivo. SIGNIFICANCE STATEMENT Homeostatic synaptic scaling is thought to prevent inappropriate levels of spiking activity through compensatory adjustments in the strength of synaptic inputs. Therefore, it is thought that perturbations in spike rate trigger scaling. Here, we find that dramatic changes in spiking activity in the embryonic spinal cord have little effect on synaptic scaling; conversely, alterations in GABAA receptor activation due to action-potential-independent GABA vesicle release can trigger scaling. The findings suggest that scaling in the living embryonic spinal cord functions to maintain synaptic strength and challenge the view that scaling acts to regulate spiking activity homeostatically. Finally, the results indicate that fetal exposure to drugs that influence GABA spontaneous release, such as nicotine, could profoundly affect synaptic maturation. PMID:27383600

Chaperonin polymers in archaea: The cytoskeleton of prokaryotes?

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

Trent, J.D.; Kagawa, H.K.; Zaluzec, N.J.

Chaperonins are protein complexes that play a critical role in folding nascent polypeptides under normal conditions and refolding damaged proteins under stress conditions. In all organisms these complexes are composed of evolutionarily conserved 60-kDa proteins arranged in double-ring structures with between 7 and 9 protein subunits per ring. These double ring structures are assumed to be the functional units in vivo, although they have never been observed inside cells. Here the authors show that the purified chaperonin from the hyperthermophilic archaeon Sulfolobus shibatae, which is closely related to chaperonins in eukaryotes, has a double ring structure at low concentrations (0.1more » mg/ml), but at more physiological concentrations, the rings stack end to end to form polymers. The polymers are stable at physiological temperatures (75 C) and closely resemble structures observed inside unfixed S. shibatae cells. The authors suggest that in vivo chaperonin activity may be regulated by polymerization and that chaperonin polymers may act as a cytoskeleton-like structure in archaea and bacteria.« less

Chaperonin Polymers in Archaea: The Cytoskeleton of Prokaryotes?

DOE R&D Accomplishments Database

Trent, J. D.; Kagawa, H. K.; Zaluzec, N. J.

1997-07-01

Chaperonins are protein complexes that play a critical role in folding nascent polypeptides under normal conditions and refolding damaged proteins under stress conditions. In all organisms these complexes are composed of evolutionarily conserved 60-kDa proteins arranged in double-ring structures with between 7 and 9 protein subunits per ring. These double ring structures are assumed to be the functional units in vivo, although they have never been observed inside cells. Here the authors show that the purified chaperonin from the hyperthermophilic archaeon Sulfolobus shibatae, which is closely related to chaperonins in eukaryotes, has a double ring structure at low concentrations (0.1 mg/ml), but at more physiological concentrations, the rings stack end to end to form polymers. The polymers are stable at physiological temperatures (75 C) and closely resemble structures observed inside unfixed S. shibatae cells. The authors suggest that in vivo chaperonin activity may be regulated by polymerization and that chaperonin polymers may act as a cytoskeleton-like structure in archaea and bacteria.

Pivotal advance: CTLA-4+ T cells exhibit normal antiviral functions during acute viral infection.

PubMed

Raué, Hans-Peter; Slifka, Mark K

2007-05-01

Previous studies have shown that T cells, which are genetically deficient in CTLA-4/CD152 expression, will proliferate uncontrollably, resulting in lethal autoimmune disease. This and other evidence indicate that CTLA-4 plays a critical role in the negative regulation of effector T cell function. In contrast to expectations, BrdU incorporation experiments demonstrated that CTLA-4 expression was associated with normal or even enhanced in vivo proliferation of virus-specific CD4+ and CD8+ T cells following acute lymphocytic choriomeningitis virus or vaccinia virus infection. When compared with CTLA-4- T cells directly ex vivo, CTLA-4+ T cells also exhibited normal antiviral effector functions following stimulation with peptide-coated cells, virus-infected cells, plate-bound anti-CD3/anti-CTLA-4, or the cytokines IL-12 and IL-18. Together, this indicates that CTLA-4 does not directly inhibit antiviral T cell expansion or T cell effector functions, at least not under the normal physiological conditions associated with either of these two acute viral infections.

An automated compound screening for anti-aging effects on the function of C. elegans sensory neurons.

PubMed

Bazopoulou, Daphne; Chaudhury, Amrita R; Pantazis, Alexandros; Chronis, Nikos

2017-08-24

Discovery of molecular targets or compounds that alter neuronal function can lead to therapeutic advances that ameliorate age-related neurodegenerative pathologies. Currently, there is a lack of in vivo screening technologies for the discovery of compounds that affect the age-dependent neuronal physiology. Here, we present a high-throughput, microfluidic-based assay for automated manipulation and on-chip monitoring and analysis of stimulus-evoked calcium responses of intact C. elegans at various life stages. First, we successfully applied our technology to quantify the effects of aging and age-related genetic and chemical factors in the calcium transients of the ASH sensory neuron. We then performed a large-scale screen of a library of 107 FDA-approved compounds to identify hits that prevented the age-dependent functional deterioration of ASH. The robust performance of our assay makes it a valuable tool for future high-throughput applications based on in vivo functional imaging.

Placenta-on-a-chip: a novel platform to study the biology of the human placenta.

PubMed

Lee, Ji Soo; Romero, Roberto; Han, Yu Mi; Kim, Hee Chan; Kim, Chong Jai; Hong, Joon-Seok; Huh, Dongeun

2016-01-01

Studying the biology of the human placenta represents a major experimental challenge. Although conventional cell culture techniques have been used to study different types of placenta-derived cells, current in vitro models have limitations in recapitulating organ-specific structure and key physiological functions of the placenta. Here we demonstrate that it is possible to leverage microfluidic and microfabrication technologies to develop a microengineered biomimetic model that replicates the architecture and function of the placenta. A "Placenta-on-a-Chip" microdevice was created by using a set of soft elastomer-based microfabrication techniques known as soft lithography. This microsystem consisted of two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane. To reproduce the placental barrier in this model, human trophoblasts (JEG-3) and human umbilical vein endothelial cells (HUVECs) were seeded onto the opposite sides of the ECM membrane and cultured under dynamic flow conditions to form confluent epithelial and endothelial layers in close apposition. We tested the physiological function of the microengineered placental barrier by measuring glucose transport across the trophoblast-endothelial interface over time. The permeability of the barrier study was analyzed and compared to that obtained from acellular devices and additional control groups that contained epithelial or endothelial layers alone. Our microfluidic cell culture system provided a tightly controlled fluidic environment conducive to the proliferation and maintenance of JEG-3 trophoblasts and HUVECs on the ECM scaffold. Prolonged culture in this model produced confluent cellular monolayers on the intervening membrane that together formed the placental barrier. This in vivo-like microarchitecture was also critical for creating a physiologically relevant effective barrier to glucose transport. Quantitative investigation of barrier function was conducted by calculating permeability coefficients and metabolic rates in varying conditions of barrier structure. The rates of glucose transport and metabolism were consistent with previously reported in vivo observations. The "Placenta-on-a-Chip" microdevice described herein provides new opportunities to simulate and analyze critical physiological responses of the placental barrier. This system may be used to address the major limitations of existing placenta model systems and serve to enable research platforms for reproductive biology and medicine.

Impaired Adaptability of in Vivo Mitochondrial Energetics to Acute Oxidative Insult in Aged Skeletal Muscle

PubMed Central

Siegel, Michael P.; Wilbur, Tim; Mathis, Mark; Shankland, Eric; Trieu, Atlas; Harper, Mary-Ellen; Marcinek, David J.

2012-01-01

Periods of elevated reactive oxygen species (ROS) production are a normal part of mitochondrial physiology. However, little is known about age-related changes in the mitochondrial response to elevated ROS in vivo. Significantly, ROS-induced uncoupling of oxidative phosphorylation has received attention as a negative feedback mechanism to reduce mitochondrial superoxide production. Here we use a novel in vivo spectroscopy system to test the hypothesis that ROS-induced uncoupling is diminished in aged mitochondria. This system simultaneously acquires 31P magnetic resonance and near-infrared optical spectra to non-invasively measure phosphometabolite and O2 concentrations in mouse skeletal muscle. Using low dose paraquat to elevate intracellular ROS we assess in vivo mitochondrial function in young, middle aged, and old mice. Oxidative phosphorylation was uncoupled to the same degree in response to ROS at each age, but this uncoupling was associated with loss of phosphorylation capacity and total ATP in old mice only. Using mice lacking UCP3 we demonstrate that this in vivo uncoupling is independent of this putative uncoupler of skeletal muscle mitochondria. These data indicate that ROS-induced uncoupling persists throughout life, but that oxidative stress leads to mitochondrial deficits and loss of ATP in aged organisms that may contribute to impaired function and degeneration. PMID:22935551

Functional Involvement of Carbonic Anhydrase in the Lysosomal Response to Cadmium Exposure in Mytilus galloprovincialis Digestive Gland

PubMed Central

Caricato, Roberto; Giordano, M. Elena; Schettino, Trifone; Lionetto, M. Giulia

2018-01-01

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme, whose functions in animals span from respiration to pH homeostasis, electrolyte transport, calcification, and biosynthetic reactions. CA is sensitive to trace metals in a number of species. In mussels, a previous study demonstrated CA activity and protein expression to be enhanced in digestive gland by cadmium exposure. The aim of the present work was to investigate the functional meaning, if any, of this response. To this end the study addressed the possible involvement of CA in the lysosomal system response of digestive gland cells to metal exposure. The in vivo exposure to acetazolamide, specific CA inhibitor, significantly inhibited the acidification of the lysosomal compartment in the digestive gland cells charged with the acidotropic probe LysoSensor Green D-189, demonstrating in vivo the physiological contribution of CA to the acidification of the lysosomes. Under CdCl2 exposure, CA activity significantly increased in parallel to the increase of the fluorescence of LysoSensor Green charged cells, which is in turn indicative of proliferation and/or increase in size of lysosomes. Acetazolamide exposure was able to completely inhibit the cadmium induced Lysosensor fluorescence increase in digestive gland cells. In conclusion, our results demonstrated the functional role of CA in the lysosomal acidification of Mytilus galloprovincialis digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction could physiologically respond to a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation. PMID:29670538

In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

PubMed Central

Peng, Rong; Maklokova, Vilena I.; Chandrashekhar, Jayadevi H.; Lan, Que

2011-01-01

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosaomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes. PMID:21437205

The Contribution of L-Type Cav1.3 Channels to Retinal Light Responses

PubMed Central

Shi, Liheng; Chang, Janet Ya-An; Yu, Fei; Ko, Michael L.; Ko, Gladys Y.-P.

2017-01-01

L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Cav1.2, Cav1.3, and Cav1.4) expressed in the retina. While Cav1.2 is expressed in all retinal cells including the Müller glia and neurons, Cav1.3 and Cav1.4 are expressed in the retinal neurons with Cav1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Cav1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Cav1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Cav1.3 are not associated with severe vision impairment in humans or in Cav1.3-null (Cav1.3−/−) mice. However, a failure to regulate Cav1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Cav1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Cav1.3 in retinal physiology and function that has been undervalued thus far. Through ex vivo and in vivo electroretinogram (ERG) recordings and immunohistochemical staining, we found that Cav1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Cav1.3 decreased ex vivo ERG a- and b-wave amplitudes. In Cav1.3−/− mice, their dark-adapted ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Cav1.3−/− mice retinas. Hence, Cav1.3 plays a more prominent role in retinal physiology and function than previously reported. PMID:29259539

The Contribution of L-Type Cav1.3 Channels to Retinal Light Responses.

PubMed

Shi, Liheng; Chang, Janet Ya-An; Yu, Fei; Ko, Michael L; Ko, Gladys Y-P

2017-01-01

L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Ca v 1.2, Ca v 1.3, and Ca v 1.4) expressed in the retina. While Ca v 1.2 is expressed in all retinal cells including the Müller glia and neurons, Ca v 1.3 and Ca v 1.4 are expressed in the retinal neurons with Ca v 1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Ca v 1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Ca v 1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Ca v 1.3 are not associated with severe vision impairment in humans or in Ca v 1.3-null (Ca v 1.3 -/- ) mice. However, a failure to regulate Ca v 1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Ca v 1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Ca v 1.3 in retinal physiology and function that has been undervalued thus far. Through ex vivo and in vivo electroretinogram (ERG) recordings and immunohistochemical staining, we found that Ca v 1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Ca v 1.3 decreased ex vivo ERG a- and b-wave amplitudes. In Ca v 1.3 -/- mice, their dark-adapted ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Ca v 1.3 -/- mice retinas. Hence, Ca v 1.3 plays a more prominent role in retinal physiology and function than previously reported.

Expression, Regulation and Putative Nutrient-Sensing Function of Taste GPCRs in the Heart

PubMed Central

Foster, Simon R.; Porrello, Enzo R.; Purdue, Brooke; Chan, Hsiu-Wen; Voigt, Anja; Frenzel, Sabine; Hannan, Ross D.; Moritz, Karen M.; Simmons, David G.; Molenaar, Peter; Roura, Eugeni; Boehm, Ulrich; Meyerhof, Wolfgang; Thomas, Walter G.

2013-01-01

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and by in situ hybridization can be visualized across the myocardium in isolated cardiac cells. Tas1r1 gene-targeted mice (Tas1r1Cre/Rosa26tdRFP) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart. PMID:23696900

Interrenal dysfunction in fish from contaminated sites: In vivo and in vitro studies

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

Hontela, A.

1995-12-31

An endocrine impairment characterized by a reduced capacity to elevate plasma cortisol levels in response to an acute standardized capture stress, has been previously diagnosed in yellow perch, Perca flavescens, and in northern pike, Esox lucius, from sites contaminated by mixtures of pollutants (heavy metals, PAHs and PCBs) or by BKME. The most recent studies were designed to (1) field validate this dysfunction usable as a marker of reduced physiological competence in fish; (2) demonstrate the impairment of the interrenal tissue in fish from sites located in a mining region in Abitibi; and (3) elucidate the physiological mechanisms underlying themore » impairment of the interrenal tissue in fish chronically exposed to xenobiotics. The responsiveness of the interrenal tissue to a standardized dose of ACTH1-39 was assessed in vivo in yellow perch from contaminated and reference sites maintained in experimental enclosures, as well as in vitro using complete growth medium in a perifusion system and in microplates. The results showed that the functional impairment of the internal tissue in fish exposed to xenobiotics is an exposure related phenomenon modulated by season. The functional tests with the interrenal tissue revealed that the responsiveness to ACTH is significantly reduced and that the synthesis of cortisol is disrupted, in fish from contaminated sites. Use of the functional ACTH-tests with the interrenal tissue of fish in environmental monitoring will be discussed.« less

Homeostatic effects of exercise and sleep on metabolic processes in mice with an overexpressed skeletal muscle clock.

PubMed

Brager, Allison J; Heemstra, Lydia; Bhambra, Raman; Ehlen, J Christopher; Esser, Karyn A; Paul, Ketema N; Novak, Colleen M

2017-01-01

Brain and muscle-ARNT-like factor (Bmal1/BMAL1) is an essential transcriptional/translational factor of circadian clocks. Loss of function of Bmal1/BMAL1 is highly disruptive to physiological and behavioral processes. In light of these previous findings, we examined if transgenic overexpression of Bmal1/BMAL1 in skeletal muscle could alter metabolic processes. First, we characterized in vivo and ex vivo metabolic phenotypes of muscle overexpressed mice (male and female) compared to wild-type littermates (WT). Second, we examined in vivo and ex vivo metabolic processes in the presence of positive and negative homeostatic challenges: high-intensity treadmill running (positive) and acute sleep deprivation (negative). In vivo measures of metabolic processes included body composition, respiratory exchange ratio (RER; VCO 2 /VO 2 ), energy expenditure, total activity counts, and food intake collected from small animal indirect calorimetry. Ex vivo measure of insulin sensitivity in skeletal muscle was determined from radioassays. RER was lower for muscle overexpressed females compared to female WTs. There were no genotype-dependent differences in metabolic phenotypes for males. With homeostatic challenges, muscle overexpressed mice had lower energy expenditure after high-intensity treadmill running. Acute sleep deprivation reduced insulin sensitivity in skeletal muscle in overexpressed male mice, but not male WTs. The present study contributes to a body of evidence showing pleiotropic, non-circadian, and homeostatic effects of altered Bmal1/BMAL1 expression on metabolic processes, demonstrating a critical need to further investigate the broad and complex actions of Bmal1/BMAL1 on physiology and behavior. Published by Elsevier B.V.

Multilevel functional genomics data integration as a tool for understanding physiology: a network biology perspective.

PubMed

Davidsen, Peter K; Turan, Nil; Egginton, Stuart; Falciani, Francesco

2016-02-01

The overall aim of physiological research is to understand how living systems function in an integrative manner. Consequently, the discipline of physiology has since its infancy attempted to link multiple levels of biological organization. Increasingly this has involved mathematical and computational approaches, typically to model a small number of components spanning several levels of biological organization. With the advent of "omics" technologies, which can characterize the molecular state of a cell or tissue (intended as the level of expression and/or activity of its molecular components), the number of molecular components we can quantify has increased exponentially. Paradoxically, the unprecedented amount of experimental data has made it more difficult to derive conceptual models underlying essential mechanisms regulating mammalian physiology. We present an overview of state-of-the-art methods currently used to identifying biological networks underlying genomewide responses. These are based on a data-driven approach that relies on advanced computational methods designed to "learn" biology from observational data. In this review, we illustrate an application of these computational methodologies using a case study integrating an in vivo model representing the transcriptional state of hypoxic skeletal muscle with a clinical study representing muscle wasting in chronic obstructive pulmonary disease patients. The broader application of these approaches to modeling multiple levels of biological data in the context of modern physiology is discussed. Copyright © 2016 the American Physiological Society.

Elasticity-based development of functionally enhanced multicellular 3D liver encapsulated in hybrid hydrogel.

PubMed

Lee, Ho-Joon; Son, Myung Jin; Ahn, Jiwon; Oh, Soo Jin; Lee, Mihee; Kim, Ansoon; Jeung, Yun-Ji; Kim, Han-Gyeul; Won, Misun; Lim, Jung Hwa; Kim, Nam-Soon; Jung, Cho-Rock; Chung, Kyung-Sook

2017-12-01

Current in vitro liver models provide three-dimensional (3-D) microenvironments in combination with tissue engineering technology and can perform more accurate in vivo mimicry than two-dimensional models. However, a human cell-based, functionally mature liver model is still desired, which would provide an alternative to animal experiments and resolve low-prediction issues on species differences. Here, we prepared hybrid hydrogels of varying elasticity and compared them with a normal liver, to develop a more mature liver model that preserves liver properties in vitro. We encapsulated HepaRG cells, either alone or with supporting cells, in a biodegradable hybrid hydrogel. The elastic modulus of the 3D liver dynamically changed during culture due to the combined effects of prolonged degradation of hydrogel and extracellular matrix formation provided by the supporting cells. As a result, when the elastic modulus of the 3D liver model converges close to that of the in vivo liver (≅ 2.3 to 5.9 kPa), both phenotypic and functional maturation of the 3D liver were realized, while hepatic gene expression, albumin secretion, cytochrome p450-3A4 activity, and drug metabolism were enhanced. Finally, the 3D liver model was expanded to applications with embryonic stem cell-derived hepatocytes and primary human hepatocytes, and it supported prolonged hepatocyte survival and functionality in long-term culture. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. We provide a functionally improved 3D liver model that recapitulates in vivo liver stiffness. We have experimentally addressed the issues of orchestrated effects of mechanical compliance, controlled matrix formation by stromal cells in conjunction with hepatic differentiation, and functional maturation of hepatocytes in a dynamic 3D microenvironment. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. Additionally, recent advances in the stem-cell technologies have made the development of 3D organoid possible, and thus, our study also provides further contribution to the development of physiologically relevant stem-cell-based 3D tissues that provide an elasticity-based predefined biomimetic 3D microenvironment. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

In vivo and in vitro effects of chromium VI on anterior pituitary hormone release and cell viability.

PubMed

Quinteros, Fernanda A; Poliandri, Ariel H B; Machiavelli, Leticia I; Cabilla, Jimena P; Duvilanski, Beatriz H

2007-01-01

Hexavalent chromium (Cr VI) is a highly toxic metal and an environmental pollutant. Different studies indicate that Cr VI exposure adversely affects reproductive functions. This metal has been shown to affect several tissues and organs but Cr VI effects on pituitary gland have not been reported. Anterior pituitary hormones are central for the body homeostasis and have a fundamental role in reproductive physiology. The aim of this study was to evaluate the effect of Cr VI at the pituitary level both in vivo and in vitro. We showed that Cr VI accumulates in the pituitary and hypothalamus, and decreases serum prolactin levels in vivo but observed no effects on LH levels. In anterior pituitary cells in culture, the effect of Cr VI on hormone secretion followed the same differential pattern. Besides, lactotrophs were more sensitive to the toxicity of the metal. As a result of oxidative stress generation, Cr VI induced apoptosis evidenced by nuclear fragmentation and caspase 3 activation. Our results indicate that the anterior pituitary gland can be a target of Cr VI toxicity in vivo and in vitro, thus producing a negative impact on the hypothalamic-pituitary-gonadal axis and affecting the normal endocrine function.

Sox2 expression in Schwann cells inhibits myelination in vivo and induces influx of macrophages to the nerve

PubMed Central

Roberts, Sheridan L.; Onaitis, Mark W.; Florio, Francesca; Quattrini, Angelo; Lloyd, Alison C.; D'Antonio, Maurizio

2017-01-01

Correct myelination is crucial for the function of the peripheral nervous system. Both positive and negative regulators within the axon and Schwann cell function to ensure the correct onset and progression of myelination during both development and following peripheral nerve injury and repair. The Sox2 transcription factor is well known for its roles in the development and maintenance of progenitor and stem cell populations, but has also been proposed in vitro as a negative regulator of myelination in Schwann cells. We wished to test fully whether Sox2 regulates myelination in vivo and show here that, in mice, sustained Sox2 expression in vivo blocks myelination in the peripheral nerves and maintains Schwann cells in a proliferative non-differentiated state, which is also associated with increased inflammation within the nerve. The plasticity of Schwann cells allows them to re-myelinate regenerated axons following injury and we show that re-myelination is also blocked by Sox2 expression in Schwann cells. These findings identify Sox2 as a physiological regulator of Schwann cell myelination in vivo and its potential to play a role in disorders of myelination in the peripheral nervous system. PMID:28743796

Microworm optode sensors limit particle diffusion to enable in vivo measurements.

PubMed

Ozaydin-Ince, Gozde; Dubach, J Matthew; Gleason, Karen K; Clark, Heather A

2011-02-15

There have been a variety of nanoparticles created for in vivo uses ranging from gene and drug delivery to tumor imaging and physiological monitoring. The use of nanoparticles to measure physiological conditions while being fluorescently addressed through the skin provides an ideal method toward minimally invasive health monitoring. Here we create unique particles that have all the necessary physical characteristics to serve as in vivo reporters, but with minimized diffusion from the point of injection. These particles, called microworms, have a cylindrical shape coated with a biocompatible porous membrane that possesses a large surface-area-to-volume ratio while maintaining a large hydrodynamic radius. We use these microworms to create fluorescent sodium sensors for use as in vivo sodium concentration detectors after subcutaneous injection. However, the microworm concept has the potential to extend to the immobilization of other types of polymers for continuous physiological detection or delivery of molecules.

Orderly recruitment of motor units under optical control in vivo.

PubMed

Llewellyn, Michael E; Thompson, Kimberly R; Deisseroth, Karl; Delp, Scott L

2010-10-01

A drawback of electrical stimulation for muscle control is that large, fatigable motor units are preferentially recruited before smaller motor units by the lowest-intensity electrical cuff stimulation. This phenomenon limits therapeutic applications because it is precisely the opposite of the normal physiological (orderly) recruitment pattern; therefore, a mechanism to achieve orderly recruitment has been a long-sought goal in physiology, medicine and engineering. Here we demonstrate a technology for reliable orderly recruitment in vivo. We find that under optical control with microbial opsins, recruitment of motor units proceeds in the physiological recruitment sequence, as indicated by multiple independent measures of motor unit recruitment including conduction latency, contraction and relaxation times, stimulation threshold and fatigue. As a result, we observed enhanced performance and reduced fatigue in vivo. These findings point to an unanticipated new modality of neural control with broad implications for nervous system and neuromuscular physiology, disease research and therapeutic innovation.

CO2/HCO3−- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor*

PubMed Central

Zippin, Jonathan H.; Chen, Yanqiu; Straub, Susanne G.; Hess, Kenneth C.; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G.; Sharp, Geoffrey W. G.; Levin, Lonny R.; Buck, Jochen

2013-01-01

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo. PMID:24100033

Physiological basis of tingling paresthesia evoked by hydroxy-alpha-sanshool.

PubMed

Lennertz, Richard C; Tsunozaki, Makoto; Bautista, Diana M; Stucky, Cheryl L

2010-03-24

Hydroxy-alpha-sanshool, the active ingredient in plants of the prickly ash plant family, induces robust tingling paresthesia by activating a subset of somatosensory neurons. However, the subtypes and physiological function of sanshool-sensitive neurons remain unknown. Here we use the ex vivo skin-nerve preparation to examine the pattern and intensity with which the sensory terminals of cutaneous neurons respond to hydroxy-alpha-sanshool. We found that sanshool excites virtually all D-hair afferents, a distinct subset of ultrasensitive light-touch receptors in the skin and targets novel populations of Abeta and C fiber nerve afferents. Thus, sanshool provides a novel pharmacological tool for discriminating functional subtypes of cutaneous mechanoreceptors. The identification of sanshool-sensitive fibers represents an essential first step in identifying the cellular and molecular mechanisms underlying tingling paresthesia that accompanies peripheral neuropathy and injury.

Physiological basis of tingling paresthesia evoked by hydroxy-α-sanshool

PubMed Central

Lennertz, Richard C; Tsunozaki, Makoto; Bautista, Diana M; Stucky, Cheryl L

2010-01-01

Hydroxy-α-sanshool, the active ingredient in plants of the prickly ash plant family, induces robust tingling paresthesia by activating a subset of somatosensory neurons. However, the subtypes and physiological function of sanshool-sensitive neurons remain unknown. Here we use the ex vivo skin-nerve preparation to examine the pattern and intensity with which the sensory terminals of cutaneous neurons respond to hydroxy-α-sanshool. We found that sanshool excites virtually all D-hair afferents, a distinct subset of ultra-sensitive light touch receptors in the skin, and targets novel populations of Aβ and C-fiber nerve afferents. Thus, sanshool provides a novel pharmacological tool for discriminating functional subtypes of cutaneous mechanoreceptors. The identification of sanshool-sensitive fibers represents an essential first step in identifying the cellular and molecular mechanisms underlying tingling paresthesia that accompanies peripheral neuropathy and injury. PMID:20335471

Posttranscriptional control of neuronal development by microRNA networks.

PubMed

Gao, Fen-Biao

2008-01-01

The proper development of the nervous system requires precise spatial and temporal control of gene expression at both the transcriptional and translational levels. In different experimental model systems, microRNAs (miRNAs) - a class of small, endogenous, noncoding RNAs that control the translation and stability of many mRNAs - are emerging as important regulators of various aspects of neuronal development. Further dissection of the in vivo physiological functions of individual miRNAs promises to offer novel mechanistic insights into the gene regulatory networks that ensure the precise assembly of a functional nervous system.

Intravital imaging of dendritic spine plasticity

PubMed Central

Sau Wan Lai, Cora

2014-01-01

Abstract Dendritic spines are the postsynaptic part of most excitatory synapses in the mammalian brain. Recent works have suggested that the structural and functional plasticity of dendritic spines have been associated with information coding and memories. Advances in imaging and labeling techniques enable the study of dendritic spine dynamics in vivo. This perspective focuses on intravital imaging studies of dendritic spine plasticity in the neocortex. I will introduce imaging tools for studying spine dynamics and will further review current findings on spine structure and function under various physiological and pathological conditions. PMID:28243511

Modelling passive diastolic mechanics with quantitative MRI of cardiac structure and function.

PubMed

Wang, Vicky Y; Lam, H I; Ennis, Daniel B; Cowan, Brett R; Young, Alistair A; Nash, Martyn P

2009-10-01

The majority of patients with clinically diagnosed heart failure have normal systolic pump function and are commonly categorized as suffering from diastolic heart failure. The left ventricle (LV) remodels its structure and function to adapt to pathophysiological changes in geometry and loading conditions, which in turn can alter the passive ventricular mechanics. In order to better understand passive ventricular mechanics, a LV finite element (FE) model was customized to geometric data segmented from in vivo tagged magnetic resonance images (MRI) data and myofibre orientation derived from ex vivo diffusion tensor MRI (DTMRI) of a canine heart using nonlinear finite element fitting techniques. MRI tissue tagging enables quantitative evaluation of cardiac mechanical function with high spatial and temporal resolution, whilst the direction of maximum water diffusion in each voxel of a DTMRI directly corresponds to the local myocardial fibre orientation. Due to differences in myocardial geometry between in vivo and ex vivo imaging, myofibre orientations were mapped into the geometric FE model using host mesh fitting (a free form deformation technique). Pressure recordings, temporally synchronized to the tagging data, were used as the loading constraints to simulate the LV deformation during diastole. Simulation of diastolic LV mechanics allowed us to estimate the stiffness of the passive LV myocardium based on kinematic data obtained from tagged MRI. Integrated physiological modelling of this kind will allow more insight into mechanics of the LV on an individualized basis, thereby improving our understanding of the underlying structural basis of mechanical dysfunction under pathological conditions.

Inflammatory Responses and Barrier Function of Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

PubMed

Halaidych, Oleh V; Freund, Christian; van den Hil, Francijna; Salvatori, Daniela C F; Riminucci, Mara; Mummery, Christine L; Orlova, Valeria V

2018-05-08

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Near-infrared spectroscopy and skeletal muscle oxidative function in vivo in health and disease: a review from an exercise physiology perspective

NASA Astrophysics Data System (ADS)

Grassi, Bruno; Quaresima, Valentina

2016-09-01

In most daily activities related to work or leisure, the energy for muscle work substantially comes from oxidative metabolism. Functional limitations or impairments of this metabolism can significantly affect exercise tolerance and performance. As a method for the functional evaluation of skeletal muscle oxidative metabolism, near-infrared spectroscopy (NIRS) has important strengths but also several limitations, some of which have been overcome by recent technological developments. Skeletal muscle fractional O2 extraction, the main variable which can be noninvasively evaluated by NIRS, is the result of the dynamic balance between O2 utilization and O2 delivery; it can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise performance and exercise tolerance in healthy subjects (in normal and in altered environmental conditions) and in patients. In the right hands, NIRS can offer insights into the physiological and pathophysiological adaptations to conditions of increased O2 needs that involve, in an integrated manner, different organs and systems of the body. In terms of patient evaluation, NIRS allows determination of the evolution of the functional impairments, to identify their correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights.

Near-infrared spectroscopy and skeletal muscle oxidative function in vivo in health and disease: a review from an exercise physiology perspective.

PubMed

Grassi, Bruno; Quaresima, Valentina

2016-09-01

In most daily activities related to work or leisure, the energy for muscle work substantially comes from oxidative metabolism. Functional limitations or impairments of this metabolism can significantly affect exercise tolerance and performance. As a method for the functional evaluation of skeletal muscle oxidative metabolism, near-infrared spectroscopy (NIRS) has important strengths but also several limitations, some of which have been overcome by recent technological developments. Skeletal muscle fractional O2 extraction, the main variable which can be noninvasively evaluated by NIRS, is the result of the dynamic balance between O2 utilization and O2 delivery; it can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise performance and exercise tolerance in healthy subjects (in normal and in altered environmental conditions) and in patients. In the right hands, NIRS can offer insights into the physiological and pathophysiological adaptations to conditions of increased O2 needs that involve, in an integrated manner, different organs and systems of the body. In terms of patient evaluation, NIRS allows determination of the evolution of the functional impairments, to identify their correlations with clinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights.

Biomimetic three-dimensional tissue models for advanced high-throughput drug screening

PubMed Central

Nam, Ki-Hwan; Smith, Alec S.T.; Lone, Saifullah; Kwon, Sunghoon; Kim, Deok-Ho

2015-01-01

Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately recreate the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when utilizing such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models which accurately mimic the physiological properties of native tissue samples, and highlight the advantages of using such 3D micro-tissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based-on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models. PMID:25385716

Structure, Folding Dynamics, and Amyloidogenesis of D76N β2-Microglobulin

PubMed Central

Mangione, P. Patrizia; Esposito, Gennaro; Relini, Annalisa; Raimondi, Sara; Porcari, Riccardo; Giorgetti, Sofia; Corazza, Alessandra; Fogolari, Federico; Penco, Amanda; Goto, Yuji; Lee, Young-Ho; Yagi, Hisashi; Cecconi, Ciro; Naqvi, Mohsin M.; Gillmore, Julian D.; Hawkins, Philip N.; Chiti, Fabrizio; Rolandi, Ranieri; Taylor, Graham W.; Pepys, Mark B.; Stoppini, Monica; Bellotti, Vittorio

2013-01-01

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition. PMID:24014031

Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables

PubMed Central

Clayton, Joseph E.; Pollard, Luther W.; Sckolnick, Maria; Bookwalter, Carol S.; Hodges, Alex R.; Trybus, Kathleen M.; Lord, Matthew

2014-01-01

A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity. PMID:24196839

Ex Vivo ERG analysis of photoreceptors using an In Vivo ERG system

PubMed Central

Vinberg, Frans; Kolesnikov, Alexander V.; Kefalov, Vladimir J.

2014-01-01

The Function of the retina and effects of drugs on it can be assessed by recording transretinal voltage across isolated retina that is perfused with physiological medium. However, building ex vivo ERG apparatus requires substantial amount of time, resources and expertise. Here we adapted a commercial in vivo ERG system for transretinal ERG recordings from rod and cone photoreceptors and compared rod and cone signalling between ex vivo and in vivo environments. We found that the rod and cone a- and b-waves recorded with the transretinal ERG adapter and a standard in vivo ERG system are comparable to those obtained from live anesthetized animals. However, ex vivo responses are somewhat slower and their oscillatory potentials are suppressed as compared to those recorded in vivo. We found that rod amplification constant (A) was comparable between ex vivo and in vivo conditions, ∼10 - 30 s-2 depending on the choice of response normalization. We estimate that the A in cones is between 3 and 6 s-2 in ex vivo conditions and by assuming equal A in vivo we arrive to light funnelling factor of 3 for cones in the mouse retina. The ex vivo ERG adapter provides a simple and affordable alternative to designing a custom-built transretinal recordings setup for the study of photoreceptors. Our results provide a roadmap to the rigorous quantitative analysis of rod and cone responses made possible with such a system. PMID:24959652

Lack of collagen VI promotes neurodegeneration by impairing autophagy and inducing apoptosis during aging.

PubMed

Cescon, Matilde; Chen, Peiwen; Castagnaro, Silvia; Gregorio, Ilaria; Bonaldo, Paolo

2016-05-01

Collagen VI is an extracellular matrix (ECM) protein with a broad distribution in different tissues and mostly deposited at the close periphery of the cell surface. Previous studies revealed that collagen VI protects neurons from the toxicity of amyloid-βpeptides and from UV-induced damage. However, the physiological role of this protein in the central nervous system (CNS) remains unknown. Here, we established primary neural cultures from murine cortex and hippocampus, and carried out in vitro and in vivo studies in wild-type and collagen VI null (Col6a1-/-) mice. Col6a1-/- neural cultures displayed an increased incidence of spontaneous apoptosis and higher vulnerability to oxidative stress, accompanied by altered regulation of autophagy with increased p62 protein levels and decreased LC3 lipidation. Analysis of brain sections confirmed increased apoptosis and abnormal regulation of autophagy in the CNS of collagen VI-deficient animals. To investigate the in vivo physiological consequences of these CNS defects, we carried out functional studies and found that motor and memory task performances were impaired in aged Col6a1-/-mice. These findings indicate that lack of collagen VI leads to spontaneous apoptosis and defective autophagy in neural cells, and point at a protective role for this ECM protein in the CNS during physiological aging.

Back-Propagation of Physiological Action Potential Output in Dendrites of Slender-Tufted L5A Pyramidal Neurons

PubMed Central

Grewe, Benjamin F.; Bonnan, Audrey; Frick, Andreas

2009-01-01

Pyramidal neurons of layer 5A are a major neocortical output type and clearly distinguished from layer 5B pyramidal neurons with respect to morphology, in vivo firing patterns, and connectivity; yet knowledge of their dendritic properties is scant. We used a combination of whole-cell recordings and Ca2+ imaging techniques in vitro to explore the specific dendritic signaling role of physiological action potential patterns recorded in vivo in layer 5A pyramidal neurons of the whisker-related ‘barrel cortex’. Our data provide evidence that the temporal structure of physiological action potential patterns is crucial for an effective invasion of the main apical dendrites up to the major branch point. Both the critical frequency enabling action potential trains to invade efficiently and the dendritic calcium profile changed during postnatal development. In contrast to the main apical dendrite, the more passive properties of the short basal and apical tuft dendrites prevented an efficient back-propagation. Various Ca2+ channel types contributed to the enhanced calcium signals during high-frequency firing activity, whereas A-type K+ and BKCa channels strongly suppressed it. Our data support models in which the interaction of synaptic input with action potential output is a function of the timing, rate and pattern of action potentials, and dendritic location. PMID:20508744

Physiological Intracellular Crowdedness is Defined by the Perimeter-to-Area Ratio of Sub-Cellular Compartments

PubMed Central

Hiroi, Noriko; Okuhara, Takahiro; Kubojima, Takeshi; Iba, Keisuke; Tabira, Akito; Yamashita, Shuji; Okada, Yasunori; Kobayashi, Tetsuya J.; Funahashi, Akira

2012-01-01

The intracellular environment is known to be a crowded and inhomogeneous space. Such an in vivo environment differs from a well-diluted, homogeneous environment for biochemical reactions. However, the effects of both crowdedness and the inhomogeneity of environment on the behavior of a mobile particle have not yet been investigated sufficiently. As described in this paper, we constructed artificial reaction spaces with fractal models, which are assumed to be non-reactive solid obstacles in a reaction space with crevices that function as operating ranges for mobile particles threading the space. Because of the homogeneity of the structures of artificial reaction spaces, the models succeeded in reproducing the physiological fractal dimension of solid structures with a smaller number of non-reactive obstacles than in the physiological condition. This incomplete compatibility was mitigated when we chose a suitable condition of a perimeter-to-area ratio of the operating range to our model. Our results also show that a simulation space is partitioned into convenient reaction compartments as an in vivo environment with the exact amount of solid structures estimated from TEM images. The characteristics of these compartments engender larger mean square displacement of a mobile particle than that of particles in smaller compartments. Subsequently, the particles start to show confined particle-like behavior. These results are compatible with our previously presented results, which predicted that a physiological environment would produce quick response and slow exhaustion reactions. PMID:22936917

In vivo imaging of the neurovascular unit in CNS disease

PubMed Central

Merlini, Mario; Davalos, Dimitrios; Akassoglou, Katerina

2014-01-01

The neurovascular unit—comprised of glia, pericytes, neurons and cerebrovasculature—is a dynamic interface that ensures physiological central nervous system (CNS) functioning. In disease dynamic remodeling of the neurovascular interface triggers a cascade of responses that determine the extent of CNS degeneration and repair. The dynamics of these processes can be adequately captured by imaging in vivo, which allows the study of cellular responses to environmental stimuli and cell-cell interactions in the living brain in real time. This perspective focuses on intravital imaging studies of the neurovascular unit in stroke, multiple sclerosis (MS) and Alzheimer disease (AD) models and discusses their potential for identifying novel therapeutic targets. PMID:25197615

Ivabradine and metoprolol differentially affect cardiac glucose metabolism despite similar heart rate reduction in a mouse model of dyslipidemia.

PubMed

Vaillant, Fanny; Lauzier, Benjamin; Ruiz, Matthieu; Shi, Yanfen; Lachance, Dominic; Rivard, Marie-Eve; Bolduc, Virginie; Thorin, Eric; Tardif, Jean-Claude; Des Rosiers, Christine

2016-10-01

While heart rate reduction (HRR) is a target for the management of patients with heart disease, contradictory results were reported using ivabradine, which selectively inhibits the pacemaker I f current, vs. β-blockers like metoprolol. This study aimed at testing whether similar HRR with ivabradine vs. metoprolol differentially modulates cardiac energy substrate metabolism, a factor determinant for cardiac function, in a mouse model of dyslipidemia (hApoB +/+ ;LDLR -/- ). Following a longitudinal study design, we used 3- and 6-mo-old mice, untreated or treated for 3 mo with ivabradine or metoprolol. Cardiac function was evaluated in vivo and ex vivo in working hearts perfused with 13 C-labeled substrates to assess substrate fluxes through energy metabolic pathways. Compared with 3-mo-old, 6-mo-old dyslipidemic mice had similar cardiac hemodynamics in vivo but impaired (P < 0.001) contractile function (aortic flow: -45%; cardiac output: -34%; stroke volume: -35%) and glycolysis (-24%) ex vivo. Despite inducing a similar 10% HRR, ivabradine-treated hearts displayed significantly higher stroke volume values and glycolysis vs. their metoprolol-treated counterparts ex vivo, values for the ivabradine group being often not significantly different from 3-mo-old mice. Further analyses highlighted additional significant cardiac alterations with disease progression, namely in the total tissue level of proteins modified by O-linked N-acetylglucosamine (O-GlcNAc), whose formation is governed by glucose metabolism via the hexosamine biosynthetic pathway, which showed a similar pattern with ivabradine vs. metoprolol treatment. Collectively, our results emphasize the implication of alterations in cardiac glucose metabolism and signaling linked to disease progression in our mouse model. Despite similar HRR, ivabradine, but not metoprolol, preserved cardiac function and glucose metabolism during disease progression. Copyright © 2016 the American Physiological Society.

Lack of evidence in vivo for a remote effect of Escherichia coli heat stable enterotoxin on jejunal fluid absorption.

PubMed

Lucas, M L; Duncan, N W; o'reilly, N F; McIlvenny, T J; Nelson, Y B

2008-05-01

On contact with the mucosa, heat stable (STa) enterotoxin from Escherichia coli reduces fluid absorption in vivo in the perfused jejunum of the anaesthetized rat. The question of whether it also has a vagally mediated remote action on jejunal absorption, when instilled into the ileum, was re-examined, given contradictory findings in the literature. A standard perfused loop preparation was used to measure luminal uptake of fluid in vivo by means of volume recovery. STa in the ileum was found to have no effect on jejunal absorption, regardless of cervical or sub-diaphragmatic vagotomy and also regardless of the nature of the perfusate anion. The batches of toxin were shown in parallel experiments to reduce fluid absorption directly in the jejunum and also in the ileum. Similarly, vagal nerves prior to section had demonstrable in vivo physiological function. There was therefore no evidence for an indirect, vagally mediated ileal effect of STa on proximal fluid absorption.

An in vitro and in vivo investigation of bivalent ligands that display preferential binding and functional activity for different melanocortin receptor homodimers

PubMed Central

Lensing, Cody J.; Freeman, Katie T.; Schnell, Sathya M.; Adank, Danielle N.; Speth, Robert C.; Haskell-Luevano, Carrie

2017-01-01

Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 (CJL-1-140) possessed a functional profile that was unique from its monovalent counterparts providing evidence of the discrete effects of bivalent ligands. Lead compound 7 (CJL-1-87) significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects. PMID:26959173

Development and function of human innate immune cells in a humanized mouse model.

PubMed

Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V; Teichmann, Lino L; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A Karolina; Manz, Markus G; Flavell, Richard A

2014-04-01

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

Development and function of human innate immune cells in a humanized mouse model

PubMed Central

Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V.; Teichmann, Lino L.; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A. Karolina; Manz, Markus G.; Flavell, Richard A.

2014-01-01

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models are unable to support development of human innate immune cells, including myeloid cells and NK cells. Here we describe a mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology. PMID:24633240

Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

DOE PAGES

Zhao, Suwen; Sakai, Ayano; Zhang, Xinshuai; ...

2014-06-30

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins inmore » 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.« less

Cardiac mTOR rescues the detrimental effects of diet-induced obesity in the heart after ischemia-reperfusion.

PubMed

Aoyagi, Toshinori; Higa, Jason K; Aoyagi, Hiroko; Yorichika, Naaiko; Shimada, Briana K; Matsui, Takashi

2015-06-15

Diet-induced obesity deteriorates the recovery of cardiac function after ischemia-reperfusion (I/R) injury. While mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, the effects of cardiac mTOR in ischemic injury under metabolic syndrome remains undefined. Using cardiac-specific transgenic mice overexpressing mTOR (mTOR-Tg mice), we studied the effect of mTOR on cardiac function in both ex vivo and in vivo models of I/R injury in high-fat diet (HFD)-induced obese mice. mTOR-Tg and wild-type (WT) mice were fed a HFD (60% fat by calories) for 12 wk. Glucose intolerance and insulin resistance induced by the HFD were comparable between WT HFD-fed and mTOR-Tg HFD-fed mice. Functional recovery after I/R in the ex vivo Langendorff perfusion model was significantly lower in HFD-fed mice than normal chow diet-fed mice. mTOR-Tg mice demonstrated better cardiac function recovery and had less of the necrotic markers creatine kinase and lactate dehydrogenase in both feeding conditions. Additionally, mTOR overexpression suppressed expression of proinflammatory cytokines, including IL-6 and TNF-α, in both feeding conditions after I/R injury. In vivo I/R models showed that at 1 wk after I/R, HFD-fed mice exhibited worse cardiac function and larger myocardial scarring along myofibers compared with normal chow diet-fed mice. In both feeding conditions, mTOR overexpression preserved cardiac function and prevented myocardial scarring. These findings suggest that cardiac mTOR overexpression is sufficient to prevent the detrimental effects of diet-induced obesity on the heart after I/R, by reducing cardiac dysfunction and myocardial scarring. Copyright © 2015 the American Physiological Society.

Classification of lymphoid neoplasms: the microscope as a tool for disease discovery

PubMed Central

Harris, Nancy Lee; Stein, Harald; Isaacson, Peter G.

2008-01-01

In the past 50 years, we have witnessed explosive growth in the understanding of normal and neoplastic lymphoid cells. B-cell, T-cell, and natural killer (NK)–cell neoplasms in many respects recapitulate normal stages of lymphoid cell differentiation and function, so that they can be to some extent classified according to the corresponding normal stage. Likewise, the molecular mechanisms involved the pathogenesis of lymphomas and lymphoid leukemias are often based on the physiology of the lymphoid cells, capitalizing on deregulated normal physiology by harnessing the promoters of genes essential for lymphocyte function. The clinical manifestations of lymphomas likewise reflect the normal function of lymphoid cells in vivo. The multiparameter approach to classification adopted by the World Health Organization (WHO) classification has been validated in international studies as being highly reproducible, and enhancing the interpretation of clinical and translational studies. In addition, accurate and precise classification of disease entities facilitates the discovery of the molecular basis of lymphoid neoplasms in the basic science laboratory. PMID:19029456

Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival*

PubMed Central

Peng, Lirong; Yuan, Zhigang; Li, Yixuan; Ling, Hongbo; Izumi, Victoria; Fang, Bin; Fukasawa, Kenji; Koomen, John; Chen, Jiandong; Seto, Edward

2015-01-01

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR. PMID:25670865

Assessment of Renal Hemodynamics and Oxygenation by Simultaneous Magnetic Resonance Imaging (MRI) and Quantitative Invasive Physiological Measurements.

PubMed

Cantow, Kathleen; Arakelyan, Karen; Seeliger, Erdmann; Niendorf, Thoralf; Pohlmann, Andreas

2016-01-01

In vivo assessment of renal perfusion and oxygenation under (patho)physiological conditions by means of noninvasive diagnostic imaging is conceptually appealing. Blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) and quantitative parametric mapping of the magnetic resonance (MR) relaxation times T 2* and T 2 are thought to provide surrogates of renal tissue oxygenation. The validity and efficacy of this technique for quantitative characterization of local tissue oxygenation and its changes under different functional conditions have not been systematically examined yet and remain to be established. For this purpose, the development of an integrative multimodality approaches is essential. Here we describe an integrated hybrid approach (MR-PHYSIOL) that combines established quantitative physiological measurements with T 2* (T 2) mapping and MR-based kidney size measurements. Standardized reversible (patho)physiologically relevant interventions, such as brief periods of aortic occlusion, hypoxia, and hyperoxia, are used for detailing the relation between the MR-PHYSIOL parameters, in particular between renal T 2* and tissue oxygenation.

Impaired adaptability of in vivo mitochondrial energetics to acute oxidative insult in aged skeletal muscle.

PubMed

Siegel, Michael P; Wilbur, Tim; Mathis, Mark; Shankland, Eric G; Trieu, Atlas; Harper, Mary-Ellen; Marcinek, David J

2012-01-01

Periods of elevated reactive oxygen species (ROS) production are a normal part of mitochondrial physiology. However, little is known about age-related changes in the mitochondrial response to elevated ROS in vivo. Significantly, ROS-induced uncoupling of oxidative phosphorylation has received attention as a negative feedback mechanism to reduce mitochondrial superoxide production. Here we use a novel in vivo spectroscopy system to test the hypothesis that ROS-induced uncoupling is diminished in aged mitochondria. This system simultaneously acquires (31)P magnetic resonance and near-infrared optical spectra to non-invasively measure phosphometabolite and O(2) concentrations in mouse skeletal muscle. Using low dose paraquat to elevate intracellular ROS we assess in vivo mitochondrial function in young, middle aged, and old mice. Oxidative phosphorylation was uncoupled to the same degree in response to ROS at each age, but this uncoupling was associated with loss of phosphorylation capacity and total ATP in old mice only. Using mice lacking UCP3 we demonstrate that this in vivo uncoupling is independent of this putative uncoupler of skeletal muscle mitochondria. These data indicate that ROS-induced uncoupling persists throughout life, but that oxidative stress leads to mitochondrial deficits and loss of ATP in aged organisms that may contribute to impaired function and degeneration. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Engineering nucleic acid structures for programmable molecular circuitry and intracellular biocomputation

NASA Astrophysics Data System (ADS)

Li, Jiang; Green, Alexander A.; Yan, Hao; Fan, Chunhai

2017-11-01

Nucleic acids have attracted widespread attention due to the simplicity with which they can be designed to form discrete structures and programmed to perform specific functions at the nanoscale. The advantages of DNA/RNA nanotechnology offer numerous opportunities for in-cell and in-vivo applications, and the technology holds great promise to advance the growing field of synthetic biology. Many elegant examples have revealed the potential in integrating nucleic acid nanostructures in cells and in vivo where they can perform important physiological functions. In this Review, we summarize the current abilities of DNA/RNA nanotechnology to realize applications in live cells and then discuss the key problems that must be solved to fully exploit the useful properties of nanostructures. Finally, we provide viewpoints on how to integrate the tools provided by DNA/RNA nanotechnology and related new technologies to construct nucleic acid nanostructure-based molecular circuitry for synthetic biology.

Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro.

PubMed

Torisawa, Yu-suke; Spina, Catherine S; Mammoto, Tadanori; Mammoto, Akiko; Weaver, James C; Tat, Tracy; Collins, James J; Ingber, Donald E

2014-06-01

Current in vitro hematopoiesis models fail to demonstrate the cellular diversity and complex functions of living bone marrow; hence, most translational studies relevant to the hematologic system are conducted in live animals. Here we describe a method for fabricating 'bone marrow-on-a-chip' that permits culture of living marrow with a functional hematopoietic niche in vitro by first engineering new bone in vivo, removing it whole and perfusing it with culture medium in a microfluidic device. The engineered bone marrow (eBM) retains hematopoietic stem and progenitor cells in normal in vivo-like proportions for at least 1 week in culture. eBM models organ-level marrow toxicity responses and protective effects of radiation countermeasure drugs, whereas conventional bone marrow culture methods do not. This biomimetic microdevice offers a new approach for analysis of drug responses and toxicities in bone marrow as well as for study of hematopoiesis and hematologic diseases in vitro.

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

PubMed Central

Riggs, Daniel L.; Roberts, Patricia J.; Chirillo, Samantha C.; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F.

2003-01-01

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. PMID:12606580

Intercellular Ca2+ Waves: Mechanisms and Function

PubMed Central

Sanderson, Michael J.

2012-01-01

Intercellular calcium (Ca2+) waves (ICWs) represent the propagation of increases in intracellular Ca2+ through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca2+ from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra- and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs. PMID:22811430

Effects of novel muscarinic M3 receptor ligand C1213 in pulmonary arterial hypertension models.

PubMed

Ahmed, Mohamed; VanPatten, Sonya; Lakshminrusimha, Satyan; Patel, Hardik; Coleman, Thomas R; Al-Abed, Yousef

2016-12-01

Pulmonary hypertension (PH) is a complex disease comprising a pathologic remodeling and thickening of the pulmonary vessels causing an after load on the right heart ventricle that can result in ventricular failure. Triggered by oxidative stress, episodes of hypoxia, and other undetermined causes, PH is associated with poor outcomes and a high rate of morbidity. In the neonate, this disease has a similar etiology but is further complicated by the transition to breathing after birth, which requires a reduction in vascular resistance. Persistent pulmonary hypertension of the newborn (PPHN) is one form of PH that is frequently unresponsive to current therapies including inhaled nitric oxide (due to lack of proper absorption and diffusion), and other therapeutics targeting signaling mediators in vascular endothelium and smooth muscle. The need for novel agents, which target distinct pathways in pulmonary hypertension, remains. Herein, we investigated the therapeutic effects of novel muscarinic receptor ligand C1213 in models of PH We demonstrated that via M3 muscarinic receptors, C1213 induced activating- eNOS phosphorylation (serine-1177), which is known to lead to nitric oxide (NO) production in endothelial cells. Using signaling pathway inhibitors, we discovered that AKT and calcium signaling contributed to eNOS phosphorylation induced by C1213. As expected for an eNOS-stimulating agent, in ex vivo and in vivo models, C1213 triggered pulmonary vasodilation and induced both pulmonary artery and systemic blood pressure reductions demonstrating its potential value in PH and PPHN In brief, this proof-of-concept study provides evidence that an M3 muscarinic receptor functionally selective ligand stimulates downstream pathways leading to antihypertensive effects using in vitro, ex vivo, and in vivo models of PH. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

A novel organotypic 3D sweat gland model with physiological functionality

PubMed Central

Grüdl, Sabine; Banowski, Bernhard; Giesen, Melanie; Sättler, Andrea; Proksch, Peter; Welss, Thomas; Förster, Thomas

2017-01-01

Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism. PMID:28796813

In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish.

PubMed

Olt, Jennifer; Johnson, Stuart L; Marcotti, Walter

2014-05-15

Hair cells detect and process sound and movement information, and transmit this with remarkable precision and efficiency to afferent neurons via specialized ribbon synapses. The zebrafish is emerging as a powerful model for genetic analysis of hair cell development and function both in vitro and in vivo. However, the full exploitation of the zebrafish is currently limited by the difficulty in obtaining systematic electrophysiological recordings from hair cells under physiological recording conditions. Thus, the biophysical properties of developing and adult zebrafish hair cells are largely unknown. We investigated potassium and calcium currents, voltage responses and synaptic activity in hair cells from the lateral line and inner ear in vivo and using near-physiological in vitro recordings. We found that the basolateral current profile of hair cells from the lateral line becomes more segregated with age, and that cells positioned in the centre of the neuromast show more mature characteristics and those towards the edge retain a more immature phenotype. The proportion of mature-like hair cells within a given neuromast increased with zebrafish development. Hair cells from the inner ear showed a developmental change in current profile between the juvenile and adult stages. In lateral line hair cells from juvenile zebrafish, exocytosis also became more efficient and required less calcium for vesicle fusion. In hair cells from mature zebrafish, the biophysical characteristics of ion channels and exocytosis resembled those of hair cells from other lower vertebrates and, to some extent, those in the immature mammalian vestibular and auditory systems. We show that although the zebrafish provides a suitable animal model for studies on hair cell physiology, it is advisable to consider that the age at which the majority of hair cells acquire a mature-type configuration is reached only in the juvenile lateral line and in the inner ear from >2 months after hatching. © 2014 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Leptin deficiency in vivo enhances the ability of splenic dendritic cells to activate T cells

PubMed Central

Ramirez, Oscar

2014-01-01

Leptin is a pleiotropic adipokine that is critical for regulating food intake and energy expenditure and also participates in functions of the immune system, including those of antigen-presenting cells. Here, we assess the effect of leptin deficiency on the function splenic dendritic cells (sDC). sDC from leptin-deficient mice (Lepob) were evaluated ex vivo for phenotype, ability to respond to inflammatory stimuli, to acquire and process antigens and to activate T cells. The data show that Lepob sDC express activation markers similar to controls and respond similarly to LPS activation or anti-CD40 cross-linking. In addition, antigen acquisition and processing by Lepob sDC was similar to controls. However, Lepob sDC elicited higher production of IFN-γ in mixed lymphocyte reactions and increased production of IL-2 by antigen-specific T-cell hybridoma relative to controls. To assess Lepob sDC activation of T cells in vivo, Lepob and control mice were infected systemically with Mycobacterium avium. Lepob mice were significantly better at neutralizing the infection as measured by splenic bacterial load over time. This was mirrored with an increased percentage of activated T cells in M. avium-infected Lepob mice. Thus, although no changes were detected in sDC phenotype, activation, antigen processing or presentation, these DC surprisingly presented an enhanced ability to activate T cells ex vivo and in vivo. These data demonstrate that leptin can modulate DC function and suggest that leptin may dampen T-cell responsiveness in the physiological setting. PMID:24966213

Mineralized three-dimensional bone constructs

NASA Technical Reports Server (NTRS)

Pellis, Neal R. (Inventor); Clarke, Mark S. F. (Inventor); Sundaresan, Alamelu (Inventor)

2011-01-01

The present disclosure provides ex vivo-derived mineralized three-dimensional bone constructs. The bone constructs are obtained by culturing osteoblasts and osteoclast precursors under randomized gravity vector conditions. Preferably, the randomized gravity vector conditions are obtained using a low shear stress rotating bioreactor, such as a High Aspect Ratio Vessel (HARV) culture system. The bone constructs of the disclosure have utility in physiological studies of bone formation and bone function, in drug discovery, and in orthopedics.

Mineralized Three-Dimensional Bone Constructs

NASA Technical Reports Server (NTRS)

Clarke, Mark S. F. (Inventor); Sundaresan, Alamelu (Inventor); Pellis, Neal R. (Inventor)

2013-01-01

The present disclosure provides ex vivo-derived mineralized three-dimensional bone constructs. The bone constructs are obtained by culturing osteoblasts and osteoclast precursors under randomized gravity vector conditions. Preferably, the randomized gravity vector conditions are obtained using a low shear stress rotating bioreactor, such as a High Aspect Ratio Vessel (HARV) culture system. The bone constructs of the disclosure have utility in physiological studies of bone formation and bone function, in drug discovery, and in orthopedics.

Development of diabetes does not alter behavioral and molecular circadian rhythms in a transgenic rat model of type 2 diabetes mellitus.

PubMed

Qian, Jingyi; Thomas, Anthony P; Schroeder, Analyne M; Rakshit, Kuntol; Colwell, Christopher S; Matveyenko, Aleksey V

2017-08-01

Metabolic state and circadian clock function exhibit a complex bidirectional relationship. Circadian disruption increases propensity for metabolic dysfunction, whereas common metabolic disorders such as obesity and type 2 diabetes (T2DM) are associated with impaired circadian rhythms. Specifically, alterations in glucose availability and glucose metabolism have been shown to modulate clock gene expression and function in vitro; however, to date, it is unknown whether development of diabetes imparts deleterious effects on the suprachiasmatic nucleus (SCN) circadian clock and SCN-driven outputs in vivo. To address this question, we undertook studies in aged diabetic rats transgenic for human islet amyloid polypeptide, an established nonobese model of T2DM (HIP rat), which develops metabolic defects closely recapitulating those present in patients with T2DM. HIP rats were also cross-bred with a clock gene reporter rat model (Per1:luciferase transgenic rat) to permit assessment of the SCN and the peripheral molecular clock function ex vivo. Utilizing these animal models, we examined effects of diabetes on 1 ) behavioral circadian rhythms, 2 ) photic entrainment of circadian activity, 3 ) SCN and peripheral tissue molecular clock function, and 4 ) melatonin secretion. We report that circadian activity, light-induced entrainment, molecular clockwork, as well as melatonin secretion are preserved in the HIP rat model of T2DM. These results suggest that despite the well-characterized ability of glucose to modulate circadian clock gene expression acutely in vitro, SCN clock function and key behavioral and physiological outputs appear to be preserved under chronic diabetic conditions characteristic of nonobese T2DM. Copyright © 2017 the American Physiological Society.

Electrophysiology of glioma: a Rho GTPase-activating protein reduces tumor growth and spares neuron structure and function.

PubMed

Vannini, Eleonora; Olimpico, Francesco; Middei, Silvia; Ammassari-Teule, Martine; de Graaf, Erik L; McDonnell, Liam; Schmidt, Gudula; Fabbri, Alessia; Fiorentini, Carla; Baroncelli, Laura; Costa, Mario; Caleo, Matteo

2016-12-01

Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Development of a rate model to investigate contributions of anatomic and physiologic determinants of in vivo skin permeation

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

Fleischer, N.M.

The skin is a heterogeneous, bi-directional impediment to chemical flux, in which the stratum corneum is a major, though not the sole, rate-limiting barrier layer to permeation. Systemic toxicity following dermal exposure to environmental chemicals and use of skin as a portal for systemic administration of drugs have led to extensive investigations of the inward flux of xenobiotics applied to the outer surface of skin. Those investigations mainly utilized in vitro experimental systems that were limited by the absence of normal physiologic functions. The objective of the present research was to investigate an in vivo skin permeation model system thatmore » was sensitive to perturbations of skin capillary physiology and stratum corneum. A [open quotes]fuzzy[close quotes] rat model system was devised that employed outward cutaneous migration of a systemically administered permeation probe, isoflurane. Specially devised, transdermal vapor collection devices were used to capture the outward flux of isoflurane through the skin. Isoflurane flux measurements, coupled with blood isoflurane concentrations, were used to calculate cutaneous permeability coefficients (K[sub p]) of isolflurane, as an index of permeation, under various conditions of normal or perturbed cutaneous physiologic states. Physiologic perturbations were performed to test the sensitivity of the model system to detect effects of minoxidil-mediated vasodilation, phenylephrine-mediated vasoconstriction, and leukotriene D[sub 4]-mediated increased capillary permeability on the outward flux of isoflurane. Tape stripping and topical ether-ethanol application produced either physical removal or chemical disruption of the stratum corneum, respectively. Minoxidil, leukotriene D[sub 4], tape stripping of stratum corneum, and topical ether-ethanol experiments produced statistically significant increases (52 to 193%) in the K[sub p's], while phenylephrine had no significant effect on isoflurane permeation.« less

Eyes Open on Sleep and Wake: In Vivo to In Silico Neural Networks

PubMed Central

Vanvinckenroye, Amaury; Vandewalle, Gilles; Chellappa, Sarah L.

2016-01-01

Functional and effective connectivity of cortical areas are essential for normal brain function under different behavioral states. Appropriate cortical activity during sleep and wakefulness is ensured by the balanced activity of excitatory and inhibitory circuits. Ultimately, fast, millisecond cortical rhythmic oscillations shape cortical function in time and space. On a much longer time scale, brain function also depends on prior sleep-wake history and circadian processes. However, much remains to be established on how the brain operates at the neuronal level in humans during sleep and wakefulness. A key limitation of human neuroscience is the difficulty in isolating neuronal excitation/inhibition drive in vivo. Therefore, computational models are noninvasive approaches of choice to indirectly access hidden neuronal states. In this review, we present a physiologically driven in silico approach, Dynamic Causal Modelling (DCM), as a means to comprehend brain function under different experimental paradigms. Importantly, DCM has allowed for the understanding of how brain dynamics underscore brain plasticity, cognition, and different states of consciousness. In a broader perspective, noninvasive computational approaches, such as DCM, may help to puzzle out the spatial and temporal dynamics of human brain function at different behavioural states. PMID:26885400

Multifunctional cell-culture platform for aligned cell sheet monitoring, transfer printing, and therapy.

PubMed

Kim, Seok Joo; Cho, Hye Rim; Cho, Kyoung Won; Qiao, Shutao; Rhim, Jung Soo; Soh, Min; Kim, Taeho; Choi, Moon Kee; Choi, Changsoon; Park, Inhyuk; Hwang, Nathaniel S; Hyeon, Taeghwan; Choi, Seung Hong; Lu, Nanshu; Kim, Dae-Hyeong

2015-03-24

While several functional platforms for cell culturing have been proposed for cell sheet engineering, a soft integrated system enabling in vitro physiological monitoring of aligned cells prior to their in vivo applications in tissue regeneration has not been reported. Here, we present a multifunctional, soft cell-culture platform equipped with ultrathin stretchable nanomembrane sensors and graphene-nanoribbon cell aligners, whose system modulus is matched with target tissues. This multifunctional platform is capable of aligning plated cells and in situ monitoring of cellular physiological characteristics during proliferation and differentiation. In addition, it is successfully applied as an in vitro muscle-on-a-chip testing platform. Finally, a simple but high-yield transfer printing mechanism is proposed to deliver cell sheets for scaffold-free, localized cell therapy in vivo. The muscle-mimicking stiffness of the platform allows the high-yield transfer printing of multiple cell sheets and results in successful therapies in diseased animal models. Expansion of current results to stem cells will provide unique opportunities for emerging classes of tissue engineering and cell therapy technologies.

Identifying Low pH Active and Lactate-Utilizing Taxa within Oral Microbiome Communities from Healthy Children Using Stable Isotope Probing Techniques

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

McLean, Jeffrey S.; Fansler, Sarah J.; Majors, Paul D.

Many human microbial infectious diseases including dental caries are polymicrobial in nature and how these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral microbes have been characterized in vitro, their physiology in vivo in the presence of the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these oral species remain uncultivated to date and little is known except their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated microorganisms will remain enigmatic despite their apparent disease correlation.more » To start addressing these knowledge gaps, we applied a novel combination of in vivo Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for temporal monitoring of carbohydrate utilization, organic acid production and identification of metabolically active and inactive bacterial species.« less

What do magnetic resonance-based measurements of Pi→ATP flux tell us about skeletal muscle metabolism?

PubMed

Kemp, Graham J; Brindle, Kevin M

2012-08-01

Magnetic resonance spectroscopy (MRS) methods offer a potentially valuable window into cellular metabolism. Measurement of flux between inorganic phosphate (Pi) and ATP using (31)P MRS magnetization transfer has been used in resting muscle to assess what is claimed to be mitochondrial ATP synthesis and has been particularly popular in the study of insulin effects and insulin resistance. However, the measured Pi→ATP flux in resting skeletal muscle is far higher than the true rate of oxidative ATP synthesis, being dominated by a glycolytically mediated Pi↔ATP exchange reaction that is unrelated to mitochondrial function. Furthermore, even if measured accurately, the ATP production rate in resting muscle has no simple relationship to mitochondrial capacity as measured either ex vivo or in vivo. We summarize the published measurements of Pi→ATP flux, concentrating on work relevant to diabetes and insulin, relate it to current understanding of the physiology of mitochondrial ATP synthesis and glycolytic Pi↔ATP exchange, and discuss some possible implications of recently reported correlations between Pi→ATP flux and other physiological measures.

Development of Microplatforms to Mimic the In Vivo Architecture of CNS and PNS Physiology and Their Diseases.

PubMed

Saliba, John; Daou, Arij; Damiati, Samar; Saliba, Jessica; El-Sabban, Marwan; Mhanna, Rami

2018-06-06

Understanding the mechanisms that govern nervous tissues function remains a challenge. In vitro two-dimensional (2D) cell culture systems provide a simplistic platform to evaluate systematic investigations but often result in unreliable responses that cannot be translated to pathophysiological settings. Recently, microplatforms have emerged to provide a better approximation of the in vivo scenario with better control over the microenvironment, stimuli and structure. Advances in biomaterials enable the construction of three-dimensional (3D) scaffolds, which combined with microfabrication, allow enhanced biomimicry through precise control of the architecture, cell positioning, fluid flows and electrochemical stimuli. This manuscript reviews, compares and contrasts advances in nervous tissues-on-a-chip models and their applications in neural physiology and disease. Microplatforms used for neuro-glia interactions, neuromuscular junctions (NMJs), blood-brain barrier (BBB) and studies on brain cancer, metastasis and neurodegenerative diseases are addressed. Finally, we highlight challenges that can be addressed with interdisciplinary efforts to achieve a higher degree of biomimicry. Nervous tissue microplatforms provide a powerful tool that is destined to provide a better understanding of neural health and disease.

Physiological importance of RNA and protein mobility in the cell nucleus

PubMed Central

2007-01-01

Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways. PMID:17994245

Mechanisms and functions of extracellular vesicle release in vivo-What we can learn from flies and worms.

PubMed

Beer, Katharina B; Wehman, Ann Marie

2017-03-04

Cells from bacteria to man release extracellular vesicles (EVs) that contain signaling molecules like proteins, lipids, and nucleic acids. The content, formation, and signaling roles of these conserved vesicles are diverse, but the physiological relevance of EV signaling in vivo is still debated. Studies in classical genetic model organisms like C. elegans and Drosophila have begun to reveal the developmental and behavioral roles for EVs. In this review, we discuss the emerging evidence for the in vivo signaling roles of EVs. Significant effort has also been made to understand the mechanisms behind the formation and release of EVs, specifically of exosomes derived from exocytosis of multivesicular bodies and of microvesicles derived from plasma membrane budding called ectocytosis. In this review, we detail the impact of flies and worms on understanding the proteins and lipids involved in EV biogenesis and highlight the open questions in the field.

In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs

PubMed Central

Langlois, Ryan A; Shapiro, Jillian S; Pham, Alissa M; tenOever, Benjamin R

2012-01-01

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery. PMID:22086233

Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

NASA Astrophysics Data System (ADS)

Varela, Juan A.; Dupuis, Julien P.; Etchepare, Laetitia; Espana, Agnès; Cognet, Laurent; Groc, Laurent

2016-03-01

Single-molecule imaging has changed the way we understand many biological mechanisms, particularly in neurobiology, by shedding light on intricate molecular events down to the nanoscale. However, current single-molecule studies in neuroscience have been limited to cultured neurons or organotypic slices, leaving as an open question the existence of fast receptor diffusion in intact brain tissue. Here, for the first time, we targeted dopamine receptors in vivo with functionalized quantum dots and were able to perform single-molecule tracking in acute rat brain slices. We propose a novel delocalized and non-inflammatory way of delivering nanoparticles (NPs) in vivo to the brain, which allowed us to label and track genetically engineered surface dopamine receptors in neocortical neurons, revealing inherent behaviour and receptor activity regulations. We thus propose a NP-based platform for single-molecule studies in the living brain, opening new avenues of research in physiological and pathological animal models.

Red-Emitting Fluorescent Probe for Detection of γ-Glutamyltranspeptidase and Its Application of Real-Time Imaging under Oxidative Stress in Cells and in Vivo.

PubMed

Liu, Feiyan; Wang, Zhen; Wang, Wenli; Luo, Jian-Guang; Kong, Lingyi

2018-06-19

γ-Glutamyltranspeptidase (GGT) plays critical roles in regulating various physiological/pathophysiological processes including the intracellular redox homeostasis. However, an effective fluorescent probe for dissecting the relationships between GGT and oxidative stress in vivo remains largely unexplored. Herein, we present a light-up fluorescent probe (DCDHF-Glu) with long wavelength emission (613 nm) for the highly sensitive and selective detection of GGT using dicyanomethylenedihydrofuran derivative as the fluorescent reporter and γ-glutamyl group as the enzyme-active trigger. DCDHF-Glu is competent to real-time image endogenous GGT in live cells and mice. In particular, DCDHF-Glu enables the direct real-time visualization of the upregulation of GGT under drug-induced oxidative stress in the HepG2 cells and the LO2 cells, as well as in vivo, vividly implying its excellent capacity in elucidation of GGT function in GGT-related biological events.

Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis.

PubMed

McDonald, Nathan A; Vander Kooi, Craig W; Ohi, Melanie D; Gould, Kathleen L

2015-12-21

F-BAR proteins function in diverse cellular processes by linking membranes to the actin cytoskeleton. Through oligomerization, multiple F-BAR domains can bend membranes into tubules, though the physiological importance of F-BAR-to-F-BAR assemblies is not yet known. Here, we investigate the F-BAR domain of the essential cytokinetic scaffold, Schizosaccharomyces pombe Cdc15, during cytokinesis. Challenging a widely held view that membrane deformation is a fundamental property of F-BARs, we report that the Cdc15 F-BAR binds, but does not deform, membranes in vivo or in vitro, and six human F-BAR domains-including those from Fer and RhoGAP4-share this property. Nevertheless, tip-to-tip interactions between F-BAR dimers are critical for Cdc15 oligomerization and high-avidity membrane binding, stabilization of contractile ring components at the medial cortex, and the fidelity of cytokinesis. F-BAR oligomerization is also critical for Fer and RhoGAP4 physiological function, demonstrating its broad importance to F-BAR proteins that function without membrane bending. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

NASA Astrophysics Data System (ADS)

Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

2016-09-01

We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD.

Sox2 expression in Schwann cells inhibits myelination in vivo and induces influx of macrophages to the nerve.

PubMed

Roberts, Sheridan L; Dun, Xin-Peng; Doddrell, Robin D S; Mindos, Thomas; Drake, Louisa K; Onaitis, Mark W; Florio, Francesca; Quattrini, Angelo; Lloyd, Alison C; D'Antonio, Maurizio; Parkinson, David B

2017-09-01

Correct myelination is crucial for the function of the peripheral nervous system. Both positive and negative regulators within the axon and Schwann cell function to ensure the correct onset and progression of myelination during both development and following peripheral nerve injury and repair. The Sox2 transcription factor is well known for its roles in the development and maintenance of progenitor and stem cell populations, but has also been proposed in vitro as a negative regulator of myelination in Schwann cells. We wished to test fully whether Sox2 regulates myelination in vivo and show here that, in mice, sustained Sox2 expression in vivo blocks myelination in the peripheral nerves and maintains Schwann cells in a proliferative non-differentiated state, which is also associated with increased inflammation within the nerve. The plasticity of Schwann cells allows them to re-myelinate regenerated axons following injury and we show that re-myelination is also blocked by Sox2 expression in Schwann cells. These findings identify Sox2 as a physiological regulator of Schwann cell myelination in vivo and its potential to play a role in disorders of myelination in the peripheral nervous system. © 2017. Published by The Company of Biologists Ltd.

In vivo articular cartilage deformation: noninvasive quantification of intratissue strain during joint contact in the human knee

NASA Astrophysics Data System (ADS)

Chan, Deva D.; Cai, Luyao; Butz, Kent D.; Trippel, Stephen B.; Nauman, Eric A.; Neu, Corey P.

2016-01-01

The in vivo measurement of articular cartilage deformation is essential to understand how mechanical forces distribute throughout the healthy tissue and change over time in the pathologic joint. Displacements or strain may serve as a functional imaging biomarker for healthy, diseased, and repaired tissues, but unfortunately intratissue cartilage deformation in vivo is largely unknown. Here, we directly quantified for the first time deformation patterns through the thickness of tibiofemoral articular cartilage in healthy human volunteers. Magnetic resonance imaging acquisitions were synchronized with physiologically relevant compressive loading and used to visualize and measure regional displacement and strain of tibiofemoral articular cartilage in a sagittal plane. We found that compression (of 1/2 body weight) applied at the foot produced a sliding, rigid-body displacement at the tibiofemoral cartilage interface, that loading generated subject- and gender-specific and regionally complex patterns of intratissue strains, and that dominant cartilage strains (approaching 12%) were in shear. Maximum principle and shear strain measures in the tibia were correlated with body mass index. Our MRI-based approach may accelerate the development of regenerative therapies for diseased or damaged cartilage, which is currently limited by the lack of reliable in vivo methods for noninvasive assessment of functional changes following treatment.

Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

PubMed Central

Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

2016-01-01

We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD. PMID:27653664

MRI to assess renal structure and function.

PubMed

Artunc, Ferruh; Rossi, Cristina; Boss, Andreas

2011-11-01

In addition to excellent anatomical depiction, MRI techniques have expanded to study functional aspects of renal physiology, such as renal perfusion, glomerular filtration rate (GFR) or tissue oxygenation. This review will focus on current developments with an emphasis on clinical applicability. The method of GFR determination is largely heterogeneous and still has weaknesses. However, the technique of employing liver disappearance curves has been shown to be accurate in healthy persons and patients with chronic kidney disease. In potential kidney donors, complete evaluation of kidney anatomy and function can be accomplished in a single-stop investigation. Techniques without contrast media can be utilized to measure renal tissue oxygenation (blood oxygen level-dependent MRI) or perfusion (arterial spin labeling) and could aid in the diagnosis and treatment of ischemic renal diseases, such as renal artery stenosis. Diffusion imaging techniques may provide information on spatially restricted water diffusion and tumor cellularity. Functional MRI opens new horizons in studying renal physiology and pathophysiology in vivo. Although extensively utilized in research, labor-intensive postprocessing and lack of standardization currently limit the clinical applicability of functional MRI. Further studies are necessary to evaluate the clinical value of functional magnetic resonance techniques for early discovery and characterization of kidney disease.

Bioresorbable silicon electronics for transient spatiotemporal mapping of electrical activity from the cerebral cortex.

PubMed

Yu, Ki Jun; Kuzum, Duygu; Hwang, Suk-Won; Kim, Bong Hoon; Juul, Halvor; Kim, Nam Heon; Won, Sang Min; Chiang, Ken; Trumpis, Michael; Richardson, Andrew G; Cheng, Huanyu; Fang, Hui; Thomson, Marissa; Bink, Hank; Talos, Delia; Seo, Kyung Jin; Lee, Hee Nam; Kang, Seung-Kyun; Kim, Jae-Hwan; Lee, Jung Yup; Huang, Younggang; Jensen, Frances E; Dichter, Marc A; Lucas, Timothy H; Viventi, Jonathan; Litt, Brian; Rogers, John A

2016-07-01

Bioresorbable silicon electronics technology offers unprecedented opportunities to deploy advanced implantable monitoring systems that eliminate risks, cost and discomfort associated with surgical extraction. Applications include postoperative monitoring and transient physiologic recording after percutaneous or minimally invasive placement of vascular, cardiac, orthopaedic, neural or other devices. We present an embodiment of these materials in both passive and actively addressed arrays of bioresorbable silicon electrodes with multiplexing capabilities, which record in vivo electrophysiological signals from the cortical surface and the subgaleal space. The devices detect normal physiologic and epileptiform activity, both in acute and chronic recordings. Comparative studies show sensor performance comparable to standard clinical systems and reduced tissue reactivity relative to conventional clinical electrocorticography (ECoG) electrodes. This technology offers general applicability in neural interfaces, with additional potential utility in treatment of disorders where transient monitoring and modulation of physiologic function, implant integrity and tissue recovery or regeneration are required.

A tunable artificial circadian clock in clock-defective mice

PubMed Central

D'Alessandro, Matthew; Beesley, Stephen; Kim, Jae Kyoung; Chen, Rongmin; Abich, Estela; Cheng, Wayne; Yi, Paul; Takahashi, Joseph S.; Lee, Choogon

2015-01-01

Self-sustaining oscillations are essential for diverse physiological functions such as the cell cycle, insulin secretion and circadian rhythms. Synthetic oscillators using biochemical feedback circuits have been generated in cell culture. These synthetic systems provide important insight into design principles for biological oscillators, but have limited similarity to physiological pathways. Here we report the generation of an artificial, mammalian circadian clock in vivo, capable of generating robust, tunable circadian rhythms. In mice deficient in Per1 and Per2 genes (thus lacking circadian rhythms), we artificially generate PER2 rhythms and restore circadian sleep/wake cycles with an inducible Per2 transgene. Our artificial clock is tunable as the period and phase of the rhythms can be modulated predictably. This feature, and other design principles of our work, might enhance the study and treatment of circadian dysfunction and broader aspects of physiology involving biological oscillators. PMID:26617050

Ultrasonically-Induced Cavitation In Vivo Depends on the Physiological State

NASA Astrophysics Data System (ADS)

Vykhodtseva, Natalia I.; Kondrashova, Maria N.

2006-05-01

Purpose: To test the hypothesis that the ultrasound-induced cavitation can be facilitated by excitation and stress. Methods: Acoustic emission was monitored from the region of the femoral artery of non-anesthetized rats using a resonant focusing detector (RFD). The RFD consists of two transducers: an inner transmitting transducer (0.87 MHz, R/D=70/28mm), an annular receiving transducer (0.43 MHz, R=70 mm, Dint/Dext=37 /58 mm). Acoustical emission was monitored: (1) during mild immobilization stress (MIS); (2) after strong immobilization stress (SIS); (3) after succinic acid administration (SUC); (4) during negative air-ion inhalation (NAI). Results: The subharmonic emission varied as a function of the physiological states. Both strong immobilization stress and SUC, which increases adrenaline release and stimulates physiological activity of cells, increased cavitation activity. Inhalation of NAI abolished stress-induced cavitation providing a sedative effect.

Predicting EMP hazard: Lessons from studies with inhaled fibrous and non-fibrous nano- and micro-particles.

PubMed

Oberdörster, Günter; Graham, Uschi

2018-05-08

Inhalation exposure to elongated cleavage fragments occurring at mineral and rock mining and crushing operations raises important questions regarding potential health effects given their resemblance to fibers with known adverse health effects like amphibole asbestos. Thus, a major goal for establishing a toxicity profile for elongate mineral particles (EMPs) is to identify and characterize a suspected hazard and characterize a risk by examining together results of hazard and exposure assessment. This will require not only knowledge about biokinetics of inhaled EMPs but also about underlying mechanisms of effects induced by retained EMPs. In vitro toxicity assays with predictive power for in vivo effects have been established as useful screening tools for toxicological characterization of particulate materials including EMPs. Important determinants of physiological/toxicological mechanisms are physico-chemical and functional properties of inhaled particulate materials. Of the physico-chemical (intrinsic) properties, size, shape and surface characteristics are well known to affect toxicological responses; functional properties include (i) solubility/dissolution rate in physiological fluid simulants in vitro and following inhalation in vivo; (ii) ROS-inducing capacity in vitro and in vivo determined as specific particle surface reactivity; (iii) bioprocessing in vivo. A key parameter for all is the dose and duration of exposure, requiring to establish exposure-dose-response relationships. Examples of studies with fibrous and non-fibrous particles are discussed to illustrate the relevancy of evaluating extrinsic and intrinsic particle properties for predicting in vivo responses of new particulate materials. This will allow hazard and risk ranking/grouping based on a comparison to toxicologically well-characterized positive and negative benchmarks. Future efforts should be directed at developing and validating new approaches using in vitro (non-animal) studies for establishing a complete risk assessment for EMPs. Further comparative in-depth analyses with analytical and ultra-high resolution technology examining bioprocessing events at target organ sites have proven highly successful to identify biotransformations in target cells at near atomic level. In the case of EMPs, such analyses can be essential to separate benign from harmful ones. Copyright © 2018. Published by Elsevier Inc.

Modeling the lung: Design and development of tissue engineered macro- and micro-physiologic lung models for research use.

PubMed

Nichols, Joan E; Niles, Jean A; Vega, Stephanie P; Argueta, Lissenya B; Eastaway, Adriene; Cortiella, Joaquin

2014-09-01

Respiratory tract specific cell populations, or tissue engineered in vitro grown human lung, have the potential to be used as research tools to mimic physiology, toxicology, pathology, as well as infectious diseases responses of cells or tissues. Studies related to respiratory tract pathogenesis or drug toxicity testing in the past made use of basic systems where single cell populations were exposed to test agents followed by evaluations of simple cellular responses. Although these simple single-cell-type systems provided good basic information related to cellular responses, much more can be learned from cells grown in fabricated microenvironments which mimic in vivo conditions in specialized microfabricated chambers or by human tissue engineered three-dimensional (3D) models which allow for more natural interactions between cells. Recent advances in microengineering technology, microfluidics, and tissue engineering have provided a new approach to the development of 2D and 3D cell culture models which enable production of more robust human in vitro respiratory tract models. Complex models containing multiple cell phenotypes also provide a more reasonable approximation of what occurs in vivo without the confounding elements in the dynamic in vivo environment. The goal of engineering good 3D human models is the formation of physiologically functional respiratory tissue surrogates which can be used as pathogenesis models or in the case of 2D screening systems for drug therapy evaluation as well as human toxicity testing. We hope that this manuscript will serve as a guide for development of future respiratory tract model systems as well as a review of conventional models. © 2014 by the Society for Experimental Biology and Medicine.

Phosphorescent nanosensors for in vivo tracking of histamine levels.

PubMed

Cash, Kevin J; Clark, Heather A

2013-07-02

Continuously tracking bioanalytes in vivo will enable clinicians and researchers to profile normal physiology and monitor diseased states. Current in vivo monitoring system designs are limited by invasive implantation procedures and biofouling, limiting the utility of these tools for obtaining physiologic data. In this work, we demonstrate the first success in optically tracking histamine levels in vivo using a modular, injectable sensing platform based on diamine oxidase and a phosphorescent oxygen nanosensor. Our new approach increases the range of measurable analytes by combining an enzymatic recognition element with a reversible nanosensor capable of measuring the effects of enzymatic activity. We use these enzyme nanosensors (EnzNS) to monitor the in vivo histamine dynamics as the concentration rapidly increases and decreases due to administration and clearance. The EnzNS system measured kinetics that match those reported from ex vivo measurements. This work establishes a modular approach to in vivo nanosensor design for measuring a broad range of potential target analytes. Simply replacing the recognition enzyme, or both the enzyme and nanosensor, can produce a new sensor system capable of measuring a wide range of specific analytical targets in vivo.

WE-H-207A-09: Theoretical Limits to Molecular Biomarker Detection Using Magnetic Nanoparticles

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

Weaver, J; Geisel School of Medicine, Dartmouth College, Hanover, NH

Purpose: Estimate the limits of molecular biomarker detection using magnetic nanoparticle methods like in vivo ELISA. Methods: Magnetic nanoparticles in an alternating magnetic field produce a magnetization that can be detected at exceedingly low levels because the signal at the harmonic frequencies is uniquely produced by the nanoparticles. Because the magnetization can also be used to characterize the nanoparticle rotational freedom, the bound state can be found. If the nanoparticles are coated with molecules that bind the desired biomarker, the rotational freedom reflects the biomarker concentration. The irreducible noise limit is the thermal noise or Johnson noise of the tissuemore » and the contrast that can be measured must be larger than that limit. The contrast produced is a function of the applied field and depends strongly on nanoparticle volume. We have estimated the contrast using a Langevin function of a single composite variable to approximate the full stochastic Langevin equation for nanoparticle dynamics. Results: The thermal noise for a bandwidth reasonable for spectroscopy suggests mid zeptomolar (10–21) to low attomolar (10–18) concentrations can be measured in a volume that is 10cm in scale. The suggested sensitivity is far below the physiologically concentrations of almost all critical biomarkers including cytokines (picomolar), hormones (nanomolar) and heat shock proteins. Conclusion: The sensitivity of in vivo ELISA concentration measurements should be sufficient to measure physiological concentrations of critical biomarkers like cytokines in vivo. Further the sensitivity should be sufficient to measure concentrations of other biomarkers that are six to eight orders of magnitude lower in concentration than immune signaling molecules like cytokines. NIH - 1U54CA151662-01 Department of Radiology.« less

Co-potentiation of antigen recognition: A mechanism to boost weak T cell responses and provide immunotherapy in vivo

PubMed Central

Hoffmann, Michele M.; Molina-Mendiola, Carlos; Nelson, Alfreda D.; Parks, Christopher A.; Reyes, Edwin E.; Hansen, Michael J.; Rajagopalan, Govindarajan; Pease, Larry R.; Schrum, Adam G.; Gil, Diana

2015-01-01

Adaptive immunity is mediated by antigen receptors that can induce weak or strong immune responses depending on the nature of the antigen that is bound. In T lymphocytes, antigen recognition triggers signal transduction by clustering T cell receptor (TCR)/CD3 multiprotein complexes. In addition, it hypothesized that biophysical changes induced in TCR/CD3 that accompany receptor engagement may contribute to signal intensity. Nonclustering monovalent TCR/CD3 engagement is functionally inert despite the fact that it may induce changes in conformational arrangement or in the flexibility of receptor subunits. We report that the intrinsically inert monovalent engagement of TCR/CD3 can specifically enhance physiologic T cell responses to weak antigens in vitro and in vivo without stimulating antigen-unengaged T cells and without interrupting T cell responses to strong antigens, an effect that we term as “co-potentiation.” We identified Mono-7D6-Fab, which biophysically altered TCR/CD3 when bound and functionally enhanced immune reactivity to several weak antigens in vitro, including a gp100-derived peptide associated with melanoma. In vivo, Mono-7D6-Fab induced T cell antigen–dependent therapeutic responses against melanoma lung metastases, an effect that synergized with other anti-melanoma immunotherapies to significantly improve outcome and survival. We conclude that Mono-7D6-Fab directly co-potentiated TCR/CD3 engagement by weak antigens and that such concept can be translated into an immunotherapeutic design. The co-potentiation principle may be applicable to other receptors that could be regulated by otherwise inert compounds whose latent potency is only invoked in concert with specific physiologic ligands. PMID:26601285

Androgen Effects on Adipose Tissue Architecture and Function in Nonhuman Primates

PubMed Central

Varlamov, Oleg; White, Ashley E.; Carroll, Julie M.; Bethea, Cynthia L.; Reddy, Arubala; Slayden, Ov; O'Rourke, Robert W.

2012-01-01

The differential association of hypoandrogenism in men and hyperandrogenism in women with insulin resistance and obesity suggests that androgens may exert sex-specific effects on adipose and other tissues, although the underlying mechanisms remain poorly understood. Moreover, recent studies also suggest that rodents and humans may respond differently to androgen imbalance. To achieve better insight into clinically relevant sex-specific mechanisms of androgen action, we used nonhuman primates to investigate the direct effects of gonadectomy and hormone replacement on white adipose tissue. We also employed a novel ex vivo approach that provides a convenient framework for understanding of adipose tissue physiology under a controlled tissue culture environment. In vivo androgen deprivation of males did not result in overt obesity or insulin resistance but did induce the appearance of very small, multilocular white adipocytes. Testosterone replacement restored normal cell size and a unilocular phenotype and stimulated adipogenic gene transcription and improved insulin sensitivity of male adipose tissue. Ex vivo studies demonstrated sex-specific effects of androgens on adipocyte function. Female adipose tissue treated with androgens displayed elevated basal but reduced insulin-dependent fatty acid uptake. Androgen-stimulated basal uptake was greater in adipose tissue of ovariectomized females than in adipose tissue of intact females and ovariectomized females replaced with estrogen and progesterone in vivo. Collectively, these data demonstrate that androgens are essential for normal adipogenesis in males and can impair essential adipocyte functions in females, thus strengthening the experimental basis for sex-specific effects of androgens in adipose tissue. PMID:22547568

Metabolic engineering pathways for rare sugars biosynthesis, physiological functionalities, and applications-a review.

PubMed

Bilal, Muhammad; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

2017-06-29

Biomolecules like rare sugars and their derivatives are referred to as monosaccharides particularly uncommon in nature. Remarkably, many of them have various known physiological functions and biotechnological applications in cosmetics, nutrition, and pharmaceutical industries. Also, they can be exploited as starting materials for synthesizing fascinating natural bioproducts with significant biological activities. Regrettably, most of the rare sugars are quite expensive, and their synthetic chemical routes are both limited and economically unfeasible due to expensive raw materials. On the other hand, their production by enzymatic means often suffers from low space-time yields and high catalyst costs due to hasty enzyme denaturation/degradation. In this context, biosynthesis of rare sugars with industrial importance is receiving renowned scientific attention, across the globe. Moreover, the utilization of renewable resources as energy sources via microbial fermentation or microbial metabolic engineering has appeared a new tool. This article presents a comprehensive review of physiological functions and biotechnological applications of rare ketohexoses and aldohexoses, including D-psicose, D-tagatose, L-tagatose, D-sorbose, L-fructose, D-allose, L-glucose, D-gulose, L-talose, L-galactose, and L-fucose. Novel in-vivo recombination pathways based on aldolase and phosphatase for the biosynthesis of rare sugars, particularly D-psicose and D-sorbose using robust microbial strains are also deliberated.

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

What have we learned about GPER function in physiology and disease from knockout mice?

PubMed

Prossnitz, Eric R; Hathaway, Helen J

2015-09-01

Estrogens, predominantly 17β-estradiol, exert diverse effects throughout the body in both normal and pathophysiology, during development and in reproductive, metabolic, endocrine, cardiovascular, nervous, musculoskeletal and immune systems. Estrogen and its receptors also play important roles in carcinogenesis and therapy, particularly for breast cancer. In addition to the classical nuclear estrogen receptors (ERα and ERβ) that traditionally mediate predominantly genomic signaling, the G protein-coupled estrogen receptor GPER has become recognized as a critical mediator of rapid signaling in response to estrogen. Mouse models, and in particular knockout (KO) mice, represent an important approach to understand the functions of receptors in normal physiology and disease. Whereas ERα KO mice display multiple significant defects in reproduction and mammary gland development, ERβ KO phenotypes are more limited, and GPER KO exhibit no reproductive deficits. However, the study of GPER KO mice over the last six years has revealed that GPER deficiency results in multiple physiological alterations including obesity, cardiovascular dysfunction, insulin resistance and glucose intolerance. In addition, the lack of estrogen-mediated effects in numerous tissues of GPER KO mice, studied in vivo or ex vivo, including those of the cardiovascular, endocrine, nervous and immune systems, reveals GPER as a genuine mediator of estrogen action. Importantly, GPER KO mice have also demonstrated roles for GPER in breast carcinogenesis and metastasis. In combination with the supporting effects of GPER-selective ligands and GPER knockdown approaches, GPER KO mice demonstrate the therapeutic potential of targeting GPER activity in diseases as diverse as obesity, diabetes, multiple sclerosis, hypertension, atherosclerosis, myocardial infarction, stroke and cancer. Copyright © 2015. Published by Elsevier Ltd.

Properties of the vastus lateralis muscle in relation to age and physiological function in master cyclists aged 55-79 years.

PubMed

Pollock, Ross D; O'Brien, Katie A; Daniels, Lorna J; Nielsen, Kathrine B; Rowlerson, Anthea; Duggal, Niharika A; Lazarus, Norman R; Lord, Janet M; Philp, Andrew; Harridge, Stephen D R

2018-04-01

In this study, results are reported from the analyses of vastus lateralis muscle biopsy samples obtained from a subset (n = 90) of 125 previously phenotyped, highly active male and female cyclists aged 55-79 years in regard to age. We then subsequently attempted to uncover associations between the findings in muscle and in vivo physiological functions. Muscle fibre type and composition (ATPase histochemistry), size (morphometry), capillary density (immunohistochemistry) and mitochondrial protein content (Western blot) in relation to age were determined in the biopsy specimens. Aside from an age-related change in capillary density in males (r = -.299; p = .02), no other parameter measured in the muscle samples showed an association with age. However, in males type I fibres and capillarity (p < .05) were significantly associated with training volume, maximal oxygen uptake, oxygen uptake kinetics and ventilatory threshold. In females, the only association observed was between capillarity and training volume (p < .05). In males, both type II fibre proportion and area (p < .05) were associated with peak power during sprint cycling and with maximal rate of torque development during a maximal voluntary isometric contraction. Mitochondrial protein content was not associated with any cardiorespiratory parameter in either males or females (p > .05). We conclude in this highly active cohort, selected to mitigate most of the effects of inactivity, that there is little evidence of age-related changes in the properties of VL muscle across the age range studied. By contrast, some of these muscle characteristics were correlated with in vivo physiological indices. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium.

PubMed

Wang, Yuli; Gunasekara, Dulan B; Reed, Mark I; DiSalvo, Matthew; Bultman, Scott J; Sims, Christopher E; Magness, Scott T; Allbritton, Nancy L

2017-06-01

The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Distribution, function and physiological role of melatonin in the lower gut

PubMed Central

Chen, Chun-Qiu; Fichna, Jakub; Bashashati, Mohammad; Li, Yong-Yu; Storr, Martin

2011-01-01

Melatonin is a hormone with endocrine, paracrine and autocrine actions. It is involved in the regulation of multiple functions, including the control of the gastrointestinal (GI) system under physiological and pathophysiological conditions. Since the gut contains at least 400 times more melatonin than the pineal gland, a review of the functional importance of melatonin in the gut seems useful, especially in the context of recent clinical trials. Melatonin exerts its physiological effects through specific membrane receptors, named melatonin-1 receptor (MT1), MT2 and MT3. These receptors can be found in the gut and their involvement in the regulation of GI motility, inflammation and pain has been reported in numerous basic and clinical studies. Stable levels of melatonin in the lower gut that are unchanged following a pinealectomy suggest local synthesis and, furthermore, implicate physiological importance of endogenous melatonin in the GI tract. Presently, only a small number of human studies report possible beneficial and also possible harmful effects of melatonin in case reports and clinical trials. These human studies include patients with lower GI diseases, especially patients with irritable bowel syndrome, inflammatory bowel disease and colorectal cancer. In this review, we summarize the presently available information on melatonin effects in the lower gut and discuss available in vitro and in vivo data. We furthermore aim to evaluate whether melatonin may be useful in future treatment of symptoms or diseases involving the lower gut. PMID:22025877

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.

PubMed

Fornaro, Michele; Sharthiya, Harsh; Tiwari, Vaibhav

2018-03-09

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

Microcirculation in the murine liver: a computational fluid dynamic model based on 3D reconstruction from in vivo microscopy.

PubMed

Piergiovanni, Monica; Bianchi, Elena; Capitani, Giada; Li Piani, Irene; Ganzer, Lucia; Guidotti, Luca G; Iannacone, Matteo; Dubini, Gabriele

2017-10-03

The liver is organized in hexagonal functional units - termed lobules - characterized by a rather peculiar blood microcirculation, due to the presence of a tangled network of capillaries - termed sinusoids. A better understanding of the hemodynamics that governs liver microcirculation is relevant to clinical and biological studies aimed at improving our management of liver diseases and transplantation. Herein, we built a CFD model of a 3D sinusoidal network, based on in vivo images of a physiological mouse liver obtained with a 2-photon microscope. The CFD model was developed with Fluent 16.0 (ANSYS Inc., Canonsburg, PA), particular care was taken in imposing the correct boundary conditions representing a physiological state. To account for the remaining branches of the sinusoids, a lumped parameter model was used to prescribe the correct pressure at each outlet. The effect of an adhered cell on local hemodynamics is also investigated for different occlusion degrees. The model here proposed accurately reproduces the fluid dynamics in a portion of the sinusoidal network in mouse liver. Mean velocities and mass flow rates are in agreement with literature values from in vivo measurements. Our approach provides details on local phenomena, hardly described by other computational studies, either focused on the macroscopic hepatic vasculature or based on homogeneous porous medium model. Copyright © 2017 Elsevier Ltd. All rights reserved.

A high performance, cost-effective, open-source microscope for scanning two-photon microscopy that is modular and readily adaptable.

PubMed

Rosenegger, David G; Tran, Cam Ha T; LeDue, Jeffery; Zhou, Ning; Gordon, Grant R

2014-01-01

Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems.

A High Performance, Cost-Effective, Open-Source Microscope for Scanning Two-Photon Microscopy that Is Modular and Readily Adaptable

PubMed Central

Rosenegger, David G.; Tran, Cam Ha T.; LeDue, Jeffery; Zhou, Ning; Gordon, Grant R.

2014-01-01

Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems. PMID:25333934

The Constraints, Construction, and Verification of a Strain-Specific Physiologically Based Pharmacokinetic Rat Model.

PubMed

Musther, Helen; Harwood, Matthew D; Yang, Jiansong; Turner, David B; Rostami-Hodjegan, Amin; Jamei, Masoud

2017-09-01

The use of in vitro-in vivo extrapolation (IVIVE) techniques, mechanistically incorporated within physiologically based pharmacokinetic (PBPK) models, can harness in vitro drug data and enhance understanding of in vivo pharmacokinetics. This study's objective was to develop a user-friendly rat (250 g, male Sprague-Dawley) IVIVE-linked PBPK model. A 13-compartment PBPK model including mechanistic absorption models was developed, with required system data (anatomical, physiological, and relevant IVIVE scaling factors) collated from literature and analyzed. Overall, 178 system parameter values for the model are provided. This study also highlights gaps in available system data required for strain-specific rat PBPK model development. The model's functionality and performance were assessed using previous literature-sourced in vitro properties for diazepam, metoprolol, and midazolam. The results of simulations were compared against observed pharmacokinetic rat data. Predicted and observed concentration profiles in 10 tissues for diazepam after a single intravenous (i.v.) dose making use of either observed i.v. clearance (CL iv ) or in vitro hepatocyte intrinsic clearance (CL int ) for simulations generally led to good predictions in various tissue compartments. Overall, all i.v. plasma concentration profiles were successfully predicted. However, there were challenges in predicting oral plasma concentration profiles for metoprolol and midazolam, and the potential reasons and according solutions are discussed. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Engineering Visual Arrestin-1 with Special Functional Characteristics*

PubMed Central

Vishnivetskiy, Sergey A.; Chen, Qiuyan; Palazzo, Maria C.; Brooks, Evan K.; Altenbach, Christian; Iverson, Tina M.; Hubbell, Wayne L.; Gurevich, Vsevolod V.

2013-01-01

Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations. PMID:23250748

Methods of in-vivo mouse lung micro-CT

NASA Astrophysics Data System (ADS)

Recheis, Wolfgang A.; Nixon, Earl; Thiesse, Jacqueline; McLennan, Geoffrey; Ross, Alan; Hoffman, Eric

2005-04-01

Micro-CT will have a profound influence on the accumulation of anatomical and physiological phenotypic changes in natural and transgenetic mouse models. Longitudinal studies will be greatly facilitated, allowing for a more complete and accurate description of events if in-vivo studies are accomplished. The purpose of the ongoing project is to establish a feasible and reproducible setup for in-vivo mouse lung micro-computed tomography (μCT). We seek to use in-vivo respiratory-gated μCT to follow mouse models of lung disease with subsequent recovery of the mouse. Methodologies for optimizing scanning parameters and gating for the in-vivo mouse lung are presented. A Scireq flexiVent ventilated the gas-anesthetized mice at 60 breaths/minute, 30 cm H20 PEEP, 30 ml/kg tidal volume and provided a respiratory signal to gate a Skyscan 1076 μCT. Physiologic monitoring allowed the control of vital functions and quality of anesthesia, e.g. via ECG monitoring. In contrary to longer exposure times with ex-vivo scans, scan times for in-vivo were reduced using 35μm pixel size, 158ms exposure time and 18μm pixel size, 316ms exposure time to reduce motion artifacts. Gating via spontaneous breathing was also tested. Optimal contrast resolution was achieved at 50kVp, 200μA, applying an aluminum filter (0.5mm). There were minimal non-cardiac related motion artifacts. Both 35μm and 1μm voxel size images were suitable for evaluation of the airway lumen and parenchymal density. Total scan times were 30 and 65 minutes respectively. The mice recovered following scanning protocols. In-vivo lung scanning with recovery of the mouse delivered reasonable image quality for longitudinal studies, e.g. mouse asthma models. After examining 10 mice, we conclude μCT is a feasible tool evaluating mouse models of lung pathology in longitudinal studies with increasing anatomic detail available for evaluation as one moves from in-vivo to ex-vivo studies. Further developments include automated bronchial tree segmentation and airway wall thickness measurement tools. Improvements in Hounsfield unit calibration have to be performed when the interest of the study lies in determining and quantifying parenchymal changes and rely on estimating partial volume contributions of underlying structures to voxel densities.

Heteromeric MT1/MT2 Melatonin Receptors Modulate Photoreceptor Function

PubMed Central

Baba, Kenkichi; Benleulmi-Chaachoua, Abla; Journé, Anne-Sophie; Kamal, Maud; Guillaume, Jean-Luc; Dussaud, Sébastien; Gbahou, Florence; Yettou, Katia; Liu, Cuimei; Contreras-Alcantara, Susana; Jockers, Ralf; Tosini, Gianluca

2013-01-01

The formation of G protein-coupled receptor (GPCR) heteromers elicits signaling diversification and holds great promise for improved drug selectivity. Most studies have been conducted in heterologous expression systems; however, in vivo validation is missing from most cases thus questioning the physiological significance of GPCR heteromerization. Melatonin MT1 and MT2 receptors have been shown to exist as homo- and heteromers in vitro. We show here that the effect of melatonin on rod photoreceptor light sensitivity is mediated by melatonin MT1/MT2 receptor heteromers. This effect involves activation of the heteromer-specific PLC/PKC pathway and is abolished in MT1−/− and MT2−/− mice as well as in mice overexpressing a non-functional MT2 receptor mutant that competes with the formation of functional MT1/MT2 heteromers in photoreceptor cells. This study establishes the essential role of melatonin receptor heteromers in retinal function and supports the physiological importance of GPCR heteromerization. Finally, our work may have important therapeutic implications, as the heteromer complex may provide a unique pharmacological target to improve photoreceptor functioning and to extend the viability of photoreceptors during aging. PMID:24106342

Emission Computed Tomography: A New Technique for the Quantitative Physiologic Study of Brain and Heart in Vivo

DOE R&D Accomplishments Database

Phelps, M. E.; Hoffman, E. J.; Huang, S. C.; Schelbert, H. R.; Kuhl, D. E.

1978-01-01

Emission computed tomography can provide a quantitative in vivo measurement of regional tissue radionuclide tracer concentrations. This facility when combined with physiologic models and radioactively labeled physiologic tracers that behave in a predictable manner allow measurement of a wide variety of physiologic variables. This integrated technique has been referred to as Physiologic Tomography (PT). PT requires labeled compounds which trace physiologic processes in a known and predictable manner, and physiologic models which are appropriately formulated and validated to derive physiologic variables from ECT data. In order to effectively achieve this goal, PT requires an ECT system that is capable of performing truly quantitative or analytical measurements of tissue tracer concentrations and which has been well characterized in terms of spatial resolution, sensitivity and signal to noise ratios in the tomographic image. This paper illustrates the capabilities of emission computed tomography and provides examples of physiologic tomography for the regional measurement of cerebral and myocardial metabolic rate for glucose, regional measurement of cerebral blood volume, gated cardiac blood pools and capillary perfusion in brain and heart. Studies on patients with stroke and myocardial ischemia are also presented.

Evaluation of a 125I-labelled benzazepinone derived voltage-gated sodium channel blocker for imaging with SPECT.

PubMed

Pérez-Medina, Carlos; Patel, Niral; Robson, Mathew; Badar, Adam; Lythgoe, Mark F; Årstad, Erik

2012-12-21

Voltage-gated sodium channels (VGSCs) are a family of transmembrane proteins that mediate fast neurotransmission, and are integral to sustain physiological conditions and higher cognitive functions. Imaging of VGSCs in vivo holds promise as a tool to elucidate operational functions in the brain and to aid the treatment of a wide range of neurological diseases. To assess the suitability of 1-benzazepin-2-one derived VGSC blockers for imaging, we have prepared a (125)I-labelled analogue of BNZA and evaluated the tracer in vivo. In an automated patch-clamp assay, a diastereomeric mixture of the non-radioactive compound blocked the Na(v)1.2 and Na(v)1.7 VGSC isoforms with IC(50) values of 4.1 ± 1.5 μM and 0.25 ± 0.07 μM, respectively. [(3)H]BTX displacement studies revealed a three-fold difference in affinity between the two diastereomers. Iodo-destannylation of a tin precursor with iodine-125 afforded the two diastereomerically pure tracers, which were used to assess binding to VGSCs in vivo by comparing their tissue distributions in mice. Whilst the results point to a lack of VGSC binding in vivo, SPECT imaging revealed highly localized uptake in the interscapular region, an area typically associated with brown adipose tissue, which in addition to high metabolic stability of the iodinated tracer, demonstrate the potential of 1-benzazepin-2-ones for in vivo imaging.

Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

PubMed Central

Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

2016-01-01

In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs.

PubMed

O'Leary, Brendan; Park, Joonho; Plaxton, William C

2011-05-15

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.

Application of Physiologically Based Absorption Modeling to Characterize the Pharmacokinetic Profiles of Oral Extended Release Methylphenidate Products in Adults

PubMed Central

Yang, Xiaoxia; Duan, John; Fisher, Jeffrey

2016-01-01

A previously presented physiologically-based pharmacokinetic model for immediate release (IR) methylphenidate (MPH) was extended to characterize the pharmacokinetic behaviors of oral extended release (ER) MPH formulations in adults for the first time. Information on the anatomy and physiology of the gastrointestinal (GI) tract, together with the biopharmaceutical properties of MPH, was integrated into the original model, with model parameters representing hepatic metabolism and intestinal non-specific loss recalibrated against in vitro and in vivo kinetic data sets with IR MPH. A Weibull function was implemented to describe the dissolution of different ER formulations. A variety of mathematical functions can be utilized to account for the engineered release/dissolution technologies to achieve better model performance. The physiological absorption model tracked well the plasma concentration profiles in adults receiving a multilayer-release MPH formulation or Metadate CD, while some degree of discrepancy was observed between predicted and observed plasma concentration profiles for Ritalin LA and Medikinet Retard. A local sensitivity analysis demonstrated that model parameters associated with the GI tract significantly influenced model predicted plasma MPH concentrations, albeit to varying degrees, suggesting the importance of better understanding the GI tract physiology, along with the intestinal non-specific loss of MPH. The model provides a quantitative tool to predict the biphasic plasma time course data for ER MPH, helping elucidate factors responsible for the diverse plasma MPH concentration profiles following oral dosing of different ER formulations. PMID:27723791

WINCS Harmoni: Closed-loop dynamic neurochemical control of therapeutic interventions

NASA Astrophysics Data System (ADS)

Lee, Kendall H.; Lujan, J. Luis; Trevathan, James K.; Ross, Erika K.; Bartoletta, John J.; Park, Hyung Ook; Paek, Seungleal Brian; Nicolai, Evan N.; Lee, Jannifer H.; Min, Hoon-Ki; Kimble, Christopher J.; Blaha, Charles D.; Bennet, Kevin E.

2017-04-01

There has been significant progress in understanding the role of neurotransmitters in normal and pathologic brain function. However, preclinical trials aimed at improving therapeutic interventions do not take advantage of real-time in vivo neurochemical changes in dynamic brain processes such as disease progression and response to pharmacologic, cognitive, behavioral, and neuromodulation therapies. This is due in part to a lack of flexible research tools that allow in vivo measurement of the dynamic changes in brain chemistry. Here, we present a research platform, WINCS Harmoni, which can measure in vivo neurochemical activity simultaneously across multiple anatomical targets to study normal and pathologic brain function. In addition, WINCS Harmoni can provide real-time neurochemical feedback for closed-loop control of neurochemical levels via its synchronized stimulation and neurochemical sensing capabilities. We demonstrate these and other key features of this platform in non-human primate, swine, and rodent models of deep brain stimulation (DBS). Ultimately, systems like the one described here will improve our understanding of the dynamics of brain physiology in the context of neurologic disease and therapeutic interventions, which may lead to the development of precision medicine and personalized therapies for optimal therapeutic efficacy.

Tissue vascularization through 3D printing: Will technology bring us flow?

PubMed

Paulsen, S J; Miller, J S

2015-05-01

Though in vivo models provide the most physiologically relevant environment for studying tissue function, in vitro studies provide researchers with explicit control over experimental conditions and the potential to develop high throughput testing methods. In recent years, advancements in developmental biology research and imaging techniques have significantly improved our understanding of the processes involved in vascular development. However, the task of recreating the complex, multi-scale vasculature seen in in vivo systems remains elusive. 3D bioprinting offers a potential method to generate controlled vascular networks with hierarchical structure approaching that of in vivo networks. Bioprinting is an interdisciplinary field that relies on advances in 3D printing technology along with advances in imaging and computational modeling, which allow researchers to monitor cellular function and to better understand cellular environment within the printed tissue. As bioprinting technologies improve with regards to resolution, printing speed, available materials, and automation, 3D printing could be used to generate highly controlled vascularized tissues in a high throughput manner for use in regenerative medicine and the development of in vitro tissue models for research in developmental biology and vascular diseases. © 2015 Wiley Periodicals, Inc.

Functional microorganisms for functional food quality.

PubMed

Gobbetti, M; Cagno, R Di; De Angelis, M

2010-09-01

Functional microorganisms and health benefits represent a binomial with great potential for fermented functional foods. The health benefits of fermented functional foods are expressed either directly through the interactions of ingested live microorganisms with the host (probiotic effect) or indirectly as the result of the ingestion of microbial metabolites synthesized during fermentation (biogenic effect). Since the importance of high viability for probiotic effect, two major options are currently pursued for improving it--to enhance bacterial stress response and to use alternative products for incorporating probiotics (e.g., ice cream, cheeses, cereals, fruit juices, vegetables, and soy beans). Further, it seems that quorum sensing signal molecules released by probiotics may interact with human epithelial cells from intestine thus modulating several physiological functions. Under optimal processing conditions, functional microorganisms contribute to food functionality through their enzyme portfolio and the release of metabolites. Overproduction of free amino acids and vitamins are two classical examples. Besides, bioactive compounds (e.g., peptides, γ-amino butyric acid, and conjugated linoleic acid) may be released during food processing above the physiological threshold and they may exert various in vivo health benefits. Functional microorganisms are even more used in novel strategies for decreasing phenomenon of food intolerance (e.g., gluten intolerance) and allergy. By a critical approach, this review will aim at showing the potential of functional microorganisms for the quality of functional foods.

Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

PubMed Central

Rezania, Alireza; Riedel, Michael J.; Wideman, Rhonda D.; Karanu, Francis; Ao, Ziliang; Warnock, Garth L.; Kieffer, Timothy J.

2011-01-01

OBJECTIVE Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells. PMID:20971966

Recovery of the immune system after exercise.

PubMed

Peake, Jonathan M; Neubauer, Oliver; Walsh, Neil P; Simpson, Richard J

2017-05-01

The notion that prolonged, intense exercise causes an "open window" of immunodepression during recovery after exercise is well accepted. Repeated exercise bouts or intensified training without sufficient recovery may increase the risk of illness. However, except for salivary IgA, clear and consistent markers of this immunodepression remain elusive. Exercise increases circulating neutrophil and monocyte counts and reduces circulating lymphocyte count during recovery. This lymphopenia results from preferential egress of lymphocyte subtypes with potent effector functions [e.g., natural killer (NK) cells, γδ T cells, and CD8 + T cells]. These lymphocytes most likely translocate to peripheral sites of potential antigen encounter (e.g., lungs and gut). This redeployment of effector lymphocytes is an integral part of the physiological stress response to exercise. Current knowledge about changes in immune function during recovery from exercise is derived from assessment at the cell population level of isolated cells ex vivo or in blood. This assessment can be biased by large changes in the distribution of immune cells between blood and peripheral tissues during and after exercise. Some evidence suggests that reduced immune cell function in vitro may coincide with changes in vivo and rates of illness after exercise, but more work is required to substantiate this notion. Among the various nutritional strategies and physical therapies that athletes use to recover from exercise, carbohydrate supplementation is the most effective for minimizing immune disturbances during exercise recovery. Sleep is an important aspect of recovery, but more research is needed to determine how sleep disruption influences the immune system of athletes. Copyright © 2017 the American Physiological Society.

The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors

PubMed Central

Hoestgaard-Jensen, K; O'Connor, R M; Dalby, N O; Simonsen, C; Finger, B C; Golubeva, A; Hammer, H; Bergmann, M L; Kristiansen, U; Krogsgaard-Larsen, P; Bräuner-Osborne, H; Ebert, B; Frølund, B; Cryan, J F; Jensen, A A

2013-01-01

BACKGROUND AND PURPOSE Explorations into the heterogeneous population of native GABA type A receptors (GABAARs) and the physiological functions governed by the multiple GABAAR subtypes have for decades been hampered by the lack of subtype-selective ligands. EXPERIMENTAL APPROACH The functional properties of the orthosteric GABAA receptor ligand 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) have been investigated in vitro, ex vivo and in vivo. KEY RESULTS Thio-4-PIOL displayed substantial partial agonist activity at the human extrasynaptic GABAAR subtypes expressed in Xenopus oocytes, eliciting maximal responses of up to ∼30% of that of GABA at α5β3γ2S, α4β3δ and α6β3δ and somewhat lower efficacies at the corresponding α5β2γ2S, α4β2δ and α6β2δ subtypes (maximal responses of 4–12%). In contrast, it was an extremely low efficacious agonist at the α1β3γ2S, α1β2γ2S, α2β2γ2S, α2β3γ2S, α3β2γ2S and α3β3γ2S GABAARs (maximal responses of 0–4%). In concordance with its agonism at extrasynaptic GABAARs and its de facto antagonism at the synaptic receptors, Thio-4-PIOL elicited robust tonic currents in electrophysiological recordings on slices from rat CA1 hippocampus and ventrobasal thalamus and antagonized phasic currents in hippocampal neurons. Finally, the observed effects of Thio-4-PIOL in rat tests of anxiety, locomotion, nociception and spatial memory were overall in good agreement with its in vitro and ex vivo properties. CONCLUSION AND IMPLICATIONS The diverse signalling characteristics of Thio-4-PIOL at GABAARs represent one of the few examples of a functionally subtype-selective orthosteric GABAAR ligand reported to date. We propose that Thio-4-PIOL could be a useful pharmacological tool in future studies exploring the physiological roles of native synaptic and extrasynaptic GABAARs. PMID:23957253

Gut feedback mechanisms and food intake: a physiological approach to slow carbohydrate bioavailability.

PubMed

Zhang, Genyi; Hasek, Like Y; Lee, Byung-Hoo; Hamaker, Bruce R

2015-04-01

Glycemic carbohydrates in foods are an important macronutrient providing the biological fuel of glucose for a variety of physiological processes. A classification of glycemic carbohydrates into rapidly digestible carbohydrate (RDC) and slowly digestible carbohydrate (SDC) has been used to specify their nutritional quality related to glucose homeostasis that is essential to normal functioning of the brain and critical to life. Although there have been many studies and reviews on slowly digestible starch (SDS) and SDC, the mechanisms of their slow digestion and absorption were mostly investigated from the material side without considering the physiological processes of their in vivo digestion, absorption, and most importantly interactions with other food components and the gastrointestinal tract. In this article, the physiological processes modulating the bioavailability of carbohydrates, specifically the rate and extent of their digestion and absorption as well as the related locations, in a whole food context, will be discussed by focusing on the activities of the gastrointestinal tract including glycolytic enzymes and glucose release, sugar sensing, gut hormones, and neurohormonal negative feedback mechanisms. It is hoped that a deep understanding of these physiological processes will facilitate the development of innovative dietary approaches to achieve desired carbohydrate or glucose bioavailability for improved health.

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate.

PubMed

Tyrakis, Petros A; Palazon, Asis; Macias, David; Lee, Kian L; Phan, Anthony T; Veliça, Pedro; You, Jia; Chia, Grace S; Sim, Jingwei; Doedens, Andrew; Abelanet, Alice; Evans, Colin E; Griffiths, John R; Poellinger, Lorenz; Goldrath, Ananda W; Johnson, Randall S

2016-12-08

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8 + T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8 + T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8 + T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic-epigenetic axis, to immune fate and function.

The immunometabolite S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate

PubMed Central

Tyrakis, Petros A.; Palazon, Asis; Macias, David; Lee, Kian. L.; Phan, Anthony. T.; Veliça, Pedro; You, Jia; Chia, Grace S.; Sim, Jingwei; Doedens, Andrew; Abelanet, Alice; Evans, Colin E.; Griffiths, John R.; Poellinger, Lorenz; Goldrath, Ananda. W.; Johnson, Randall S.

2016-01-01

R-2-hydroxyglutarate accumulates to millimolar levels in cancers with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both R- and S-2-hydroxyglutarate, the other enantiomer of this metabolite, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that CD8+ T-lymphocytes accumulate 2-hydroxyglutarate in response to T-cell receptor triggering. This increases to millimolar levels in physiological oxygen conditions, via a hypoxia inducible factor 1 alpha-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-lymphocyte differentiation in response to this metabolite. Modulation of histone and DNA demethylation as well as hypoxia inducible factor 1 alpha stability mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T-lymphocytes. Thus S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, via a metabolic-epigenetic axis, to immune fate and function. PMID:27798602

Impaired angiogenesis in aminopeptidase N-null mice

PubMed Central

Rangel, Roberto; Sun, Yan; Guzman-Rojas, Liliana; Ozawa, Michael G.; Sun, Jessica; Giordano, Ricardo J.; Van Pelt, Carolyn S.; Tinkey, Peggy T.; Behringer, Richard R.; Sidman, Richard L.; Arap, Wadih; Pasqualini, Renata

2007-01-01

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports of in vivo inactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases. PMID:17360568

Four-dimensional in vivo X-ray microscopy with projection-guided gating

NASA Astrophysics Data System (ADS)

Mokso, Rajmund; Schwyn, Daniel A.; Walker, Simon M.; Doube, Michael; Wicklein, Martina; Müller, Tonya; Stampanoni, Marco; Taylor, Graham K.; Krapp, Holger G.

2015-03-01

Visualizing fast micrometer scale internal movements of small animals is a key challenge for functional anatomy, physiology and biomechanics. We combine phase contrast tomographic microscopy (down to 3.3 μm voxel size) with retrospective, projection-based gating (in the order of hundreds of microseconds) to improve the spatiotemporal resolution by an order of magnitude over previous studies. We demonstrate our method by visualizing 20 three-dimensional snapshots through the 150 Hz oscillations of the blowfly flight motor.

Intradiscal pressure variation under spontaneous ventilation

NASA Astrophysics Data System (ADS)

Roriz, Paulo; Ferreira, J.; Potes, J. C.; Oliveira, M. T.; Santos, J. L.; Simões, J. A.; Frazão, O.

2014-05-01

The pressure measured in the intervertebral discs is a response to the loads acting on the spine. External loads, such as the reaction forces resulting from locomotion, manual handling and collisions are probably the most relevant in studying spine trauma. However, the physiological functions such as breathing and hearth rate also participate in subtle variations of intradiscal pressure that can be observed only in vivo at resting. Present work is an effort to measure the effect of breathing on intradiscal pressure of an anesthetized sheep.

Large-Animal Biventricular Working Heart Perfusion System with Low Priming Volume-Comparison between in vivo and ex vivo Cardiac Function.

PubMed

Abicht, Jan-Michael; Mayr, Tanja Axinja Jelena; Jauch, Judith; Guethoff, Sonja; Buchholz, Stefan; Reichart, Bruno; Bauer, Andreas

2018-01-01

Existing large-animal, ex vivo, cardiac perfusion models are restricted in their ability to establish an ischemia/reperfusion condition as seen in cardiac surgery or transplantation. Other working heart systems only challenge one ventricle or require a substantially larger priming volume. We describe a novel biventricular cardiac perfusion system with reduced priming volume. Juvenile pig hearts were cardiopleged, explanted, and reperfused ex vivo after 150 minutes of cold ischemia. Autologous whole blood was used as perfusate (minimal priming volume 350 mL). After 15 minutes of Langendorff perfusion (LM), the system was switched into a biventricular working mode (WM) and studied for 3 hours. During reperfusion, complete unloading of both ventricles and constant-pressure coronary perfusion was achieved. During working mode perfusion, the preload and afterload pressure of both ventricles was controlled within the targeted physiologic range. Functional parameters such as left ventricular work index were reduced in ex vivo working mode (in vivo: 787 ± 186 vs. 1 h WM 498 ± 66 mm Hg·mL/g·min; p  < 0.01), but remained stable throughout the following study period (3 h WM 517 ± 103 mm Hg·mL/g·min; p  = 0.63). Along with the elevated workload during WM, myocardial metabolism and oxygen consumption increased compared with LM (0.021 ± 0.08 vs. 0.06 ± 0.01 mL/min/g; 1 h after reperfusion). Histologic examination of the myocardium revealed no structural damage. In the ex vivo perfusion system, stable hemodynamic and metabolic conditions can be established for a period of 3 hours while functional and blood parameters are easily accessible. Moreover, because of the minimal priming volume, the novel ex vivo cardiac perfusion circuit allows for autologous perfusion, using the limited amount of blood available from the organ donating animal. Georg Thieme Verlag KG Stuttgart · New York.

A novel single walled carbon nanotube (SWCNT) functionalization agent facilitating in vivo combined chemo/thermo therapy

NASA Astrophysics Data System (ADS)

Zhang, Liwen; Rong, Pengfei; Chen, Minglong; Gao, Shi; Zhu, Lei

2015-10-01

Carbon nanotubes (CNTs) have shown intriguing applications in biotechnological and biomedical fields due to their unique shape and properties. However, the fact that unmodified CNTs are prone to aggregation, stunts CNTs applications under physiological conditions. In this research, we found that as little as 1/5th the single walled carbon nanotube (SWCNT) weight of Evans Blue (EB) is capable of dispersing SWCNT as well as facilitating SWCNT functionalization. In view of the binding between EB and albumin, the yielding product (SWCNT/EB) demonstrated extreme stability for weeks under physiological conditions and it can be endowed with a therapeutic ability by simply mixing SWCNT/EB with an albumin based drug. Specifically, the formed SWCNT/EB/albumin/PTX nanocomplex exhibits strong near-infrared (NIR) absorbance, and can serve as an agent for chemo/thermal therapeutic purposes. Our in vivo result reveals that SWCNT/EB/albumin/PTX after being administered into the MDA-MB-435 tumor would effectively ablate the tumor by chemo and photothermal therapy. Such a combined treatment strategy provides remarkable therapeutic outcomes in restraining tumor growth compared to chemo or photothermal therapy alone. Overall, our strategy of dispersing SWCNTs by EB can be used as a platform for carrying other drugs or functional genes with the aid of albumin to treat diseases. The present study opens new opportunities in surface modification of SWCNTs for future clinical disease treatment.Carbon nanotubes (CNTs) have shown intriguing applications in biotechnological and biomedical fields due to their unique shape and properties. However, the fact that unmodified CNTs are prone to aggregation, stunts CNTs applications under physiological conditions. In this research, we found that as little as 1/5th the single walled carbon nanotube (SWCNT) weight of Evans Blue (EB) is capable of dispersing SWCNT as well as facilitating SWCNT functionalization. In view of the binding between EB and albumin, the yielding product (SWCNT/EB) demonstrated extreme stability for weeks under physiological conditions and it can be endowed with a therapeutic ability by simply mixing SWCNT/EB with an albumin based drug. Specifically, the formed SWCNT/EB/albumin/PTX nanocomplex exhibits strong near-infrared (NIR) absorbance, and can serve as an agent for chemo/thermal therapeutic purposes. Our in vivo result reveals that SWCNT/EB/albumin/PTX after being administered into the MDA-MB-435 tumor would effectively ablate the tumor by chemo and photothermal therapy. Such a combined treatment strategy provides remarkable therapeutic outcomes in restraining tumor growth compared to chemo or photothermal therapy alone. Overall, our strategy of dispersing SWCNTs by EB can be used as a platform for carrying other drugs or functional genes with the aid of albumin to treat diseases. The present study opens new opportunities in surface modification of SWCNTs for future clinical disease treatment. Electronic supplementary information (ESI) available: Characterization of EB dispersed SWCNT; chemical structures of dyes applied for SWCNT dispersion; spectrum of EB/albumin; PTX loading efficiency onto albumin at different ratios. See DOI: 10.1039/c5nr03752b

Magnetic resonance-compatible model of isolated working heart from large animal for multimodal assessment of cardiac function, electrophysiology, and metabolism.

PubMed

Vaillant, Fanny; Magat, Julie; Bour, Pierre; Naulin, Jérôme; Benoist, David; Loyer, Virginie; Vieillot, Delphine; Labrousse, Louis; Ritter, Philippe; Bernus, Olivier; Dos Santos, Pierre; Quesson, Bruno

2016-05-15

To provide a model close to the human heart, and to study intrinsic cardiac function at the same time as electromechanical coupling, we developed a magnetic resonance (MR)-compatible setup of isolated working perfused pig hearts. Hearts from pigs (40 kg, n = 20) and sheep (n = 1) were blood perfused ex vivo in the working mode with and without loaded right ventricle (RV), for 80 min. Cardiac function was assessed by measuring left intraventricular pressure and left ventricular (LV) ejection fraction (LVEF), aortic and mitral valve dynamics, and native T1 mapping with MR imaging (1.5 Tesla). Potential myocardial alterations were assessed at the end of ex vivo perfusion from late-Gadolinium enhancement T1 mapping. The ex vivo cardiac function was stable across the 80 min of perfusion. Aortic flow and LV-dP/dtmin were significantly higher (P < 0.05) in hearts perfused with loaded RV, without differences for heart rate, maximal and minimal LV pressure, LV-dP/dtmax, LVEF, and kinetics of aortic and mitral valves. T1 mapping analysis showed a spatially homogeneous distribution over the LV. Simultaneous recording of hemodynamics, LVEF, and local cardiac electrophysiological signals were then successfully performed at baseline and during electrical pacing protocols without inducing alteration of MR images. Finally, (31)P nuclear MR spectroscopy (9.4 T) was also performed in two pig hearts, showing phosphocreatine-to-ATP ratio in accordance with data previously reported in vivo. We demonstrate the feasibility to perfuse isolated pig hearts in the working mode, inside an MR environment, allowing simultaneous assessment of cardiac structure, mechanics, and electrophysiology, illustrating examples of potential applications. Copyright © 2016 the American Physiological Society.

Enhanced dopamine detection sensitivity by PEDOT/graphene oxide coating on in vivo carbon fiber electrodes.

PubMed

Taylor, I Mitch; Robbins, Elaine M; Catt, Kasey A; Cody, Patrick A; Happe, Cassandra L; Cui, Xinyan Tracy

2017-03-15

Dopamine (DA) is a monoamine neurotransmitter responsible for regulating a variety of vital life functions. In vivo detection of DA poses a challenge due to the low concentration and high speed of physiological signaling. Fast scan cyclic voltammetry at carbon fiber microelectrodes (CFEs) is an effective method to monitor real-time in vivo DA signaling, however the sensitivity is somewhat limited. Electrodeposition of poly(3,4-ethylene dioxythiophene) (PEDOT)/graphene oxide (GO) onto the CFE surface is shown to increase the sensitivity and lower the limit of detection for DA compared to bare CFEs. Thicker PEDOT/GO coatings demonstrate higher sensitivities for DA, but display the negative drawback of slow adsorption and electron transfer kinetics. The moderate thickness resulting from 25 s electrodeposition of PEDOT/GO produces the optimal electrode, exhibiting an 880% increase in sensitivity, a 50% decrease in limit of detection and minimally altered electrode kinetics. PEDOT/GO coated electrodes rapidly and robustly detect DA, both in solution and in the rat dorsal striatum. This increase in DA sensitivity is likely due to increasing the electrode surface area with a PEDOT/GO coating and improved adsorption of DA's oxidation product (DA-o-quinone). Increasing DA sensitivity without compromising electrode kinetics is expected to significantly improve our understanding of the DA function in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced Dopamine Detection Sensitivity by PEDOT/Graphene Oxide Coating on in vivo Carbon Fiber Electrodes

PubMed Central

Taylor, I. Mitch; Robbins, Elaine M.; Catt, Kasey A.; Cody, Patrick A.; Weaver, Cassandra L.; Cui, Xinyan Tracy

2016-01-01

Dopamine (DA) is a monoamine neurotransmitter responsible for regulating a variety of vital life functions. In vivo detection of DA poses a challenge due to the low concentration and high speed of physiological signaling. Fast scan cyclic voltammetry at carbon fiber microelectrodes (CFEs) is an effective method to monitor real-time in vivo DA signaling, however the sensitivity is somewhat limited. Electrodeposition of poly(3,4-ethylene dioxythiophene) (PEDOT)/graphene oxide (GO) onto the CFE surface is shown to increase the sensitivity and lower the limit of detection for DA compared to bare CFEs. Thicker PEDOT/GO coatings demonstrate higher sensitivities for DA, but display the negative drawback of slow adsorption and electron transfer kinetics. The moderate thickness resulting from 25 s electrodeposition of PEDOT/GO produces the optimal electrode, exhibiting an 880% increase in sensitivity, a 50% decrease in limit of detection and minimally altered electrode kinetics. PEDOT/GO coated electrodes rapidly and robustly detect DA, both in solution and in the rat dorsal striatum. This increase in DA sensitivity is likely due to increasing the electrode surface area with a PEDOT/GO coating and improved adsorption of DA’s oxidation product (DA-o-quinone). Increasing DA sensitivity without compromising electrode kinetics is expected to significantly improve our understanding of the DA function in vivo. PMID:27268013

PDT dose dosimetry for Photofrin-mediated pleural photodynamic therapy (pPDT)

NASA Astrophysics Data System (ADS)

Ong, Yi Hong; Kim, Michele M.; Finlay, Jarod C.; Dimofte, Andreea; Singhal, Sunil; Glatstein, Eli; Cengel, Keith A.; Zhu, Timothy C.

2018-01-01

Photosensitizer fluorescence excited by photodynamic therapy (PDT) treatment light can be used to monitor the in vivo concentration of the photosensitizer and its photobleaching. The temporal integral of the product of in vivo photosensitizer concentration and light fluence is called PDT dose, which is an important dosimetry quantity for PDT. However, the detected photosensitizer fluorescence may be distorted by variations in the absorption and scattering of both excitation and fluorescence light in tissue. Therefore, correction of the measured fluorescence for distortion due to variable optical properties is required for absolute quantification of photosensitizer concentration. In this study, we have developed a four-channel PDT dose dosimetry system to simultaneously acquire light dosimetry and photosensitizer fluorescence data. We measured PDT dose at four sites in the pleural cavity during pleural PDT. We have determined an empirical optical property correction function using Monte Carlo simulations of fluorescence for a range of physiologically relevant tissue optical properties. Parameters of the optical property correction function for Photofrin fluorescence were determined experimentally using tissue-simulating phantoms. In vivo measurements of photosensitizer fluorescence showed negligible photobleaching of Photofrin during the PDT treatment, but large intra- and inter-patient heterogeneities of in vivo Photofrin concentration are observed. PDT doses delivered to 22 sites in the pleural cavity of 8 patients were different by 2.9 times intra-patient and 8.3 times inter-patient.

Use of ex vivo and in vitro cultures of the human respiratory tract to study the tropism and host responses of highly pathogenic avian influenza A (H5N1) and other influenza viruses.

PubMed

Chan, Renee W Y; Chan, Michael C W; Nicholls, John M; Malik Peiris, J S

2013-12-05

The tropism of influenza viruses for the human respiratory tract is a key determinant of host-range, and consequently, of pathogenesis and transmission. Insights can be obtained from clinical and autopsy studies of human disease and relevant animal models. Ex vivo cultures of the human respiratory tract and in vitro cultures of primary human cells can provide complementary information provided they are physiologically comparable in relevant characteristics to human tissues in vivo, e.g. virus receptor distribution, state of differentiation. We review different experimental models for their physiological relevance and summarize available data using these cultures in relation to highly pathogenic avian influenza H5N1, in comparison where relevant, with other influenza viruses. Transformed continuous cell-lines often differ in important ways to the corresponding tissues in vivo. The state of differentiation of primary human cells (respiratory epithelium, macrophages) can markedly affect virus tropism and host responses. Ex vivo cultures of human respiratory tissues provide a close resemblance to tissues in vivo and may be used to risk assess animal viruses for pandemic threat. Physiological factors (age, inflammation) can markedly affect virus receptor expression and virus tropism. Taken together with data from clinical studies on infected humans and relevant animal models, data from ex vivo and in vitro cultures of human tissues and cells can provide insights into virus transmission and pathogenesis and may provide understanding that leads to novel therapeutic interventions. Copyright © 2013 Elsevier B.V. All rights reserved.

Use Of Transgenic Mice In UDP-Glucuronosyltransferase (UGT) Studies

PubMed Central

Ou, Zhimin; Huang, Min; Zhao, Lizi; Xie, Wen

2009-01-01

Transgenic mouse models are useful to understand the function and regulation of drug metabolizing enzymes in vivo. This article is intended to describe the general strategies and to discuss specific examples on how to use transgenic, gene knockout, and humanized mice to study the function as well as genetic and pharmacological regulation of UDP-glucuronosyltransferases (UGTs). The physiological and pharmacological implications of transcription factor-mediated UGT regulation will also be discussed. The UGT-regulating transcription factors to be discussed in this article include nuclear hormone receptors (NRs), aryl hydrocarbon receptor (AhR), and nuclear factor erythroid 2-related factor 2 (Nrf2). PMID:20070245

Caloric restriction impedes age-related decline of mitochondrial function and neuronal activity

PubMed Central

Lin, Ai-Ling; Coman, Daniel; Jiang, Lihong; Rothman, Douglas L; Hyder, Fahmeed

2014-01-01

Caloric restriction (CR) prolongs lifespan and retards many detrimental effects of aging, but its effect on brain mitochondrial function and neuronal activity—especially in healthy aging—remains unexplored. Here we measured rates of neuronal glucose oxidation and glutamate–glutamine neurotransmitter cycling in young control, old control (i.e., healthy aging), and old CR rats using in vivo nuclear magnetic resonance spectroscopy. We found that, compared with the young control, neuronal energy production and neurotransmission rates were significantly reduced in healthy aging, but were preserved in old CR rats. The results suggest that CR mitigated the age-related deceleration of brain physiology. PMID:24984898

Spices as functional foods.

PubMed

Viuda-Martos, M; Ruiz-Navajas, Y; Fernández-López, J; Pérez-Alvarez, J A

2011-01-01

Spices and aromatic herbs have been used since antiquity as preservatives, colorants, and flavor enhancers. Spices, which have long been the basis of traditional medicine in many countries, have also been the subject of study, particularly by the chemical, pharmaceutical, and food industries, because of their potential use for improving health. Both in vitro and in vivo studies have demonstrated how these substances act as antioxidants, digestive stimulants, and hypolipidemics and show antibacterial, anti-inflammatory, antiviral, and anticancerigenic activities. These beneficial physiological effects may also have possible preventative applications in a variety of pathologies. The aim of this review is to present an overview of the potential of spices and aromatic herbs as functional foods.

Engineering intracellular active transport systems as in vivo biomolecular tools.

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

Bachand, George David; Carroll-Portillo, Amanda

2006-11-01

Active transport systems provide essential functions in terms of cell physiology and metastasis. These systems, however, are also co-opted by invading viruses, enabling directed transport of the virus to and from the cell's nucleus (i.e., the site of virus replication). Based on this concept, fundamentally new approaches for interrogating and manipulating the inner workings of living cells may be achievable by co-opting Nature's active transport systems as an in vivo biomolecular tool. The overall goal of this project was to investigate the ability to engineer kinesin-based transport systems for in vivo applications, specifically the collection of effector proteins (e.g., transcriptionalmore » regulators) within single cells. In the first part of this project, a chimeric fusion protein consisting of kinesin and a single chain variable fragment (scFv) of an antibody was successfully produced through a recombinant expression system. The kinesin-scFv retained both catalytic and antigenic functionality, enabling selective capture and transport of target antigens. The incorporation of a rabbit IgG-specific scFv into the kinesin established a generalized system for functionalizing kinesin with a wide range of target-selective antibodies raised in rabbits. The second objective was to develop methods of isolating the intact microtubule network from live cells as a platform for evaluating kinesin-based transport within the cytoskeletal architecture of a cell. Successful isolation of intact microtubule networks from two distinct cell types was demonstrated using glutaraldehyde and methanol fixation methods. This work provides a platform for inferring the ability of kinesin-scFv to function in vivo, and may also serve as a three-dimensional scaffold for evaluating and exploiting kinesin-based transport for nanotechnological applications. Overall, the technology developed in this project represents a first-step in engineering active transport system for in vivo applications. Further development could potentially enable selective capture of intracellular antigens, targeted delivery of therapeutic agents, or disruption of the transport systems and consequently the infection and pathogenesis cycle of biothreat agents.« less

Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

PubMed

Uchida, Sae; Kagitani, Fusako

2017-05-12

The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

Hypothalamic PGC-1α Protects Against High Fat Diet Exposure by Regulating ERα

PubMed Central

Morselli, Eugenia; Fuente-Martin, Esther; Finan, Brian; Kim, Min; Frank, Aaron; Garcia-Caceres, Cristina; Navas, Carlos Rodriguez; Gordillo, Ruth; Neinast, Michael; Kalainayakan, Sarada P.; Gao, Yuanqing; Yi, Chun-Xia; Hahner, Lisa; Palmer, Biff F.; Tschöp, Matthias H.; Clegg, Deborah J.

2014-01-01

Summary High fat diets (HFD) lead to obesity and inflammation in the central nervous system. Estrogens and Estrogen Receptor alpha (ERα) protect premenopausal females from the metabolic complications of inflammation and obesity related disease. Here we demonstrate that hypothalamic PGC-1α regulates ERα and inflammation in vivo. HFD significantly increased palmitic acid (PA) and sphingolipids in the CNS of males when compared to female mice. PA, in vitro, and HFD, in vivo, reduced PGC-1α and ERα in hypothalamic neurons and astrocytes of male mice and promoted inflammation. PGC-1α depletion with ERα overexpression significantly inhibited PA-induced inflammation, confirming that ERα is a critical determinant of the anti-inflammatory response. Physiologic relevance of ERα-regulated inflammation was demonstrated by reduced myocardial function in male but not female mice following chronic HFD exposure. Our findings show for the first time that HFD/PA reduces PGC-1α and ERα, promoting inflammation and decrements in myocardial function in a sex-specific way. PMID:25373903

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling

PubMed Central

Ho, Chia Chi M.; Chhabra, Akanksha; Starkl, Philipp; Schnorr, Peter-John; Wilmes, Stephan; Moraga, Ignacio; Kwon, Hye-Sook; Gaudenzio, Nicolas; Sibilano, Riccardo; Wehrman, Tom S.; Gakovic, Milica; Sockolosky, Jonathan T.; Tiffany, Matthew R.; Ring, Aaron M.; Piehler, Jacob; Weissman, Irving L.; Galli, Stephen J.; Shizuru, Judith A.; Garcia, K. Christopher

2017-01-01

SUMMARY Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems. PMID:28283060

Bioengineered vocal fold mucosa for voice restoration*

PubMed Central

Ling, Changying; Li, Qiyao; Brown, Matthew E.; Kishimoto, Yo; Toya, Yutaka; Devine, Erin E.; Choi, Kyeong-Ok; Nishimoto, Kohei; Norman, Ian G.; Tsegyal, Tenzin; Jiang, Jack J.; Burlingham, William J.; Gunasekaran, Sundaram; Smith, Lloyd M.; Frey, Brian L.; Welham, Nathan V.

2015-01-01

Patients with voice impairment caused by advanced vocal fold (VF) fibrosis or tissue loss have few treatment options. A transplantable, bioengineered VF mucosa would address the individual and societal costs of voice-related communication loss. Such a tissue must be biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration, and physiologically capable of maintaining a barrier against the airway lumen. Here, we isolated primary human VF fibroblasts and epithelial cells and cocultured them under organotypic conditions. The resulting engineered mucosae showed morphologic features of native tissue, proteome-level evidence of mucosal morphogenesis and emerging extracellular matrix complexity, and rudimentary barrier function in vitro. When grafted into canine larynges ex vivo, the mucosae generated vibratory behavior and acoustic output that were indistinguishable from those of native VF tissue. When grafted into humanized mice in vivo, the mucosae survived and were well tolerated by the human adaptive immune system. This tissue engineering approach has the potential to restore voice function in patients with otherwise untreatable VF mucosal disease. PMID:26582902

Hypothalamic PGC-1α protects against high-fat diet exposure by regulating ERα.

PubMed

Morselli, Eugenia; Fuente-Martin, Esther; Finan, Brian; Kim, Min; Frank, Aaron; Garcia-Caceres, Cristina; Navas, Carlos Rodriguez; Gordillo, Ruth; Neinast, Michael; Kalainayakan, Sarada P; Li, Dan L; Gao, Yuanqing; Yi, Chun-Xia; Hahner, Lisa; Palmer, Biff F; Tschöp, Matthias H; Clegg, Deborah J

2014-10-23

High-fat diets (HFDs) lead to obesity and inflammation in the central nervous system (CNS). Estrogens and estrogen receptor α (ERα) protect premenopausal females from the metabolic complications of inflammation and obesity-related disease. Here, we demonstrate that hypothalamic PGC-1α regulates ERα and inflammation in vivo. HFD significantly increased palmitic acid (PA) and sphingolipids in the CNS of male mice when compared to female mice. PA, in vitro, and HFD, in vivo, reduced PGC-1α and ERα in hypothalamic neurons and astrocytes of male mice and promoted inflammation. PGC-1α depletion with ERα overexpression significantly inhibited PA-induced inflammation, confirming that ERα is a critical determinant of the anti-inflammatory response. Physiologic relevance of ERα-regulated inflammation was demonstrated by reduced myocardial function in male, but not female, mice following chronic HFD exposure. Our findings show that HFD/PA reduces PGC-1α and ERα, promoting inflammation and decrements in myocardial function in a sex-specific way.

Gravitational physiology of human immune cells: a review of in vivo, ex vivo and in vitro studies

NASA Technical Reports Server (NTRS)

Cogoli, A.

1996-01-01

The study of the function of immune cells in microgravity has been studied for more than 20 years in several laboratories. It is clear today that the immune system is depressed in more than 50% of the astronauts during and after space flight and that the activation of T lymphocytes by mitogens in vitro changes dramatically. This article gives an overview of the gravitational studies conducted by our laboratory in Spacelab, in MIR station, in sounding rockets and on the ground in the clinostat and the centrifuge. Three experimental approaches are followed in our work: (i) Ex vivo studies are performed with blood samples drawn from astronauts; (ii) in vivo studies are based on the application of seven antigens to the skin of the astronauts; (iii) in vitro studies are carried out with immune cells purified from the blood of healthy donors (not astronauts). The data from our in vivo and ex vivo studies are in agreement with those of other laboratories and show that the immunological function is depressed in the majority of astronauts as a consequence of the stress of space flight rather than by a direct influence of gravity on the cell. Immune depression may become a critical hazard on long duration flights on space stations or to other planets. In vitro experiments show that cultures of free-floating lymphocytes and monocytes undergo a dramatic depression of activation by the mitogen concanavalin A, while activation is more than doubled when the cells are attached to microcarrier beads. Such effects may be attributed to both direct and indirect effects of gravitational unloading on basic biological mechanisms of the cell. While the in vitro data are very important to clarify certain aspects of the biological mechanism of T cells activation, they are not descriptive of the changes of the immunological function of the astronauts.

Gravitational physiology of human immune cells: a review of in vivo, ex vivo and in vitro studies.

PubMed

Cogoli, A

1996-04-01

The study of the function of immune cells in microgravity has been studied for more than 20 years in several laboratories. It is clear today that the immune system is depressed in more than 50% of the astronauts during and after space flight and that the activation of T lymphocytes by mitogens in vitro changes dramatically. This article gives an overview of the gravitational studies conducted by our laboratory in Spacelab, in MIR station, in sounding rockets and on the ground in the clinostat and the centrifuge. Three experimental approaches are followed in our work: (i) Ex vivo studies are performed with blood samples drawn from astronauts; (ii) in vivo studies are based on the application of seven antigens to the skin of the astronauts; (iii) in vitro studies are carried out with immune cells purified from the blood of healthy donors (not astronauts). The data from our in vivo and ex vivo studies are in agreement with those of other laboratories and show that the immunological function is depressed in the majority of astronauts as a consequence of the stress of space flight rather than by a direct influence of gravity on the cell. Immune depression may become a critical hazard on long duration flights on space stations or to other planets. In vitro experiments show that cultures of free-floating lymphocytes and monocytes undergo a dramatic depression of activation by the mitogen concanavalin A, while activation is more than doubled when the cells are attached to microcarrier beads. Such effects may be attributed to both direct and indirect effects of gravitational unloading on basic biological mechanisms of the cell. While the in vitro data are very important to clarify certain aspects of the biological mechanism of T cells activation, they are not descriptive of the changes of the immunological function of the astronauts.

The Functions of Metallothionein and ZIP and ZnT Transporters: An Overview and Perspective

PubMed Central

Kimura, Tomoki; Kambe, Taiho

2016-01-01

Around 3000 proteins are thought to bind zinc in vivo, which corresponds to ~10% of the human proteome. Zinc plays a pivotal role as a structural, catalytic, and signaling component that functions in numerous physiological processes. It is more widely used as a structural element in proteins than any other transition metal ion, is a catalytic component of many enzymes, and acts as a cellular signaling mediator. Thus, it is expected that zinc metabolism and homeostasis have sophisticated regulation, and elucidating the underlying molecular basis of this is essential to understanding zinc functions in cellular physiology and pathogenesis. In recent decades, an increasing amount of evidence has uncovered critical roles of a number of proteins in zinc metabolism and homeostasis through influxing, chelating, sequestrating, coordinating, releasing, and effluxing zinc. Metallothioneins (MT) and Zrt- and Irt-like proteins (ZIP) and Zn transporters (ZnT) are the proteins primarily involved in these processes, and their malfunction has been implicated in a number of inherited diseases such as acrodermatitis enteropathica. The present review updates our current understanding of the biological functions of MTs and ZIP and ZnT transporters from several new perspectives. PMID:26959009

The effect of mechanical loads on the degradation of aliphatic biodegradable polyesters.

PubMed

Li, Ying; Chu, Zhaowei; Li, Xiaoming; Ding, Xili; Guo, Meng; Zhao, Haoran; Yao, Jie; Wang, Lizhen; Cai, Qiang; Fan, Yubo

2017-06-01

Aliphatic biodegradable polyesters have been the most widely used synthetic polymers for developing biodegradable devices as alternatives for the currently used permanent medical devices. The performances during biodegradation process play crucial roles for final realization of their functions. Because physiological and biochemical environment in vivo significantly affects biodegradation process, large numbers of studies on effects of mechanical loads on the degradation of aliphatic biodegradable polyesters have been launched during last decades. In this review article, we discussed the mechanism of biodegradation and several different mechanical loads that have been reported to affect the biodegradation process. Other physiological and biochemical factors related to mechanical loads were also discussed. The mechanical load could change the conformational strain energy and morphology to weaken the stability of the polymer. Besides, the load and pattern could accelerate the loss of intrinsic mechanical properties of polymers. This indicated that investigations into effects of mechanical loads on the degradation should be indispensable. More combination condition of mechanical loads and multiple factors should be considered in order to keep the degradation rate controllable and evaluate the degradation process in vivo accurately. Only then can the degradable devise achieve the desired effects and further expand the special applications of aliphatic biodegradable polyesters.

Evaluation of Eu(II) -based positive contrast enhancement after intravenous, intraperitoneal, and subcutaneous injections.

PubMed

Ekanger, Levi A; Polin, Lisa A; Shen, Yimin; Haacke, E Mark; Allen, Matthew J

2016-07-01

Eu(II) -based contrast agents offer physiologically relevant, metal-based redox sensing that is unachievable with Gd(III) -based contrast agents. To evaluate the in vivo contrast enhancement of Eu(II) as a function of injection type, we performed intravenous, intraperitoneal, and subcutaneous injections in mice. Our data reveal a correlation between reported oxygen content and expected rates of diffusion with the persistence of Eu(II) -based contrast enhancement. Biodistribution studies revealed europium clearance through the liver and kidneys for intravenous and intraperitoneal injections, but no contrast enhancement was observed in organs associated with clearance. These data represent a step toward understanding the behavior of Eu(II) -based complexes in vivo. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Real-time high dynamic range laser scanning microscopy

NASA Astrophysics Data System (ADS)

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-04-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach.

PubMed

Opalińska, Magdalena; Parys, Katarzyna; Jańska, Hanna

2017-11-18

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i -AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4's in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4's physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

PubMed Central

Parys, Katarzyna; Jańska, Hanna

2017-01-01

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4’s physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome. PMID:29156584

Calcitonin and calcitonin receptors: bone and beyond

PubMed Central

Pondel, Marc

2000-01-01

Calcitonin (CT), a 32 amino acid peptide hormone produced primarily by the thyroid, and its receptor (CTR) are well known for their ability to regulate osteoclast mediated bone resorption and enhance Ca2+ excretion by the kidney. However, recent studies now suggest that CT and CTRs may play an important role in a variety of processes as wide ranging as embryonic/foetal development and sperm function/physiology. In this review article, CT and CTR gene transcription, signal transduction and function are addressed. The effects of CT on the physiology of a variety of organ systems are discussed and the relationship between polymorphisms in the CTR gene and bone mineral density (BMD)/osteoporosis is examined. Recent studies demonstrating the ability of receptor activity modifying proteins (RAMPs) to post-translationally modify the calcitonin receptor-like receptor (CRLR) are detailed and studies employing transgenic mouse technology to determine the temporal and tissue specific transcriptional activity of the CTR gene in vivo are discussed. PMID:11298188

Eccentric Contraction-Induced Muscle Injury: Reproducible, Quantitative, Physiological Models to Impair Skeletal Muscle’s Capacity to Generate Force

PubMed Central

Call, Jarrod A.; Lowe, Dawn A.

2018-01-01

In order to investigate the molecular and cellular mechanisms of muscle regeneration an experimental injury model is required. Advantages of eccentric contraction-induced injury are that it is a controllable, reproducible, and physiologically relevant model to cause muscle injury, with injury being defined as a loss of force generating capacity. While eccentric contractions can be incorporated into conscious animal study designs such as downhill treadmill running, electrophysiological approaches to elicit eccentric contractions and examine muscle contractility, for example before and after the injurious eccentric contractions, allows researchers to circumvent common issues in determining muscle function in a conscious animal (e.g., unwillingness to participate). Herein, we describe in vitro and in vivo methods that are reliable, repeatable, and truly maximal because the muscle contractions are evoked in a controlled, quantifiable manner independent of subject motivation. Both methods can be used to initiate eccentric contraction-induced injury and are suitable for monitoring functional muscle regeneration hours to days to weeks post-injury. PMID:27492161

Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS

PubMed Central

Martínez-Silva, María de Lourdes; Imhoff-Manuel, Rebecca D; Sharma, Aarti; Heckman, CJ; Shneider, Neil A; Roselli, Francesco; Zytnicki, Daniel

2018-01-01

Hyperexcitability has been suggested to contribute to motoneuron degeneration in amyotrophic lateral sclerosis (ALS). If this is so, and given that the physiological type of a motor unit determines the relative susceptibility of its motoneuron in ALS, then one would expect the most vulnerable motoneurons to display the strongest hyperexcitability prior to their degeneration, whereas the less vulnerable should display a moderate hyperexcitability, if any. We tested this hypothesis in vivo in two unrelated ALS mouse models by correlating the electrical properties of motoneurons with their physiological types, identified based on their motor unit contractile properties. We found that, far from being hyperexcitable, the most vulnerable motoneurons become unable to fire repetitively despite the fact that their neuromuscular junctions were still functional. Disease markers confirm that this loss of function is an early sign of degeneration. Our results indicate that intrinsic hyperexcitability is unlikely to be the cause of motoneuron degeneration. PMID:29580378

Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology.

PubMed

Kiang, J G; Tsokos, G C

1998-11-01

Heat shock proteins (HSPs) are detected in all cells, prokaryotic and eukaryotic. In vivo and in vitro studies have shown that various stressors transiently increase production of HSPs as protection against harmful insults. Increased levels of HSPs occur after environmental stresses, infection, normal physiological processes, and gene transfer. Although the mechanisms by which HSPs protect cells are not clearly understood, their expression can be modulated by cell signal transducers, such as changes in intracellular pH, cyclic AMP, Ca2+, Na+, inositol trisphosphate, protein kinase C, and protein phosphatases. Most of the HSPs interact with other proteins in cells and alter their function. These and other protein-protein interactions may mediate the little understood effects of HSPs on various cell functions. In this review, we focus on the structure of the HSP-70 family (HSP-70s), regulation of HSP-70 gene expression, their cytoprotective effects, and the possibility of regulating HSP-70 expression through modulation of signal transduction pathways. The clinical importance and therapeutic potential of HSPs are discussed.

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

PubMed Central

Hofmann, Sebastian; Braun, Attila; Pozgaj, Rastislav; Morowski, Martina; Vögtle, Timo; Nieswandt, Bernhard

2014-01-01

Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. Objective The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. Methods and Results We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84−/− mice. In vivo, Cd84−/− mice exhibited unaltered hemostatic function and arterial thrombus formation. Conclusion These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions. PMID:25551754

Dietary nitrate does not reduce oxygen cost of exercise or improve muscle mitochondrial function in patients with mitochondrial myopathy.

PubMed

Nabben, Miranda; Schmitz, Joep P J; Ciapaite, Jolita; le Clercq, Carlijn M P; van Riel, Natal A; Haak, Harm R; Nicolay, Klaas; de Coo, Irenaeus F M; Smeets, Hubert; Praet, Stephan F; van Loon, Luc J; Prompers, Jeanine J

2017-05-01

Muscle weakness and exercise intolerance negatively affect the quality of life of patients with mitochondrial myopathy. Short-term dietary nitrate supplementation has been shown to improve exercise performance and reduce oxygen cost of exercise in healthy humans and trained athletes. We investigated whether 1 wk of dietary inorganic nitrate supplementation decreases the oxygen cost of exercise and improves mitochondrial function in patients with mitochondrial myopathy. Ten patients with mitochondrial myopathy (40 ± 5 yr, maximal whole body oxygen uptake = 21.2 ± 3.2 ml·min -1 ·kg body wt -1 , maximal work load = 122 ± 26 W) received 8.5 mg·kg body wt -1 ·day -1 inorganic nitrate (~7 mmol) for 8 days. Whole body oxygen consumption at 50% of the maximal work load, in vivo skeletal muscle oxidative capacity (evaluated from postexercise phosphocreatine recovery using 31 P-magnetic resonance spectroscopy), and ex vivo mitochondrial oxidative capacity in permeabilized skinned muscle fibers (measured with high-resolution respirometry) were determined before and after nitrate supplementation. Despite a sixfold increase in plasma nitrate levels, nitrate supplementation did not affect whole body oxygen cost during submaximal exercise. Additionally, no beneficial effects of nitrate were found on in vivo or ex vivo muscle mitochondrial oxidative capacity. This is the first time that the therapeutic potential of dietary nitrate for patients with mitochondrial myopathy was evaluated. We conclude that 1 wk of dietary nitrate supplementation does not reduce oxygen cost of exercise or improve mitochondrial function in the group of patients tested. Copyright © 2017 the American Physiological Society.

Platelet bioreactor-on-a-chip

PubMed Central

Mazutis, Linas; Wu, Stephen; Sylman, Joanna L.; Ehrlicher, Allen; Machlus, Kellie R.; Feng, Qiang; Lu, Shijiang; Lanza, Robert; Neeves, Keith B.; Weitz, David A.; Italiano, Joseph E.

2014-01-01

Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. PMID:25606631

Silver-loaded seaweed-based cellulosic fiber improves epidermal skin physiology in atopic dermatitis: safety assessment, mode of action and controlled, randomized single-blinded exploratory in vivo study.

PubMed

Fluhr, Joachim W; Breternitz, Maria; Kowatzki, Doreen; Bauer, Andrea; Bossert, Joerg; Elsner, Peter; Hipler, Uta-Christina

2010-08-01

The epidermal part of the skin is the major interface between the internal body and the external environment. The skin has a specific physiology and is to different degrees adapted for protection against multiple exogenous stress factors. Clothing is the material with the longest and most intensive contact to human skin. It plays a critical role especially in inflammatory dermatoses or skin conditions with an increased susceptibility of bacterial and fungal infections like atopic dermatitis. Previously, we have shown a dose-dependent antibacterial and antifungal activity of silver-loaded seaweed-based cellulosic fibres. We studied the mode of action of silver-loaded seaweed-based cellulosic fiber and performed a broad safety assessment. The principal aim was to analyse the effects of wearing the textile on epidermal skin physiology in 37 patients with atopic dermatitis in a controlled, randomized single-blinded in vivo study. Furthermore, the sensitization potential was tested in a patch test in 111 panellists. We could demonstrate in vitro a dose-dependent scavenging of induced reactive oxygen species by silver-loaded seaweed-based cellulosic fibers. Safety assessment of these fibres showed no detectable release of silver ions. Furthermore, ex vivo assessment after 24 h application both in healthy volunteers and patients with atopic dermatitis by sequential tape stripping and subsequently raster electron microscopy and energy dispersive microanalysis analysis revealed no detectable amounts of silver in any of stratum corneum layers. Serum analysis of silver showed no detectable levels. The in vivo patch testing of 111 volunteers revealed no sensitization against different SeaCell Active (SeaCell GmbH, Rudolstadt, Germany) containing fabrics. The in vivo study on 37 patients with known atopic dermatitis and mild-to-moderate eczema on their arms were randomly assigned to either silver-loaded seaweed fibre T-shirts or to cotton T-shirts for 8 weeks. A significant reduction in Staphylococcus aureus colonization was detectable for the silver T-shirts compared with cotton T-shirts without any changes in non-pathogenic surface bacteria colonization. Furthermore, a more pronounced improvement in barrier function (transepidermal water loss) was observed in mildly involved eczema areas during the first 4 weeks of the study. Stratum corneum hydration and surface pH improved in both treatment groups over time. The tested silver-loaded seaweed fibre can be regarded as safe and seams to be suited for application in bio-active textiles in atopic dermatitis based on its positive in vivo activity.

Non-invasive pH determination adjacent to degradable biomaterials in vivo.

PubMed

Bartsch, Ivonne; Willbold, Elmar; Rosenhahn, Bodo; Witte, Frank

2014-01-01

An appropriate pH level is an important prerequisite for the physiologal functioning of cells and tissues. Changes in the extracellular pH often lead to specific cellular reactions and an altered metabolism of cells and tissues influences the extracellular pH range. Thus a method to monitor the extracellular pH is a valuable tool to track specific tissue reactions. In this article we describe a method for the determination of the pH range adjacent to degradable biomaterials using wireless in vivo imaging. Using hairless but immunocompetent mice the fluorophor 5-(6)-carboxy SNARF-1 and the in vivo fluorescence and multispectral acquisition and analysis system Maestro it is possible to track shifts in pH in small living animals over a longer period of time. This method is especially suitable for studies which focus on the interaction of degrading biomaterials with their adjacent tissues. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research.

PubMed

Choy, Garry; Choyke, Peter; Libutti, Steven K

2003-10-01

Recently, there has been tremendous interest in developing techniques such as MRI, micro-CT, micro-PET, and SPECT to image function and processes in small animals. These technologies offer deep tissue penetration and high spatial resolution, but compared with noninvasive small animal optical imaging, these techniques are very costly and time consuming to implement. Optical imaging is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. In vivo optical imaging is the result of a coalescence of technologies from chemistry, physics, and biology. The development of highly sensitive light detection systems has allowed biologists to use imaging in studying physiological processes. Over the last few decades, biochemists have also worked to isolate and further develop optical reporters such as GFP, luciferase, and cyanine dyes. This article reviews the common types of fluorescent and bioluminescent optical imaging, the typical system platforms and configurations, and the applications in the investigation of cancer biology.

Satellite cells in human skeletal muscle plasticity

PubMed Central

Snijders, Tim; Nederveen, Joshua P.; McKay, Bryon R.; Joanisse, Sophie; Verdijk, Lex B.; van Loon, Luc J. C.; Parise, Gianni

2015-01-01

Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models. PMID:26557092

Multidimensional profiling platforms reveal metabolic dysregulation caused by organophosphorus pesticides.

PubMed

Medina-Cleghorn, Daniel; Heslin, Ann; Morris, Patrick J; Mulvihill, Melinda M; Nomura, Daniel K

2014-02-21

We are environmentally exposed to countless synthetic chemicals on a daily basis, with an increasing number of these chemical exposures linked to adverse health effects. However, our understanding of the (patho)physiological effects of these chemicals remains poorly understood, due in part to a general lack of effort to systematically and comprehensively identify the direct interactions of environmental chemicals with biological macromolecules in mammalian systems in vivo. Here, we have used functional chemoproteomic and metabolomic platforms to broadly identify direct enzyme targets that are inhibited by widely used organophosphorus (OP) pesticides in vivo in mice and to determine metabolic alterations that are caused by these chemicals. We find that these pesticides directly inhibit over 20 serine hydrolases in vivo leading to widespread disruptions in lipid metabolism. Through identifying direct biological targets of OP pesticides, we show heretofore unrecognized modes of toxicity that may be associated with these agents and underscore the utility of using multidimensional profiling approaches to obtain a more complete understanding of toxicities associated with environmental chemicals.

Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung.

PubMed

Oh, Phil; Borgström, Per; Witkiewicz, Halina; Li, Yan; Borgström, Bengt J; Chrastina, Adrian; Iwata, Koji; Zinn, Kurt R; Baldwin, Richard; Testa, Jacqueline E; Schnitzer, Jan E

2007-03-01

How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital fluorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body gamma-scintigraphic imaging shows rapid, specific delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafficking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. 'In vivo proteomic imaging' as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.

Activating mitochondrial function and haemoglobin expression with EH-201, an inducer of erythropoietin in neuronal cells, reverses memory impairment.

PubMed

Horng, Lin-Yea; Hsu, Pei-Lun; Chen, Li-Wen; Tseng, Wang-Zou; Hsu, Kai-Tin; Wu, Chia-Ling; Wu, Rong-Tsun

2015-10-01

Memory impairment can be progressive in neurodegenerative diseases, and physiological ageing or brain injury, mitochondrial dysfunction and oxidative stress are critical components of these issues. An early clinical study has demonstrated cognitive improvement during erythropoietin treatment in patients with chronic renal failure. As erythropoietin cannot freely cross the blood-brain barrier, we tested EH-201 (2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside, also known as TSG), a low MW inducer of erythropoietin, for its therapeutic effects on memory impairment in models of neurodegenerative diseases, physiological ageing or brain injury. The effects of EH-201 were investigated in astrocytes and PC12 neuronal-like cells. In vivo, we used sleep-deprived (SD) mice as a stress model, amyloid-β (Aβ)-injected mice as a physiological ageing model and kainic acid (KA)-injected mice as a brain damage model to assess the therapeutic effects of EH-201. EH-201 induced expression of erythropoietin, PPAR-γ coactivator 1α (PGC-1α) and haemoglobin in astrocytes and PC12 neuronal-like cells. In vivo, EH-201 treatment restored memory impairment, as assessed by the passive avoidance test, in SD, Aβ and KA mouse models. In the hippocampus of mice given EH-201 in their diet, levels of erythropoietin, PGC-1α and haemoglobin were increased The induction of endogenous erythropoietin in neuronal cells by inducers such as EH-201 might be a therapeutic strategy for memory impairment in neurodegenerative disease, physiological ageing or traumatic brain injury. © 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

Inducible Knockout of the Cyclin-Dependent Kinase 5 Activator p35 Alters Hippocampal Spatial Coding and Neuronal Excitability

PubMed Central

Kamiki, Eriko; Boehringer, Roman; Polygalov, Denis; Ohshima, Toshio; McHugh, Thomas J.

2018-01-01

p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5), a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice. PMID:29867369

Altered LARK Expression Perturbs Development and Physiology of the Drosophila PDF Clock Neurons

PubMed Central

Huang, Yanmei; Howlett, Eric; Stern, Michael; Jackson, F. Rob

2009-01-01

The LARK RNA-binding protein (RBP) has well documented roles in the circadian systems of Drosophila and mammals. Recent studies have demonstrated that the Drosophila LARK RBP is associated with many mRNA targets, in vivo, including those that regulate either neurophysiology or development of the nervous system. In the present study, we have employed conditional expression techniques to distinguish developmental and physiological functions of LARK for a defined class of neurons: the Pigment Dispersing Factor (PDF)-containing LNv clock neurons. We found that increased LARK expression during development dramatically alters the small LNv class of neurons with no obvious effects on the large LNv cells. Conversely, conditional expression of LARK at the adult stage results in altered clock protein rhythms and circadian locomotor activity, even though neural morphology is normal in such animals. Electrophysiological analyses at the larval neuromuscular junction indicate a role for LARK in regulating neuronal excitability. Altogether, our results demonstrate that LARK activity is critical for neuronal development and physiology. PMID:19303442

Brain physiological state evaluated by real-time multiparametric tissue spectroscopy in vivo

NASA Astrophysics Data System (ADS)

Mayevsky, Avraham; Barbiro-Michaely, Efrat; Kutai-Asis, Hofit; Deutsch, Assaf; Jaronkin, Alex

2004-07-01

The significance of normal mitochondrial function in cellular energy homeostasis as well as its involvement in acute and chronic neurodegenerative disease was reviewed recently (Nicholls & Budd. Physiol Rev. 80: 315-360, 2000). Nevertheless, monitoring of mitochondrial function in vivo and real time mode was not used by many investigators and is very rare in clinical practice. The main principle tool available for the evaluation of mitochondrial function is the monitoring of NADH fluorescence. In order to interpret correctly the changes in NADH redox state in vivo, it is necessary to correlate this signal to other parameters, reflecting O2 supply to the brain. Therefore, we have developed and applied a multiparametric optical monitoring system, by which microcirculatory blood flow and hemoglobin oxygenation is measured, together with mitochondrial NADH fluorescence. Since the calibration of these signals is not in absolute units, the simultaneous monitoring provide a practical tool for the interpretation of brain functional state under various pathophysiological conditions. The monitoring system combines a time-sharing fluorometer-reflectometer for the measurement of NADH fluorescence and hemoglobin oxygenation as well as a laser Doppler flowmeter for the recording of microcirculatory blood flow. A combined fiber optic probe was located on the surface of the brain using a skull cemented cannula. Rats and gerbils were exposed to anoxia, ischemia and spreading depression and the functional state of the brain was evaluated. The results showed a clear correlation between O2 supply/demand as well as, energy balance under the various pathophysiological conditions. This monitoring approach could be adapted to clinical monitoring of tissue vitality.

Absolute quantification of carnosine in human calf muscle by proton magnetic resonance spectroscopy

NASA Astrophysics Data System (ADS)

Özdemir, Mahir S.; Reyngoudt, Harmen; DeDeene, Yves; Sazak, Hakan S.; Fieremans, Els; Delputte, Steven; D'Asseler, Yves; Derave, Wim; Lemahieu, Ignace; Achten, Eric

2007-12-01

Carnosine has been shown to be present in the skeletal muscle and in the brain of a variety of animals and humans. Despite the various physiological functions assigned to this metabolite, its exact role remains unclear. It has been suggested that carnosine plays a role in buffering in the intracellular physiological pHi range in skeletal muscle as a result of accepting hydrogen ions released in the development of fatigue during intensive exercise. It is thus postulated that the concentration of carnosine is an indicator for the extent of the buffering capacity. However, the determination of the concentration of this metabolite has only been performed by means of muscle biopsy, which is an invasive procedure. In this paper, we utilized proton magnetic resonance spectroscopy (1H MRS) in order to perform absolute quantification of carnosine in vivo non-invasively. The method was verified by phantom experiments and in vivo measurements in the calf muscles of athletes and untrained volunteers. The measured mean concentrations in the soleus and the gastrocnemius muscles were found to be 2.81 ± 0.57/4.8 ± 1.59 mM (mean ± SD) for athletes and 2.58 ± 0.65/3.3 ± 0.32 mM for untrained volunteers, respectively. These values are in agreement with previously reported biopsy-based results. Our results suggest that 1H MRS can provide an alternative method for non-invasively determining carnosine concentration in human calf muscle in vivo.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

PubMed

Flood, P; Alvarez, L; Reynaud, E G

2016-10-11

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Indolethylamine-N-methyltransferase Polymorphisms: Genetic and Biochemical Approaches for Study of Endogenous N,N,-dimethyltryptamine.

PubMed

Dean, Jon G

2018-01-01

N,N -dimethyltryptamine (DMT) is a powerful serotonergic psychedelic whose exogenous administration elicits striking psychedelic effects in humans. Studies have identified DMT and analogous compounds (e.g., 5-hydroxy-DMT, 5-methoxy-DMT) alongside of an enzyme capable of synthesizing DMT endogenously from tryptamine, indolethylamine- N -methyltransferase (INMT), in human and several other mammalian tissues. Subsequently, multiple hypotheses for the physiological role of endogenous DMT have emerged, from proposed immunomodulatory functions to an emphasis on the overlap between the mental states generated by exogenous DMT and naturally occurring altered states of consciousness; e.g., schizophrenia. However, no clear relationship between endogenous DMT and naturally occurring altered states of consciousness has yet been established from in vivo assays of DMT in bodily fluids. The advent of genetic screening has afforded the capability to link alterations in the sequence of specific genes to behavioral and molecular phenotypes via expression of identified single nucleotide polymorphisms (SNPs) in cell and animal models. As SNPs in INMT may impact endogenous DMT synthesis and levels via changes in INMT expression and/or INMT structure and function, these combined genetic and biochemical approaches circumvent the limitations of assaying DMT in bodily fluids and may augment data from prior in vitro and in vivo work. Therefore, all reported SNPs in INMT were amassed from genetic and biochemical literature and genomic databases to consolidate a blueprint for future studies aimed at elucidating whether DMT plays a physiological role.

Fractal Patterns of Neural Activity Exist within the Suprachiasmatic Nucleus and Require Extrinsic Network Interactions

PubMed Central

Hu, Kun; Meijer, Johanna H.; Shea, Steven A.; vanderLeest, Henk Tjebbe; Pittman-Polletta, Benjamin; Houben, Thijs; van Oosterhout, Floor; Deboer, Tom; Scheer, Frank A. J. L.

2012-01-01

The mammalian central circadian pacemaker (the suprachiasmatic nucleus, SCN) contains thousands of neurons that are coupled through a complex network of interactions. In addition to the established role of the SCN in generating rhythms of ∼24 hours in many physiological functions, the SCN was recently shown to be necessary for normal self-similar/fractal organization of motor activity and heart rate over a wide range of time scales—from minutes to 24 hours. To test whether the neural network within the SCN is sufficient to generate such fractal patterns, we studied multi-unit neural activity of in vivo and in vitro SCNs in rodents. In vivo SCN-neural activity exhibited fractal patterns that are virtually identical in mice and rats and are similar to those in motor activity at time scales from minutes up to 10 hours. In addition, these patterns remained unchanged when the main afferent signal to the SCN, namely light, was removed. However, the fractal patterns of SCN-neural activity are not autonomous within the SCN as these patterns completely broke down in the isolated in vitro SCN despite persistence of circadian rhythmicity. Thus, SCN-neural activity is fractal in the intact organism and these fractal patterns require network interactions between the SCN and extra-SCN nodes. Such a fractal control network could underlie the fractal regulation observed in many physiological functions that involve the SCN, including motor control and heart rate regulation. PMID:23185285

Indolethylamine-N-methyltransferase Polymorphisms: Genetic and Biochemical Approaches for Study of Endogenous N,N,-dimethyltryptamine

PubMed Central

Dean, Jon G.

2018-01-01

N,N-dimethyltryptamine (DMT) is a powerful serotonergic psychedelic whose exogenous administration elicits striking psychedelic effects in humans. Studies have identified DMT and analogous compounds (e.g., 5-hydroxy-DMT, 5-methoxy-DMT) alongside of an enzyme capable of synthesizing DMT endogenously from tryptamine, indolethylamine-N-methyltransferase (INMT), in human and several other mammalian tissues. Subsequently, multiple hypotheses for the physiological role of endogenous DMT have emerged, from proposed immunomodulatory functions to an emphasis on the overlap between the mental states generated by exogenous DMT and naturally occurring altered states of consciousness; e.g., schizophrenia. However, no clear relationship between endogenous DMT and naturally occurring altered states of consciousness has yet been established from in vivo assays of DMT in bodily fluids. The advent of genetic screening has afforded the capability to link alterations in the sequence of specific genes to behavioral and molecular phenotypes via expression of identified single nucleotide polymorphisms (SNPs) in cell and animal models. As SNPs in INMT may impact endogenous DMT synthesis and levels via changes in INMT expression and/or INMT structure and function, these combined genetic and biochemical approaches circumvent the limitations of assaying DMT in bodily fluids and may augment data from prior in vitro and in vivo work. Therefore, all reported SNPs in INMT were amassed from genetic and biochemical literature and genomic databases to consolidate a blueprint for future studies aimed at elucidating whether DMT plays a physiological role. PMID:29740267

Terahertz time-lapse imaging of hydration in physiological tissues

NASA Astrophysics Data System (ADS)

Bennett, David B.; Taylor, Zachary D.; Bajwa, Neha; Tewari, Priyamvada; Maccabi, Ashkan; Sung, Shijun; Singh, Rahul S.; Culjat, Martin O.; Grundfest, Warren S.; Brown, Elliott R.

2011-02-01

This study describes terahertz (THz) imaging of hydration changes in physiological tissues with high water concentration sensitivity. A fast-scanning, pulsed THz imaging system (centered at 525 GHz; 125 GHz bandwidth) was utilized to acquire a 35 mm x 35 mm field-of-view with 0.5 mm x 0.5 mm pixels in less than two minutes. THz time-lapsed images were taken on three sample systems: (1) a simple binary system of water evaporating from a polypropylene towel, (2) the accumulation of fluid at the site of a sulfuric acid burn on ex vivo porcine skin, and (3) the evaporative dehydration of an ex vivo porcine cornea. The diffusion-regulating behavior of corneal tissue is elucidated, and the correlation of THz reflectivity with tissue hydration is measured using THz spectroscopy on four ex vivo corneas. We conclude that THz imaging can discern small differences in the distribution of water in physiological tissues and is a good candidate for burn and corneal imaging.

Renaissance of morphological studies: the examination of functional structures in living animal organs using the in vivo cryotechnique.

PubMed

Ohno, Shinichi; Saitoh, Yurika; Ohno, Nobuhiko; Terada, Nobuo

2017-01-01

Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living animal organs, as well as the locations of their molecular components. Recently, the live imaging of animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living animal organs are compatible with those obtained from live images of the same organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living animal organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components-such as rapidly changing signaling molecules, membrane channels, or receptors-in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.

Extra permeability is required to model dynamic oxygen measurements: evidence for functional recruitment?

PubMed Central

Barrett, Matthew JP; Suresh, Vinod

2013-01-01

Neural activation triggers a rapid, focal increase in blood flow and thus oxygen delivery. Local oxygen consumption also increases, although not to the same extent as oxygen delivery. This ‘uncoupling' enables a number of widely-used functional neuroimaging techniques; however, the physiologic mechanisms that govern oxygen transport under these conditions remain unclear. Here, we explore this dynamic process using a new mathematical model. Motivated by experimental observations and previous modeling, we hypothesized that functional recruitment of capillaries has an important role during neural activation. Using conventional mechanisms alone, the model predictions were inconsistent with in vivo measurements of oxygen partial pressure. However, dynamically increasing net capillary permeability, a simple description of functional recruitment, led to predictions consistent with the data. Increasing permeability in all vessel types had the same effect, but two alternative mechanisms were unable to produce predictions consistent with the data. These results are further evidence that conventional models of oxygen transport are not sufficient to predict dynamic experimental data. The data and modeling suggest that it is necessary to include a mechanism that dynamically increases net vascular permeability. While the model cannot distinguish between the different possibilities, we speculate that functional recruitment could have this effect in vivo. PMID:23673433

Steps for the autologous ex vivo perfused porcine liver-kidney experiment.

PubMed

Chung, Wen Yuan; Eltweri, Amar M; Isherwood, John; Haqq, Jonathan; Ong, Seok Ling; Gravante, Gianpiero; Lloyd, David M; Metcalfe, Matthew S; Dennison, Ashley R

2013-12-18

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems.

PubMed

Lee-Montiel, Felipe T; George, Subin M; Gough, Albert H; Sharma, Anup D; Wu, Juanfang; DeBiasio, Richard; Vernetti, Lawrence A; Taylor, D Lansing

2017-10-01

This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we developed and present a method for computationally modeling and measuring oxygen that can easily be implemented in all MPS models. We have applied this method in a liver MPS in which we are then able to control oxygenation in separate devices and demonstrate that zonation-dependent hepatocyte functions in the MPS recapitulate what is known about in vivo liver physiology. We believe that this advance allows a deep experimental investigation on the role of zonation in liver metabolism and disease. In addition, modeling and measuring oxygen tension will be required as investigators migrate from PDMS to plastic and glass devices.

Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems

PubMed Central

Lee-Montiel, Felipe T; George, Subin M; Sharma, Anup D; Wu, Juanfang; DeBiasio, Richard; Vernetti, Lawrence A; Taylor, D Lansing

2017-01-01

This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2′-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we developed and present a method for computationally modeling and measuring oxygen that can easily be implemented in all MPS models. We have applied this method in a liver MPS in which we are then able to control oxygenation in separate devices and demonstrate that zonation-dependent hepatocyte functions in the MPS recapitulate what is known about in vivo liver physiology. We believe that this advance allows a deep experimental investigation on the role of zonation in liver metabolism and disease. In addition, modeling and measuring oxygen tension will be required as investigators migrate from PDMS to plastic and glass devices. PMID:28409533

In vivo Visuotopic Brain Mapping with Manganese-Enhanced MRI and Resting-State Functional Connectivity MRI

PubMed Central

Chan, Kevin C.; Fan, Shu-Juan; Chan, Russell W.; Cheng, Joe S.; Zhou, Iris Y.; Wu, Ed X.

2014-01-01

The rodents are an increasingly important model for understanding the mechanisms of development, plasticity, functional specialization and disease in the visual system. However, limited tools have been available for assessing the structural and functional connectivity of the visual brain network globally, in vivo and longitudinally. There are also ongoing debates on whether functional brain connectivity directly reflects structural brain connectivity. In this study, we explored the feasibility of manganese-enhanced MRI (MEMRI) via 3 different routes of Mn2+ administration for visuotopic brain mapping and understanding of physiological transport in normal and visually deprived adult rats. In addition, resting-state functional connectivity MRI (RSfcMRI) was performed to evaluate the intrinsic functional network and structural-functional relationships in the corresponding anatomical visual brain connections traced by MEMRI. Upon intravitreal, subcortical, and intracortical Mn2+ injection, different topographic and layer-specific Mn enhancement patterns could be revealed in the visual cortex and subcortical visual nuclei along retinal, callosal, cortico-subcortical, transsynaptic and intracortical horizontal connections. Loss of visual input upon monocular enucleation to adult rats appeared to reduce interhemispheric polysynaptic Mn2+ transfer but not intra- or inter-hemispheric monosynaptic Mn2+ transport after Mn2+ injection into visual cortex. In normal adults, both structural and functional connectivity by MEMRI and RSfcMRI was stronger interhemispherically between bilateral primary/secondary visual cortex (V1/V2) transition zones (TZ) than between V1/V2 TZ and other cortical nuclei. Intrahemispherically, structural and functional connectivity was stronger between visual cortex and subcortical visual nuclei than between visual cortex and other subcortical nuclei. The current results demonstrated the sensitivity of MEMRI and RSfcMRI for assessing the neuroarchitecture, neurophysiology and structural-functional relationships of the visual brains in vivo. These may possess great potentials for effective monitoring and understanding of the basic anatomical and functional connections in the visual system during development, plasticity, disease, pharmacological interventions and genetic modifications in future studies. PMID:24394694

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid.

PubMed

Tokunaga, Tomohisa; Watanabe, Bunta; Sato, Sho; Kawamoto, Jun; Kurihara, Tatsuo

2017-08-16

Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring of its in vivo behavior without abolishing its native functions has not yet been developed. Here, we designed and synthesized an ω-ethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function of EPA. eEPA has an ω-ethynyl group tag in place of the ω-methyl group of EPA. An ethynyl group has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover, this group can specifically react in situ with azide compounds, such as those with fluorescent group, via click chemistry. In this study, we first synthesized eEPA efficiently based on the following well-known strategies. To introduce four C-C double bonds, a coupling reaction between terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and stereoselective partial hydrogenation with P-2 nickel, the triple bonds were converted to cis double bonds. One double bond and an ω-terminal C-C triple bond were introduced by Wittig reaction with a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the resulting probe by using an EPA-producing bacterium, Shewanella livingstonensis Ac10. This cold-adapted bacterium inducibly produces EPA at low temperatures, and the EPA-deficient mutant (ΔEPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was exogenously supplemented to ΔEPA, eEPA was incorporated into the membrane phospholipids as an acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation with eEPA, the growth retardation and abnormal morphology of ΔEPA were almost completely suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo behavior of EPA.

Cardiovascular function in term fetal sheep conceived, gestated and studied in the hypobaric hypoxia of the Andean altiplano.

PubMed

Herrera, Emilio A; Rojas, Rodrigo T; Krause, Bernardo J; Ebensperger, Germán; Reyes, Roberto V; Giussani, Dino A; Parer, Julian T; Llanos, Aníbal J

2016-03-01

High-altitude hypoxia causes intrauterine growth restriction and cardiovascular programming. However, adult humans and animals that have evolved at altitude show certain protection against the effects of chronic hypoxia. Whether the highland fetus shows similar protection against high altitude gestation is unclear. We tested the hypothesis that high-altitude fetal sheep have evolved cardiovascular compensatory mechanisms to withstand chronic hypoxia that are different from lowland sheep. We studied seven high-altitude (HA; 3600 m) and eight low-altitude (LA; 520 m) pregnant sheep at ∼90% gestation. Pregnant ewes and fetuses were instrumented for cardiovascular investigation. A three-period experimental protocol was performed in vivo: 30 min of basal, 1 h of acute superimposed hypoxia (∼10% O2) and 30 min of recovery. Further, we determined ex vivo fetal cerebral and femoral arterial function. HA pregnancy led to chronic fetal hypoxia, growth restriction and altered cardiovascular function. During acute superimposed hypoxia, LA fetuses redistributed blood flow favouring the brain, heart and adrenals, whereas HA fetuses showed a blunted cardiovascular response. Importantly, HA fetuses have a marked reduction in umbilical blood flow versus LA. Isolated cerebral arteries from HA fetuses showed a higher contractile capacity but a diminished response to catecholamines. In contrast, femoral arteries from HA fetuses showed decreased contractile capacity and increased adrenergic contractility. The blunting of the cardiovascular responses to hypoxia in fetuses raised in the Alto Andino may indicate a change in control strategy triggered by chronic hypoxia, switching towards compensatory mechanisms that are more cost-effective in terms of oxygen uptake. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Tiny giants of gene regulation: experimental strategies for microRNA functional studies

PubMed Central

Steinkraus, Bruno R.; Toegel, Markus

2016-01-01

The discovery over two decades ago of short regulatory microRNAs (miRNAs) has led to the inception of a vast biomedical research field dedicated to understanding these powerful orchestrators of gene expression. Here we aim to provide a comprehensive overview of the methods and techniques underpinning the experimental pipeline employed for exploratory miRNA studies in animals. Some of the greatest challenges in this field have been uncovering the identity of miRNA–target interactions and deciphering their significance with regard to particular physiological or pathological processes. These endeavors relied almost exclusively on the development of powerful research tools encompassing novel bioinformatics pipelines, high‐throughput target identification platforms, and functional target validation methodologies. Thus, in an unparalleled manner, the biomedical technology revolution unceasingly enhanced and refined our ability to dissect miRNA regulatory networks and understand their roles in vivo in the context of cells and organisms. Recurring motifs of target recognition have led to the creation of a large number of multifactorial bioinformatics analysis platforms, which have proved instrumental in guiding experimental miRNA studies. Subsequently, the need for discovery of miRNA–target binding events in vivo drove the emergence of a slew of high‐throughput multiplex strategies, which now provide a viable prospect for elucidating genome‐wide miRNA–target binding maps in a variety of cell types and tissues. Finally, deciphering the functional relevance of miRNA post‐transcriptional gene silencing under physiological conditions, prompted the evolution of a host of technologies enabling systemic manipulation of miRNA homeostasis as well as high‐precision interference with their direct, endogenous targets. WIREs Dev Biol 2016, 5:311–362. doi: 10.1002/wdev.223 For further resources related to this article, please visit the WIREs website. PMID:26950183

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons.

PubMed

Juárez-Morales, José L; Martinez-De Luna, Reyna I; Zuber, Michael E; Roberts, Alan; Lewis, Katharine E

2017-09-01

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo. We have used an islet1 enhancer to label Rohon-Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis, suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007-1020, 2017. © 2017 Wiley Periodicals, Inc.

Long-term in vivo left ventricular assist device study with a titanium centrifugal pump.

PubMed

Ohtsuka, G; Nakata, K; Yoshikawa, M; Mueller, J; Takano, T; Yamane, S; Gronau, N; Glueck, J; Takami, Y; Sueoka, A; Letsou, G; Schima, H; Schmallegger, H; Wolner, E; Koyanagi, H; Fujisawa, A; Baldwin, J C; Nosé, Y

1998-01-01

A totally implantable centrifugal artificial heart has been developed. The plastic prototype, Gyro PI 601, passed 2 day hemodynamic tests as a functional total artificial heart, 2 week screening tests for antithrombogenicity, and 1 month system feasibility. Based on these results, a metallic prototype, Gyro PI 702, was subjected to in vivo left ventricular assist device (LVAD) studies. The pump system employed the Gyro PI 702, which has the same inner dimensions and the same characteristics as the Gyro PI 601, including an eccentric inlet port, a double pivot bearing system, and a magnet coupling system. The PI 702 is driven with the Vienna DC brushless motor actuator. For the in vivo LVAD study, the pump actuator package was implanted in the preperitoneal space in two calves, from the left ventricular apex to the descending aorta. Case 1 achieved greater than 9 month survival without any complications, at an average flow rate of 6.6 L/min with 10.2 W input power. Case 2 was killed early due to the excessive growth of the calf, which caused functional obstruction of the inlet port. There was no blood clot inside the pump. During these periods, neither case exhibited any physiologic abnormalities. The PI 702 pump gives excellent results as a long-term implantable LVAD.

Single histidine button in cardiac troponin I sustains heart performance in response to severe hypercapnic respiratory acidosis in vivo.

PubMed

Palpant, Nathan J; D'Alecy, Louis G; Metzger, Joseph M

2009-05-01

Intracellular acidosis is a profound negative regulator of myocardial performance. We hypothesized that titrating myofilament calcium sensitivity by a single histidine substituted cardiac troponin I (A164H) would protect the whole animal physiological response to acidosis in vivo. To experimentally induce severe hypercapnic acidosis, mice were exposed to a 40% CO(2) challenge. By echocardiography, it was found that systolic function and ventricular geometry were maintained in cTnI A164H transgenic (Tg) mice. By contrast, non-Tg (Ntg) littermates experienced rapid and marked cardiac decompensation during this same challenge. For detailed hemodymanic assessment, Millar pressure-conductance catheterization was performed while animals were treated with a beta-blocker, esmolol, during a severe hypercapnic acidosis challenge. Survival and load-independent measures of contractility were significantly greater in Tg vs. Ntg mice. This assay showed that Ntg mice had 100% mortality within 5 min of acidosis. By contrast, systolic and diastolic function were protected in Tg mice during acidosis, and they had 100% survival. This study shows that, independent of any beta-adrenergic compensation, myofilament-based molecular manipulation of inotropy by histidine-modified troponin I maintains cardiac inotropic and lusitropic performance and markedly improves survival during severe acidosis in vivo.

Sulforaphane reduces vascular inflammation in mice and prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway.

PubMed

Nallasamy, Palanisamy; Si, Hongwei; Babu, Pon Velayutham Anandh; Pan, Dengke; Fu, Yu; Brooke, Elizabeth A S; Shah, Halley; Zhen, Wei; Zhu, Hong; Liu, Dongmin; Li, Yunbo; Jia, Zhenquan

2014-08-01

Sulforaphane, a naturally occurring isothiocyanate present in cruciferous vegetables, has received wide attention for its potential to improve vascular function in vitro. However, its effect in vivo and the molecular mechanism of sulforaphane at physiological concentrations remain unclear. Here, we report that a sulforaphane concentration as low as 0.5 μM significantly inhibited tumor necrosis factor-α (TNF-α)-induced adhesion of monocytes to human umbilical vein endothelial cells, a key event in the pathogenesis of atherosclerosis both in static and under flow conditions. Such physiological concentrations of sulforaphane also significantly suppressed TNF-α-induced production of monocyte chemotactic protein-1 and adhesion molecules including soluble vascular adhesion molecule-1 and soluble E-selectin, key mediators in the regulation of enhanced endothelial cell-monocyte interaction. Furthermore, sulforaphane inhibited TNF-α-induced nuclear factor (NF)-κB transcriptional activity, Inhibitor of NF-κB alpha (IκBα) degradation and subsequent NF-κB p65 nuclear translocation in endothelial cells, suggesting that sulforaphane can inhibit inflammation by suppressing NF-κB signaling. In an animal study, sulforaphane (300 ppm) in a mouse diet significantly abolished TNF-α-increased ex vivo monocyte adhesion and circulating adhesion molecules and chemokines in C57BL/6 mice. Histology showed that sulforaphane treatment significantly prevented the eruption of endothelial lining in the intima layer of the aorta and preserved elastin fibers' delicate organization, as shown by Verhoeff-van Gieson staining. Immunohistochemistry studies showed that sulforaphane treatment also reduced vascular adhesion molecule-1 and monocyte-derived F4/80-positive macrophages in the aorta of TNF-α-treated mice. In conclusion, sulforaphane at physiological concentrations protects against TNF-α-induced vascular endothelial inflammation, in both in vitro and in vivo models. This anti-inflammatory effect of sulforaphane may be, at least in part, associated with interfering with the NF-κB pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Sulforaphane reduces vascular inflammation in mice and prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway

PubMed Central

Nallasamy, Palanisamy; Si, Hongwei; Babu, Pon Velayutham Anandh; Pan, Dengke; Fu, Yu; Brooke, Elizabeth A.S.; Shah, Halley; Zhen, Wei; Zhu, Hong; Liu, Dongmin; Li, Yunbo; Jia, Zhenquan

2014-01-01

Sulforaphane, a naturally-occurring isothiocyanate present in cruciferous vegetables, has received wide attention for its potential to improve vascular function in vitro. However, its effect in vivo and the molecular mechanism of sulforaphane at physiological concentrations remain unclear. Here, we report that a sulforaphane concentration as low as 0.5 μM significantly inhibited TNF-α-induced adhesion of monocytes to human umbilical vein endothelial cells (HUVECs), a key event in the pathogenesis of atherosclerosis both in static and under flow conditions. Such physiological concentrations of sulforaphane also significantly suppressed TNF-α-induced production of monocyte chemotactic protein-1 (MCP-1), adhesion molecule sVCAM-1 and sE-Selectin, key mediators in the regulation of enhanced endothelial cell-monocyte interaction. Furthermore, sulforaphane inhibited TNF-α-induced NF-κB transcriptional activity, IκBα degradation and subsequent NF-κB p65 nuclear translocation in endothelial cells, suggesting that sulforaphane can inhibit inflammation by suppressing NF-κB signaling. In an animal study, sulforaphane (300 ppm) in a mouse diet significantly abolished TNF-α-increased ex vivo monocyte adhesion and circulating adhesion molecules and chemokines in C57BL/6 mice. Histology showed that sulforaphane treatment significantly prevented the eruption of endothelial lining in the intima layer of the aorta and preserved elastin fibers’ delicate organization as shown by Verhoeff-van Gieson staining. Immunohistochemistry studies showed that sulforaphane treatment also reduced VCAM-1 and monocytes-derived F4/80-positive macrophages in the aorta of TNF-α-treated mice. In conclusion, sulforaphane at physiological concentrations protects against TNF-α-induced vascular endothelial inflammation, in both in vitro and in vivo models. This anti-inflammatory effect of sulforaphane may be, at least in part, associated with interfering with the NF-κB pathway. PMID:24880493

HDAC9 promotes glioblastoma growth via TAZ-mediated EGFR pathway activation.

PubMed

Yang, Rui; Wu, Yanan; Wang, Mei; Sun, Zhongfeng; Zou, Jiahua; Zhang, Yundong; Cui, Hongjuan

2015-04-10

Histone deacetylase 9 (HDAC9), a member of class II HDACs, regulates a wide variety of normal and abnormal physiological functions. We found that HDAC9 is over-expressed in prognostically poor glioblastoma patients. Knockdown HDAC9 decreased proliferation in vitro and tumor formation in vivo. HDAC9 accelerated cell cycle in part by potentiating the EGFR signaling pathway. Also, HDAC9 interacted with TAZ, a key downstream effector of Hippo pathway. Knockdown of HDAC9 decreased the expression of TAZ. We found that overexpressed TAZ in HDAC9-knockdown cells abrogated the effects induced by HDAC9 silencing both in vitro and in vivo. We demonstrated that HDAC9 promotes tumor formation of glioblastoma via TAZ-mediated EGFR pathway activation, and provide the evidence for promising target for the treatment of glioblastoma.

In vivo functional photoacoustic tomography of traumatic brain injury in rats

NASA Astrophysics Data System (ADS)

Oh, Jung-Taek; Song, Kwang-Hyung; Li, Meng-Lin; Stoica, George; Wang, Lihong V.

2006-02-01

In this study, we demonstrate the potential of photoacoustic tomography for the study of traumatic brain injury (TBI) in rats in vivo. Based on spectroscopic photoacoustic tomography that can detect the absorption rates of oxy- and deoxy-hemoglobins, the blood oxygen saturation and total blood volume in TBI rat brains were visualized. Reproducible cerebral trauma was induced using a fluid percussion TBI device. The time courses of the hemodynamic response following the trauma initiation were imaged with multi-wavelength photoacoustic tomography with bandwidth-limited spatial resolution through the intact skin and skull. In the pilot set of experiments, trauma induced hematomas and blood oxygen saturation level changes were detected, a finding consistent with the known physiological responses to TBI. This new imaging method will be useful for future studies on TBI-related metabolic activities and the effects of therapeutic agents.

Monte Carlo modeling of fluorescence in semi-infinite turbid media

NASA Astrophysics Data System (ADS)

Ong, Yi Hong; Finlay, Jarod C.; Zhu, Timothy C.

2018-02-01

The incident field size and the interplay of absorption and scattering can influence the in-vivo light fluence rate distribution and complicate the absolute quantification of fluorophore concentration in-vivo. In this study, we use Monte Carlo simulations to evaluate the effect of incident beam radius and optical properties to the fluorescence signal collected by isotropic detector placed on the tissue surface. The optical properties at the excitation and emission wavelengths are assumed to be identical. We compute correction factors to correct the fluorescence intensity for variations due to incident field size and optical properties. The correction factors are fitted to a 4-parameters empirical correction function and the changes in each parameter are compared for various beam radius over a range of physiologically relevant tissue optical properties (μa = 0.1 - 1 cm-1 , μs'= 5 - 40 cm-1 ).

Rational chemical design of the next generation of molecular imaging probes based on physics and biology: mixing modalities, colors and signals

PubMed Central

Longmire, Michelle R.; Ogawa, Mikako; Choyke, Peter L.

2012-01-01

In recent years, numerous in vivo molecular imaging probes have been developed. As a consequence, much has been published on the design and synthesis of molecular imaging probes focusing on each modality, each type of material, or each target disease. More recently, second generation molecular imaging probes with unique, multi-functional, or multiplexed characteristics have been designed. This critical review focuses on (i) molecular imaging using combinations of modalities and signals that employ the full range of the electromagnetic spectra, (ii) optimized chemical design of molecular imaging probes for in vivo kinetics based on biology and physiology across a range of physical sizes, (iii) practical examples of second generation molecular imaging probes designed to extract complementary data from targets using multiple modalities, color, and comprehensive signals (277 references). PMID:21607237

Real-time high dynamic range laser scanning microscopy

PubMed Central

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-01-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging. PMID:27032979

Organomatics and organometrics: Novel platforms for long-term whole-organ culture

PubMed Central

Bruinsma, Bote G.; Yarmush, Martin L.; Uygun, Korkut

2014-01-01

Organ culture systems are instrumental as experimental whole-organ models of physiology and disease, as well as preservation modalities facilitating organ replacement therapies such as transplantation. Nevertheless, a coordinated system of machine perfusion components and integrated regulatory control has yet to be fully developed to achieve long-term maintenance of organ function ex vivo. Here we outline current strategies for organ culture, or organomatics, and how these systems can be regulated by means of computational algorithms, or organometrics, to achieve the organ culture platforms anticipated in modern-day biomedicine. PMID:25035864

What Are the bona fide GSK3 Substrates?

PubMed

Sutherland, Calum

2011-01-01

Nearly 100 proteins are proposed to be substrates for GSK3, suggesting that this enzyme is a fundamental regulator of almost every process in the cell, in every tissue in the body. However, it is not certain how many of these proposed substrates are regulated by GSK3 in vivo. Clearly, the identification of the physiological functions of GSK3 will be greatly aided by the identification of its bona fide substrates, and the development of GSK3 as a therapeutic target will be highly influenced by this range of actions, hence the need to accurately establish true GSK3 substrates in cells. In this paper the evidence that proposed GSK3 substrates are likely to be physiological targets is assessed, highlighting the key cellular processes that could be modulated by GSK3 activity and inhibition.

Characterizing the Interaction between tartrazine and two serum albumins by a hybrid spectroscopic approach.

PubMed

Pan, Xingren; Qin, Pengfei; Liu, Rutao; Wang, Jing

2011-06-22

Tartrazine is an artificial azo dye commonly used in food products. The present study evaluated the interaction of tartrazine with two serum albumins (SAs), human serum albumin (HSA) and bovine serum albumin (BSA), under physiological conditions by means of fluorescence, three-dimensional fluorescence, UV-vis absorption, and circular dichroism (CD) techniques. The fluorescence data showed that tartrazine could bind to the two SAs to form a complex. The binding process was a spontaneous molecular interaction procedure, in which van der Waals and hydrogen bond interactions played a major role. Additionally, as shown by the UV-vis absorption, three-dimensional fluorescence, and CD results, tartrazine could lead to conformational and some microenvironmental changes of both SAs, which may affect the physiological functions of SAs. The work provides important insight into the mechanism of toxicity of tartrazine in vivo.

In vivo sodium concentration continuously monitored with fluorescent sensors.

PubMed

Dubach, J Matthew; Lim, Edward; Zhang, Ning; Francis, Kevin P; Clark, Heather

2011-02-01

Sodium balance is vital to maintaining normal physiological function. Imbalances can occur in a variety of diseases, during certain surgical operations or during rigorous exercise. There is currently no method to continuously monitor sodium concentration in patients who may be susceptible to hyponatremia. Our approach was to design sodium specific fluorescent sensors capable of measuring physiological fluctuations in sodium concentration. The sensors are submicron plasticized polymer particles containing sodium recognition components that are coated with biocompatible poly(ethylene) glycol. Here, the sensors were brought up in saline and placed in the subcutaneous area of the skin of mice by simple injection. The fluorescence was monitored in real time using a whole animal imager to track changes in sodium concentrations. This technology could be used to monitor certain disease states or warn against dangerously low levels of sodium during exercise.

Rodent models of insomnia: a review of experimental procedures that induce sleep disturbances.

PubMed

Revel, Florent G; Gottowik, Juergen; Gatti, Sylvia; Wettstein, Joseph G; Moreau, Jean-Luc

2009-06-01

Insomnia, the most common sleep disorder, is characterized by persistent difficulty in falling or staying asleep despite adequate opportunity to sleep, leading to daytime fatigue and mental dysfunction. As sleep is a sophisticated physiological process generated by a network of neuronal systems that cannot be reproduced in-vitro, pre-clinical development of hypnotic drugs requires in-vivo investigations. Accordingly, this review critically evaluates current and putative rodent models of insomnia which could be used to screen novel hypnotics. Only few valid insomnia models are currently available, although many experimental conditions lead to disturbance of physiological sleep. We categorized these conditions as a function of the procedure used to induce perturbation of sleep, and we discuss their respective advantages and pitfalls with respect to validity, feasibility and translational value to human research.

Fibrous scaffolds fabricated by emulsion electrospinning: from hosting capacity to in vivo biocompatibility

NASA Astrophysics Data System (ADS)

Spano, F.; Quarta, A.; Martelli, C.; Ottobrini, L.; Rossi, R. M.; Gigli, G.; Blasi, L.

2016-04-01

Electrospinning is a versatile method for preparing functional three-dimensional scaffolds. Synthetic and natural polymers have been used to produce micro- and nanofibers that mimic extracellular matrices. Here, we describe the use of emulsion electrospinning to prepare blended fibers capable of hosting aqueous species and releasing them in solution. The existence of an aqueous and a non-aqueous phase allows water-soluble molecules to be introduced without altering the structure and the degradation of the fibers, and means that their release properties under physiological conditions can be controlled. To demonstrate the loading capability and flexibility of the blend, various species were introduced, from magnetic nanoparticles and quantum rods to biological molecules. Cellular studies showed the spontaneous adhesion and alignment of cells along the fibers. Finally, in vivo experiments demonstrated the high biocompatibility and safety of the scaffolds up to 21 days post-implantation.Electrospinning is a versatile method for preparing functional three-dimensional scaffolds. Synthetic and natural polymers have been used to produce micro- and nanofibers that mimic extracellular matrices. Here, we describe the use of emulsion electrospinning to prepare blended fibers capable of hosting aqueous species and releasing them in solution. The existence of an aqueous and a non-aqueous phase allows water-soluble molecules to be introduced without altering the structure and the degradation of the fibers, and means that their release properties under physiological conditions can be controlled. To demonstrate the loading capability and flexibility of the blend, various species were introduced, from magnetic nanoparticles and quantum rods to biological molecules. Cellular studies showed the spontaneous adhesion and alignment of cells along the fibers. Finally, in vivo experiments demonstrated the high biocompatibility and safety of the scaffolds up to 21 days post-implantation. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00782a

In vitro immune functions in thiamine-replete and -depleted lake trout (Salvelinus namaycush).

PubMed

Ottinger, Christopher A; Honeyfield, Dale C; Densmore, Christine L; Iwanowicz, Luke R

2014-05-01

In this study we examined the impacts of in vivo thiamine deficiency on lake trout leukocyte function measured in vitro. When compared outside the context of individual-specific thiamine concentrations no significant differences were observed in leukocyte bactericidal activity or in concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) stimulated leukocyte proliferation. Placing immune functions into context with the ratio of in vivo liver thiamine monophosphate (TMP--biologically inactive form) to thiamine pyrophosphate (TPP--biologically active form) proved to be the best indicator of thiamine depletion impacts as determined using regression modeling. These observed relationships indicated differential effects on the immune measures with bactericidal activity exhibiting an inverse relationship with TMP to TPP ratios, Con A stimulated mitogenesis exhibiting a positive relationship with TMP to TPP ratios and PHA-P stimulated mitogenesis exhibiting no significant relationships. In addition, these relationships showed considerable complexity which included the consistent observation of a thiamine-replete subgroup with characteristics similar to those seen in the leukocytes from thiamine-depleted fish. When considered together, our observations indicate that lake trout leukocytes experience cell-type specific impacts as well as an altered physiologic environment when confronted with a thiamine-limited state. Published by Elsevier Ltd.

In vitro immune functions in thiamine-replete and -depleted lake trout (Salvelinus namaycush)

USGS Publications Warehouse

Ottinger, Christopher A.; Honeyfield, Dale C.; Densmore, Christine L.; Iwanowicz, Luke R.

2014-01-01

In this study we examined the impacts of in vivo thiamine deficiency on lake trout leukocyte function measured in vitro. When compared outside the context of individual-specific thiamine concentrations no significant differences were observed in leukocyte bactericidal activity or in concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) stimulated leukocyte proliferation. Placing immune functions into context with the ratio of in vivo liver thiamine monophosphate (TMP – biologically inactive form) to thiamine pyrophosphate (TPP – biologically active form) proved to be the best indicator of thiamine depletion impacts as determined using regression modeling. These observed relationships indicated differential effects on the immune measures with bactericidal activity exhibiting an inverse relationship with TMP to TPP ratios, Con A stimulated mitogenesis exhibiting a positive relationship with TMP to TPP ratios and PHA-P stimulated mitogenesis exhibiting no significant relationships. In addition, these relationships showed considerable complexity which included the consistent observation of a thiamine-replete subgroup with characteristics similar to those seen in the leukocytes from thiamine-depleted fish. When considered together, our observations indicate that lake trout leukocytes experience cell-type specific impacts as well as an altered physiologic environment when confronted with a thiamine-limited state.

Hyperoxia reduces insulin release and induces mitochondrial dysfunction with possible implications for hyperoxic treatment of neonates.

PubMed

Hals, Ingrid; Ohki, Tsuyoshi; Singh, Rinku; Ma, Zuheng; Björklund, Anneli; Balasuriya, Chandima; Scholz, Hanne; Grill, Valdemar

2017-10-01

We previously showed that hyperoxia in vitro negatively affects beta cells of the rat. Here, we tested for possible clinical significance as well as mitochondrial interactions by hyperoxia, using human islets (function and viability), INS-1 832/13 cells (mitochondrial metabolism), and mouse neonates (effects in vivo). Lastly, we assessed relevant parameters in a cohort of individuals born preterm and then exposed to hyperoxia. Human islets and INS-1 832/13 cells were exposed to 24 h of hyperoxia (90-92% oxygen). Mouse neonates were subjected to 5 days of continuous hyperoxia. Individuals born preterm were evaluated in terms of glucose homeostasis and beta cell function by HbA1c and the HOMA2 formula. In human islets, hyperoxia significantly reduced glucose-stimulated insulin secretion by 42.2 ± 5.3% and viability assessed by MTT by 22.5 ± 5.4%. Hyperoxia down-regulated mitochondrial complex II by 21 ± 5% and upregulated complex III by 26 ± 10.1% and complex IV by 37 ± 10.6%. Partly similar effects on mitochondrial complexes were found in hyperoxia-exposed INS-1 832/13 cells. Exposure to hyperoxia swiftly reduced oxygen consumption in these cells and increased mitochondrial uncoupling. Hyperoxia transiently but significantly reduced insulin release in mouse neonates. Individuals born preterm displayed higher HbA1c versus controls, as well as insulin resistance. Thus, hyperoxia exerts negative effects in vitro on human beta cells and results indicate inhibitory effects on insulin secretion in vivo in mouse neonates. Negative effects may be lessened by the demonstrated swift and profound mitochondrial adaptability. Our findings open the possibility that hyperoxia could negatively affect beta cells of preterm human neonates. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Dynamics of the sensory response to urethral flow over multiple time scales in rat

PubMed Central

Danziger, Zachary C; Grill, Warren M

2015-01-01

The pudendal nerve carries sensory information from the urethra that controls spinal reflexes necessary to maintain continence and achieve efficient micturition. Despite the key role urethral sensory feedback plays in regulation of the lower urinary tract, there is little information about the characteristics of urethral sensory responses to physiological stimuli, and the quantitative relationship between physiological stimuli and the evoked sensory activation is unknown. Such a relation is critical to understanding the neural control of the lower urinary tract and how dysfunction arises in disease states. We systematically quantified pudendal afferent responses to fluid flow in the urethra in vivo in the rat. We characterized the sensory response across a range of stimuli, and describe a previously unreported long-term neural accommodation phenomenon. We developed and validated a compact mechanistic mathematical model capable of reproducing the pudendal sensory activity in response to arbitrary profiles of urethral flows. These results describe the properties and function of urethral afferents that are necessary to understand how sensory disruption manifests in lower urinary tract pathophysiology. Key points Sensory information from the urethra is essential to maintain continence and to achieve efficient micturition and when compromised by disease or injury can lead to substantial loss of function. Despite the key role urethral sensory information plays in the lower urinary tract, the relationship between physiological urethral stimuli, such as fluid flow, and the neural sensory response is poorly understood. This work systematically quantifies pudendal afferent responses to a range of fluid flows in the urethra in vivo and describes a previously unknown long-term neural accommodation phenomenon in these afferents. We present a compact mechanistic mathematical model that reproduces the pudendal sensory activity in response to urethral flow. These results have implications for understanding urinary tract dysfunction caused by neuropathy or nerve damage, such as urinary retention or incontinence, as well as for the development of strategies to mitigate the symptoms of these conditions. PMID:26041695

Modeling Fetal Alcohol Spectrum Disorder: validating an ex vivo primary hippocampal cell culture system

PubMed Central

Tunc-Ozcan, Elif; Ferreira, Adriana B.; Redei, Eva E.

2016-01-01

Background Fetal Alcohol Spectrum Disorder (FASD) is the leading non-genetic cause of mental retardation. There are no treatments for FASD to date. Preclinical in vivo and in vitro studies could help in identifying novel drug targets as for other diseases. Here, we describe an ex vivo model that combines the physiological advantages of prenatal ethanol (E) exposure in vivo with the uniformity of primary fetal hippocampal culture to characterize the effects of prenatal E. The insulin signaling pathways are known to be involved in hippocampal functions. Therefore, we compared the expression of insulin signaling pathway genes between fetal hippocampi (in vivo) and primary hippocampal culture (ex vivo). The similarity of prenatal E effects in these two paradigms would deem the ex vivo culture acceptable to screen possible treatments for FASD. Methods Pregnant Sprague-Dawley rats received one of three diets: ad libitum standard lab chow (control-C), isocaloric pair-fed (PF, nutritional control), and E containing liquid diets from gestational day (GD) 8. Fetal male and female hippocampi were collected either on GD21 (in vivo) or on GD18 for primary culture (ex vivo). Transcript levels of Igf2, Igf2r, Insr, Grb10, Rasgrf1 and Zac1 were measured by RT-qPCR. Results Hippocampal transcript levels differed by prenatal treatment in both males and females with sex differences observed in the expression of Igf2 and Insr. The effect of prenatal E on the hippocampal expression of the insulin pathway genes was parallel in the in vivo and the ex vivo conditions. Conclusions The similarity of gene expression changes in response to prenatal E between the in vivo and the ex vivo conditions ascertain that these effects are already set in the fetal hippocampus at GD18. This strengthens the feasibility of the ex vivo primary hippocampal culture as a tool to test and screen candidate drug targets for FASD. PMID:27162054

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma

PubMed Central

Miller, Tyler E.; Liau, Brian B.; Wallace, Lisa C.; Morton, Andrew R.; Xie, Qi; Dixit, Deobrat; Factor, Daniel C.; Kim, Leo J. Y.; Morrow, James J.; Wu, Qiulian; Mack, Stephen C.; Hubert, Christopher G.; Gillespie, Shawn M.; Flavahan, William A.; Hoffmann, Thomas; Thummalapalli, Rohit; Hemann, Michael T.; Paddison, Patrick J.; Horbinski, Craig M.; Zuber, Johannes; Scacheri, Peter C.; Bernstein, Bradley E.; Tesar, Paul J.; Rich, Jeremy N.

2017-01-01

Glioblastoma is a universally lethal cancer with a median survival of approximately 15 months1. Despite substantial efforts to define druggable targets, there are no therapeutic options that meaningfully extend glioblastoma patient lifespan. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology2–4 for use in orthotopic patient-derived xenograft (PDX) models, creating a high-throughput negative selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators necessary for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, supporting targeting the transcription elongation machinery as a therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, which could supply untapped opportunities for therapeutic intervention. PMID:28678782

Human Metabolome-derived Cofactors Are Required for the Antibacterial Activity of Siderocalin in Urine*

PubMed Central

Shields-Cutler, Robin R.; Crowley, Jan R.; Miller, Connelly D.; Stapleton, Ann E.; Cui, Weidong; Henderson, Jeffrey P.

2016-01-01

In human urinary tract infections, host cells release the antimicrobial protein siderocalin (SCN; also known as lipocalin-2, neutrophil gelatinase-associated lipocalin, or 24p3) into the urinary tract. By binding to ferric catechol complexes, SCN can sequester iron, a growth-limiting nutrient for most bacterial pathogens. Recent evidence links the antibacterial activity of SCN in human urine to iron sequestration and metabolomic variation between individuals. To determine whether these metabolomic associations correspond to functional Fe(III)-binding SCN ligands, we devised a biophysical protein binding screen to identify SCN ligands through direct analysis of human urine. This screen revealed a series of physiologic unconjugated urinary catechols that were able to function as SCN ligands of which pyrogallol in particular was positively associated with high urinary SCN activity. In a purified, defined culture system, these physiologic SCN ligands were sufficient to activate SCN antibacterial activity against Escherichia coli. In the presence of multiple SCN ligands, native mass spectrometry demonstrated that SCN may preferentially combine different ligands to coordinate iron, suggesting that availability of specific ligand combinations affects in vivo SCN antibacterial activity. These results support a mechanistic link between the human urinary metabolome and innate immune function. PMID:27780864

Analyses of a Mutant Foxp3 Allele Reveal BATF as a Critical Transcription Factor in the Differentiation and Accumulation of Tissue Regulatory T Cells.

PubMed

Hayatsu, Norihito; Miyao, Takahisa; Tachibana, Masashi; Murakami, Ryuichi; Kimura, Akihiko; Kato, Takako; Kawakami, Eiryo; Endo, Takaho A; Setoguchi, Ruka; Watarai, Hiroshi; Nishikawa, Takeshi; Yasuda, Takuwa; Yoshida, Hisahiro; Hori, Shohei

2017-08-15

Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate. Copyright © 2017 Elsevier Inc. All rights reserved.

Developing a Physiologically-Based Pharmacokinetic Model Knowledgebase in Support of Provisional Model Construction

EPA Science Inventory

Developing physiologically-based pharmacokinetic (PBPK) models for chemicals can be resource-intensive, as neither chemical-specific parameters nor in vivo pharmacokinetic data are easily available for model construction. Previously developed, well-parameterized, and thoroughly-v...

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas-permeable membranes.

PubMed

Xiao, Wenjin; Shinohara, Marie; Komori, Kikuo; Sakai, Yasuyuki; Matsui, Hitoshi; Osada, Tomoharu

2014-01-01

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%-O2 (+)] or physiological oxygen concentrations [10%-O2 (+), 5%-O2 (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas-impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS-O2 (-)]. The results indicated that the hepatocytes under 10%-O2 (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS-O2 (-) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug-metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long-term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen-permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers.

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

PubMed Central

Strulson, Christopher A.; Boyer, Joshua A.; Whitman, Elisabeth E.; Bevilacqua, Philip C.

2014-01-01

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg2+ ion concentrations are low, K+ concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo–like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg2+ (0.5–2 mM) and K+ (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution. PMID:24442612

Alternative functional in vitro models of human intestinal epithelia

PubMed Central

Kauffman, Amanda L.; Gyurdieva, Alexandra V.; Mabus, John R.; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J.

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport. PMID:23847534

Alternative functional in vitro models of human intestinal epithelia.

PubMed

Kauffman, Amanda L; Gyurdieva, Alexandra V; Mabus, John R; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

Functional characterization of Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis

PubMed Central

Zhang, Jinfang; Wan, Lixin; Dai, Xiangpeng; Sun, Yi; Wei, Wenyi

2014-01-01

The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that primarily governs cell cycle progression. APC/C is composed of at least 14 core subunits and recruits its substrates for ubiquitination via one of the two adaptor proteins, Cdc20 or Cdh1, in M or M/early G1 phase, respectively. Furthermore, recent studies have shed light on crucial functions for APC/C in maintaining genomic integrity, neuronal differentiation, cellular metabolism and tumorigenesis. To gain better insight into the in vivo physiological functions of APC/C in regulating various cellular processes, particularly development and tumorigenesis, a number of mouse models of APC/C core subunits, coactivators or inhibitors have been established and characterized. However, due to their essential role in cell cycle regulation, most of the germline knockout mice targeting the APC/C pathway are embryonic lethal, indicating the need for generating conditional knockout mouse models to assess the role in tumorigenesis for each APC/C signaling component in specific tissues. In this review, we will first provide a brief introduction of the ubiquitin-proteasome system (UPS) and the biochemical activities and cellular functions of the APC/C E3 ligase. We will then focus primarily on characterizing genetic mouse models used to understand the physiological roles of each APC/C signaling component in embryogenesis, cell proliferation, development and carcinogenesis. Finally, we discuss future research directions to further elucidate the physiological contributions of APC/C components during tumorigenesis and validate their potentials as a novel class of anti-cancer targets. PMID:24569229

In vivo neuronal calcium imaging in C. elegans.

PubMed

Chung, Samuel H; Sun, Lin; Gabel, Christopher V

2013-04-10

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon and GCaMP allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

The importance of hypoxia and extra physiologic oxygen shock/stress for collection and processing of stem and progenitor cells to understand true physiology/pathology of these cells ex vivo.

PubMed

Broxmeyer, Hal E; O'Leary, Heather A; Huang, Xinxin; Mantel, Charlie

2015-07-01

Hematopoietic stem (HSCs) and progenitor (HPCs) cells reside in a hypoxic (lowered oxygen tension) environment, in vivo. We review literature on growth of HSCs and HPCs under hypoxic and normoxic (ambient air) conditions with a focus on our recent work demonstrating the detrimental effects of collecting and processing cells in ambient air through a phenomenon termed extra physiologic oxygen shock/stress (EPHOSS), and we describe means to counteract EPHOSS for enhanced collection of HSCs. Collection and processing of bone marrow and cord blood cells in ambient air cause rapid differentiation and loss of HSCs, with increases in HPCs. This apparently irreversible EPHOSS phenomenon results from increased mitochondrial reactive oxygen species, mediated by a p53-cyclophilin D-mitochondrial permeability transition pore axis, and involves hypoxia inducing factor-1α and micro-RNA 210. EPHOSS can be mitigated by collecting and processing cells in lowered (3%) oxygen, or in ambient air in the presence of, cyclosporine A which effects the mitochondrial permeability transition pore, resulting in increased HSC collections. Our recent findings may be advantageous for HSC collection for hematopoietic cell transplantation, and likely for enhanced collection of other stem cell types. EPHOSS should be considered when ex-vivo cell analysis is utilized for personalized medicine, as metabolism of cells and their response to targeted drug treatment ex vivo may not mimic what occurs in vivo.

Obesity-induced oxidative stress, accelerated functional decline with age and increased mortality in mice.

PubMed

Zhang, Yiqiang; Fischer, Kathleen E; Soto, Vanessa; Liu, Yuhong; Sosnowska, Danuta; Richardson, Arlan; Salmon, Adam B

2015-06-15

Obesity is a serious chronic disease that increases the risk of numerous co-morbidities including metabolic syndrome, cardiovascular disease and cancer as well as increases risk of mortality, leading some to suggest this condition represents accelerated aging. Obesity is associated with significant increases in oxidative stress in vivo and, despite the well-explored relationship between oxidative stress and aging, the role this plays in the increased mortality of obese subjects remains an unanswered question. Here, we addressed this by undertaking a comprehensive, longitudinal study of a group of high fat-fed obese mice and assessed both their changes in oxidative stress and in their performance in physiological assays known to decline with aging. In female C57BL/6J mice fed a high-fat diet starting in adulthood, mortality was significantly increased as was oxidative damage in vivo. High fat-feeding significantly accelerated the decline in performance in several assays, including activity, gait, and rotarod. However, we also found that obesity had little effect on other markers of function and actually improved performance in grip strength, a marker of muscular function. Together, this first comprehensive assessment of longitudinal, functional changes in high fat-fed mice suggests that obesity may induce segmental acceleration of some of the aging process. Published by Elsevier Inc.

Vinpocetine modulates metabolic activity and function during retinal ischemia.

PubMed

Nivison-Smith, Lisa; O'Brien, Brendan J; Truong, Mai; Guo, Cindy X; Kalloniatis, Michael; Acosta, Monica L

2015-05-01

Vinpocetine protects against a range of degenerative conditions and insults of the central nervous system via multiple modes of action. Little is known, however, of its effects on metabolism. This may be highly relevant, as vinpocetine is highly protective against ischemia, a process that inhibits normal metabolic function. This study uses the ischemic retina as a model to characterize vinpocetine's effects on metabolism. Vinpocetine reduced the metabolic demand of the retina following ex vivo hypoxia and ischemia to normal levels based on lactate dehydrogenase activity. Vinpocetine delivered similar effects in an in vivo model of retinal ischemia-reperfusion, possibly through increasing glucose availability. Vinpocetine's effects on glucose also appeared to improve glutamate homeostasis in ischemic Müller cells. Other actions of vinpocetine following ischemia-reperfusion, such as reduced cell death and improved retinal function, were possibly a combination of the drug's actions on metabolism and other retinal pathways. Vinpocetine's metabolic effects appeared independent of its other known actions in ischemia, as it recovered retinal function in a separate metabolic model where the glutamate-to-glutamine metabolic pathway was inhibited in Müller cells. The results of this study indicate that vinpocetine mediates ischemic damage partly through altered metabolism and has potential beneficial effects as a treatment for ischemia of neuronal tissues. Copyright © 2015 the American Physiological Society.

Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment.

PubMed

Pillai, Viju Vijayan; Weber, Darren M; Phinney, Brett S; Selvaraj, Vimal

2017-01-01

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.

Comparative In Vivo Effects of Hemoglobin-Based Oxygen Carriers (HBOC) with Varying Prooxidant and Physiological Reactivity.

PubMed

Toma, Vlad Al; Farcaș, Anca D; Roman, Ioana; Sevastre, Bogdan; Hathazi, Denisa; Scurtu, Florina; Damian, Grigore; Silaghi-Dumitrescu, Radu

2016-01-01

A series of hemoglobin-based oxygen carrier candidates (HBOC), previously noted for their differences in prooxidative and physiological reactivity, were compared in terms of the negative effects displayed upon injection in Wistar rats. At the concentrations tested, antioxidant strategies based on albumin as well as based on rubrerythrin appear to offer observable physiological advantages.

Phosphinothricin Acetyltransferases Identified Using In Vivo, In Vitro, and Bioinformatic Analyses

PubMed Central

VanDrisse, Chelsey M.; Hentchel, Kristy L.

2016-01-01

ABSTRACT Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results of in vitro studies were consistent with the in vivo results. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases in Burkholderia xenovorans, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli. IMPORTANCE The work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role of Gcn5 N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination of in vivo, in vitro, and bioinformatics approaches reported here identified GNATs that can acetylate and detoxify phosphinothricin. PMID:27694229

The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo.

PubMed

Monet, Marie; Domenga, Valérie; Lemaire, Barbara; Souilhol, Céline; Langa, Francina; Babinet, Charles; Gridley, Thomas; Tournier-Lasserve, Elisabeth; Cohen-Tannoudji, Michel; Joutel, Anne

2007-04-15

Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy.

The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory.

PubMed

Cicvaric, Ana; Yang, Jiaye; Krieger, Sigurd; Khan, Deeba; Kim, Eun-Jung; Dominguez-Rodriguez, Manuel; Cabatic, Maureen; Molz, Barbara; Acevedo Aguilar, Juan Pablo; Milicevic, Radoslav; Smani, Tarik; Breuss, Johannes M; Kerjaschki, Dontscho; Pollak, Daniela D; Uhrin, Pavel; Monje, Francisco J

2016-12-01

Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized. Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin. Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF. This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology. Key messages Podoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions. Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation. Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well as a role for podoplanin in plasticity-related brain neuronal functions is here proposed.

In-depth physiological characterization of primary human hepatocytes in a 3D hollow-fiber bioreactor.

PubMed

Mueller, Daniel; Tascher, Georg; Müller-Vieira, Ursula; Knobeloch, Daniel; Nuessler, Andreas K; Zeilinger, Katrin; Heinzle, Elmar; Noor, Fozia

2011-08-01

As the major research focus is shifting to three-dimensional (3D) cultivation techniques, hollow-fiber bioreactors, allowing the formation of tissue-like structures, show immense potential as they permit controlled in vitro cultivation while supporting the in vivo environment. In this study we carried out a systematic and detailed physiological characterization of human liver cells in a 3D hollow-fiber bioreactor system continuously run for > 2 weeks. Primary human hepatocytes were maintained viable and functional over the whole period of cultivation. Both general cellular functions, e.g. oxygen uptake, amino acid metabolism and substrate consumption, and liver-specific functions, such as drug-metabolizing capacities and the production of liver-specific metabolites were found to be stable for > 2 weeks. As expected, donor-to-donor variability was observed in liver-specific functions, namely urea and albumin production. Moreover, we show the maintenance of primary human hepatocytes in serum-free conditions in this set-up. The stable basal cytochrome P450 activity 3 weeks after isolation of the cells demonstrates the potential of such a system for pharmacological applications. Liver cells in the presented 3D bioreactor system could eventually be used not only for long-term metabolic and toxicity studies but also for chronic repeated dose toxicity assessment. Copyright © 2011 John Wiley & Sons, Ltd.

Engineering of functional, perfusable 3D microvascular networks on a chip.

PubMed

Kim, Sudong; Lee, Hyunjae; Chung, Minhwan; Jeon, Noo Li

2013-04-21

Generating perfusable 3D microvessels in vitro is an important goal for tissue engineering, as well as for reliable modelling of blood vessel function. To date, in vitro blood vessel models have not been able to accurately reproduce the dynamics and responses of endothelial cells to grow perfusable and functional 3D vascular networks. Here we describe a microfluidic-based platform whereby we model natural cellular programs found during normal development and angiogenesis to form perfusable networks of intact 3D microvessels as well as tumor vasculatures based on the spatially controlled co-culture of endothelial cells with stromal fibroblasts, pericytes or cancer cells. The microvessels possess the characteristic morphological and biochemical markers of in vivo blood vessels, and exhibit strong barrier function and long-term stability. An open, unobstructed microvasculature allows the delivery of nutrients, chemical compounds, biomolecules and cell suspensions, as well as flow-induced mechanical stimuli into the luminal space of the endothelium, and exhibits faithful responses to physiological shear stress as demonstrated by cytoskeleton rearrangement and increased nitric oxide synthesis. This simple and versatile platform provides a wide range of applications in vascular physiology studies as well as in developing vascularized organ-on-a-chip and human disease models for pharmaceutical screening.

Physiological and Molecular Effects of in vivo and ex vivo Mild Skin Barrier Disruption.

PubMed

Pfannes, Eva K B; Weiss, Lina; Hadam, Sabrina; Gonnet, Jessica; Combardière, Béhazine; Blume-Peytavi, Ulrike; Vogt, Annika

2018-01-01

The success of topically applied treatments on skin relies on the efficacy of skin penetration. In order to increase particle or product penetration, mild skin barrier disruption methods can be used. We previously described cyanoacrylate skin surface stripping as an efficient method to open hair follicles, enhance particle penetration, and activate Langerhans cells. We conducted ex vivo and in vivo measurements on human skin to characterize the biological effect and quantify barrier disruption-related inflammation on a molecular level. Despite the known immunostimulatory effects, this barrier disruption and hair follicle opening method was well accepted and did not result in lasting changes of skin physiological parameters, cytokine production, or clinical side effects. Only in ex vivo human skin did we find a discrete increase in IP-10, TGF-β, IL-8, and GM-CSF mRNA. The data underline the safety profile of this method and demonstrate that the procedure per se does not cause substantial inflammation or skin damage, which is also of interest when applied to non-invasive sampling of biomarkers in clinical trials. © 2018 S. Karger AG, Basel.

In vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs.

PubMed

Ueki, Hiroshi; Wang, I-Hsuan; Fukuyama, Satoshi; Katsura, Hiroaki; da Silva Lopes, Tiago Jose; Neumann, Gabriele; Kawaoka, Yoshihiro

2018-06-25

The pathophysiological changes that occur in lungs infected with influenza viruses are poorly understood. Here we established an in vivo imaging system that combines two-photon excitation microscopy and fluorescent influenza viruses of different pathogenicity. This approach allowed us to monitor and correlate several parameters and physiological changes including the spread of infection, pulmonary permeability, pulmonary perfusion speed, number of recruited neutrophils in infected lungs, and neutrophil motion in the lungs of live mice. Several physiological changes were larger and occurred earlier in mice infected with a highly pathogenic H5N1 influenza virus compared with those infected with a mouse-adapted human strain. These findings demonstrate the potential of our in vivo imaging system to provide novel information about the pathophysiological consequences of virus infections.

Microencapsulated fluorescent pH probe as implantable sensor for monitoring the physiological state of fish embryos.

PubMed

Gurkov, Anton; Sadovoy, Anton; Shchapova, Ekaterina; Teh, Cathleen; Meglinski, Igor; Timofeyev, Maxim

2017-01-01

In vivo physiological measurement is a major challenge in modern science and technology, as is environment conservation at the global scale. Proper toxicological testing of widely produced mixtures of chemicals is a necessary step in the development of new products, allowing us to minimize the human impact on aquatic ecosystems. However, currently available bioassay-based techniques utilizing small aquatic organisms such as fish embryos for toxicity testing do not allow assessing in time the changes in physiological parameters in the same individual. In this study, we introduce microencapsulated fluorescent probes as a promising tool for in vivo monitoring of internal pH variation in zebrafish embryos. The pH alteration identified under stress conditions demonstrates the applicability of the microencapsulated fluorescent probes for the repeated analysis of the embryo's physiological state. The proposed approach has strong potential to simultaneously measure a range of physiological characteristics using a set of specific fluorescent probes and to finally bring toxicological bioassays and related research fields to a new level of effectiveness and sensitivity.

Concise Review: Bone Marrow Mononuclear Cells for the Treatment of Ischemic Syndromes: Medicinal Product or Cell Transplantation?

PubMed Central

Rico, Laura; Herrera, Concha

2012-01-01

In November of 2011, the Committee for Advanced Therapies (CAT) of the European Medicines Agency (EMA) published two scientific recommendations regarding the classification of autologous bone marrow-derived mononuclear cells (BM-MNCs) and autologous bone marrow-derived CD133+ cells as advanced therapy medicinal products (ATMPs), specifically tissue-engineered products, when intended for regeneration in ischemic heart tissue on the basis that they are not used for the same essential function (hematological restoration) that they fulfill in the donor. In vitro and in vivo evidence demonstrates that bone marrow cells are physiologically involved in adult neovascularization and tissue repair, making their therapeutic use for these purposes a simple exploitation of their own essential functions. Therefore, from a scientific/legal point of view, nonsubstantially manipulated BM-MNCs and CD133+ cells are not an ATMP, because they have a physiological role in the processes of postnatal neovascularization and, when used therapeutically for vascular restoration in ischemic tissues, they are carrying out one of their essential physiological functions (the legal definition recognizes that cells can have several essential functions). The consequences of classifying BM-MNCs and CD133+ cells as medicinal products instead of cellular transplantation, like bone marrow transplantation, in terms of costs and time for these products to be introduced into clinical practice, make this an issue of crucial importance. Therefore, the recommendations of EMA/CAT could be reviewed in collaboration with scientific societies, in light of organizational and economic consequences as well as scientific knowledge recently acquired about the mechanisms of postnatal neovascularization and the function of bone marrow in the regeneration of remote tissues. PMID:23197819

Comprehensive evaluation of poly(I:C) induced inflammatory response in an airway epithelial model

PubMed Central

Lever, Amanda R; Park, Hyoungshin; Mulhern, Thomas J; Jackson, George R; Comolli, James C; Borenstein, Jeffrey T; Hayden, Patrick J; Prantil-Baun, Rachelle

2015-01-01

Respiratory viruses invade the upper airway of the lung, triggering a potent immune response that often exacerbates preexisting conditions such as asthma and COPD. Poly(I:C) is a synthetic analog of viral dsRNA that induces the characteristic inflammatory response associated with viral infection, such as loss of epithelial integrity, and increased production of mucus and inflammatory cytokines. Here, we explore the mechanistic responses to poly(I:C) in a well-defined primary normal human bronchial epithelial (NHBE) model that recapitulates in vivo functions and responses. We developed functional and quantifiable methods to evaluate the physiology of our model in both healthy and inflamed states. Through gene and protein expression, we validated the differentiation state and population of essential cell subtypes (i.e., ciliated, goblet, club, and basal cells) as compared to the human lung. Assays for total mucus production, cytokine secretion, and barrier function were used to evaluate in vitro physiology and response to viral insult. Cells were treated apically with poly(I:C) and evaluated 48 h after induction. Results revealed a dose-dependent increase in goblet cell differentiation, as well as, an increase in mucus production relative to controls. There was also a dose-dependent increase in secretion of IL-6, IL-8, TNF-α, and RANTES. Epithelial barrier function, as measured by TEER, was maintained at 1501 ± 355 Ω*cm² postdifferentiation, but dropped significantly when challenged with poly(I:C). This study provides first steps toward a well-characterized model with defined functional methods for understanding dsRNA stimulated inflammatory responses in a physiologically relevant manner. PMID:25847914

Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

PubMed

Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

2007-08-03

Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi--Rhodnius prolixus interaction using confocal microscopy.

PubMed

Feder, Denise; Gomes, Suzete A O; de Thomaz, André A; Almeida, Diogo B; Faustino, Wagner M; Fontes, Adriana; Stahl, Cecília V; Santos-Mallet, Jacenir R; Cesar, Carlos L

2009-12-01

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi-R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1

PubMed Central

Tu, Xiaolin; Chen, Jianquan; Lim, Joohyun; Karner, Courtney M.; Lee, Seung-Yon; Heisig, Julia; Wiese, Cornelia; Surendran, Kameswaran; Kopan, Raphael; Gessler, Manfred; Long, Fanxin

2012-01-01

Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo. PMID:22457635

Interrenal dysfunction in fish from contaminated sites: In vivo and in vitro assessment

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

Hontela, A.

1998-01-01

Cortisol, synthesized in the interrenal cells of teleost head kidney, has a major role in the physiologic response to physical and chemical stressors. Plasma levels of cortisol increase in physiologically competent fish acutely exposed to stressors such as cadmium or mercury. The effects of chronic low level exposures are less well understood. The author has diagnosed an endocrine impairment characterized by a reduced capacity to elevate plasma cortisol levels in response to an acute standardized capture stress in yellow perch (Perca flavescens) and in northern pike (Esox lucius) sampled at sites contaminated by mixtures of pollutants (heavy metals, polycyclic aromaticmore » hydrocarbons, and polychlorinated biphenyls), by heavy metals, or by bleached kraft mill effluent. The studies with fish, as well as with amphibians at contaminated sites, demonstrated that low level chronic exposures impair secretion of corticosteroids. The author has developed new tests for assessment of the functional integrity of teleost and amphibian interrenal tissue using an adrenocorticotropic hormone (ACTH) challenge, in vivo and in vitro. The reduced ability to respond to ACTH indicates that the normal neuroendocrine response to stressors may be disrupted and that the ability to cope with biotic and abiotic stressors in the environment may be significantly reduced in the impaired animals.« less

Microsomal and Cytosolic Scaling Factors in Dog and Human Kidney Cortex and Application for In Vitro-In Vivo Extrapolation of Renal Metabolic Clearance

PubMed Central

Scotcher, Daniel; Billington, Sarah; Brown, Jay; Jones, Christopher R.; Brown, Colin D. A.; Rostami-Hodjegan, Amin

2017-01-01

In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of subcellular fractions rely on robust estimates of physiologically relevant scaling factors for the prediction of clearance in vivo. The purpose of the current study was to measure the microsomal and cytosolic protein per gram of kidney (MPPGK and CPPGK) in dog and human kidney cortex using appropriate protein recovery marker and evaluate functional activity of human cortex microsomes. Cytochrome P450 (CYP) content and glucose-6-phosphatase (G6Pase) activity were used as microsomal protein markers, whereas glutathione-S-transferase activity was a cytosolic marker. Functional activity of human microsomal samples was assessed by measuring mycophenolic acid glucuronidation. MPPGK was 33.9 and 44.0 mg/g in dog kidney cortex, and 41.1 and 63.6 mg/g in dog liver (n = 17), using P450 content and G6Pase activity, respectively. No trends were noted between kidney, liver, and intestinal scalars from the same animals. Species differences were evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g in kidney cortex (n = 38), respectively. MPPGK was 2-fold greater than the commonly used in vitro-in vivo extrapolation scalar; this difference was attributed mainly to tissue source (mixed kidney regions versus cortex). Robust human MPPGK and CPPGK scalars were measured for the first time. The work emphasized the importance of regional differences (cortex versus whole kidney–specific MPPGK, tissue weight, and blood flow) and a need to account for these to improve assessment of renal metabolic clearance and its extrapolation to in vivo. PMID:28270564

Long Non-coding RNAs and their Role in Metastasis

PubMed Central

WEIDLE, ULRICH H; BIRZELE, FABIAN; KOLLMORGEN, GWEN; RÜGER, RÜDIGER

2017-01-01

The perception of long non-coding RNAs as chunk RNA and transcriptional noise has been steadily replaced by their role as validated targets for a diverse set of physiological processes in the past few years. However, for the vast majority of lncRNAs their precise mode of action and physiological function remain to be uncovered. A large body of evidence has revealed their essential role in all stages of cancirogenesis and metastasis. In this review we focus on the role of lncRNAs in metastasis. We grouped selected lncRNAs into three categories based on in vitro and in vivo mode of action-related studies and clinical relevance for metastasis. Grouped according to their mode of action, in category I we discuss lncRNAs such as CCAT2, DREH, LET, NKILA, treRNA, HOTAIR, H19, FENDRR, lincROR, MALAT, GClnc1, BCAR4, SCHLAP1 and lncRNA ATP, all lncRNAs with in vitro and in vivo metastasis-related data and clinical significance. In category II we discuss lncRNAs CCAT1, PCAT1, PTENgp1, GPLINC, MEG3, ZEB2-AS, LCT13, ANRIL, NBAT1 and lncTCF7 all characterized by their mode of action in vitro and clinical significance, but pending or preliminary in vivo data. Finally, under category III, we discuss lncRNAs BANCR, FRLnc1, SPRY4-IT1 and LIMT with partially or poorly-resolved mode of action and varying degree of validation in clinical metastasis. Finally we discuss metastasis-related translational aspects of lncRNAs. PMID:28446530

The recovery time course of the endothelial-cell glycocalyx in vivo and its implications in vitro

PubMed Central

Potter, Daniel R.; Jiang, John; Damiano, Edward R.

2009-01-01

Compelling evidence continues to emerge suggesting that the glycocalyx surface layer on vascular endothelial cells plays a determining role in numerous physiological processes including inflammation, microvascular permeability, and endothelial mechanotransduction. Previous research has shown that enzymes degrade the glycocalyx, while inflammation causes shedding of the layer. To track the endogenous recovery of the glycocalyx in vivo, we used fluorescent micro-particle image velocimetry (µ-PIV) in mouse cremaster-muscle venules to estimate the hydrodynamically relevant glycocalyx thickness 1, 3, 5, and 7 days after enzymatic or cytokine-mediated degradation of the layer. Results indicate that after acute degradation of the glycocalyx, 5–7 days are required for the layer to endogenously restore itself to its native hydrodynamically relevant thickness in vivo. In light of these findings, and since demonstrable evidence has emerged that standard cell-culture conditions are not conducive to providing the environment and/or cellular conditions necessary to produce and maintain a physiologically relevant cell-surface glycocalyx in vitro, we sought to determine if merely the passage of time would be sufficient to promote the production of a hydrodynamically relevant glycocalyx on a confluent monolayer of human umbilical vein endothelial cells (HUVECs). Using µ-PIV, we found that the hydrodynamically relevant glycocalyx was substantially absent 7 days post-confluence on HUVEC-lined cylindrical collagen microchannels maintained under standard culture conditions. Thus it remains to be determined how a hydrodynamically relevant glycocalyx surface layer can be synthesized and maintained in culture before the endothelial-cell culture model can be used to elucidate glycocalyx-mediated mechanisms of endothelial-cell function. PMID:19443840

Gold nanoparticles as physiological markers of urine internalization into urothelial cells in vivo

PubMed Central

Hudoklin, Samo; Zupančič, Daša; Makovec, Darko; Kreft, Mateja Erdani; Romih, Rok

2013-01-01

Background Urothelial bladder is the reservoir of urine and the urothelium minimizes the exchange of urine constituents with this tissue. Our aim was to test 1.9 nm biocompatible gold nanoparticles as a novel marker of internalization into the urothelial cells under physiological conditions in vivo. Methods We compared normal and neoplastic mice urothelium. Neoplastic lesions were induced by 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water for 10 weeks. Nanoparticles, intravenously injected into normal and BBN-treated mice, were filtered through the kidneys and became constituents of the urine within 90 minutes after injection. Results Gold nanoparticles were densely accumulated in the urine, while their internalization into urothelial cells depended on the cell differentiation stage. In the terminally differentiated superficial urothelial cells of normal animals, nanoparticles were occasionally found in the endosomes, but not in the fusiform vesicles. Regions of exfoliated cells were occasionally found in the normal urothelium. Superficial urothelial cells located next to exfoliated regions contained gold nanoparticles in the endosomes and in the cytosol beneath the apical plasma membrane. The urothelium of BBN-treated animals developed fat hyperplasia with moderate dysplasia. The superficial cells of BBN-treated animals were partially differentiated as demonstrated by the lack of fusiform vesicles. These cells contained the gold nanoparticles distributed in the endosomes and throughout their cytosol. Conclusion Gold nanoparticles are a valuable marker to study urine internalization into urothelial cells in vivo. Moreover, they can be used as a sensitive marker of differentiation and functionality of urothelial cells. PMID:24143099

Iron Chelation Nanoparticles with Delayed Saturation as an Effective Therapy for Parkinson Disease.

PubMed

Wang, Nan; Jin, Xin; Guo, Dongbo; Tong, Gangsheng; Zhu, Xinyuan

2017-02-13

Iron accumulation in substantia nigra pars compacta (SNpc) has been proved to be a prominent pathophysiological feature of Parkinson's diseases (PD), which can induce the death of dopaminergic (DA) neurons, up-regulation of reactive oxygen species (ROS), and further loss of motor control. In recent years, iron chelation therapy has been demonstrated to be an effective treatment for PD, which has shown significant improvements in clinical trials. However, the current iron chelators are suboptimal due to their short circulation time, side effects, and lack of proper protection from chelation with ions in blood circulation. In this work, we designed and constructed iron chelation therapeutic nanoparticles protected by a zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) to delay the saturation of iron chelators in blood circulation and prolong the in vivo lifetime, with HIV-1 trans-activating transcriptor (TAT) served as a shuttle to enhance the blood-brain barrier (BBB) permeability. We explored and investigated whether the Parkinsonian neurodegeneration and the corresponding symptoms in behaviors and physiologies could be prevented or reversed both in vitro and in vivo. The results demonstrated that iron chelator loaded therapeutic nanoparticles could reverse functional deficits in Parkinsonian mice not only physiologically but also behaviorally. On the contrary, both untreated PD mice and non-TAT anchored nanoparticle treated PD mice showed similar loss in DA neurons and difficulties in behaviors. Therefore, with protection of zwitterionic polymer and prolonged in vivo lifetime, iron chelator loaded nanoparticles with delayed saturation provide a PD phenotype reversion therapy and significantly improve the living quality of the Parkinsonian mice.

MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo.

PubMed

Torella, Daniele; Iaconetti, Claudio; Catalucci, Daniele; Ellison, Georgina M; Leone, Angelo; Waring, Cheryl D; Bochicchio, Angela; Vicinanza, Carla; Aquila, Iolanda; Curcio, Antonio; Condorelli, Gianluigi; Indolfi, Ciro

2011-09-30

MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal-regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

PubMed Central

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

2014-01-01

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

PubMed

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

2014-03-28

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

In-vivo measurements of the tear film on a cornea and a contact lens by use of interferometry

NASA Astrophysics Data System (ADS)

Licznerski, Tomasz J.; Kasprzak, Henryk T.; Kowalik, Waldemar

1996-12-01

The tear film fulfills several important functions in the eye. Apart of its physiologic functions like maintaining a moist environment for the epithelial cells of the cornea and conjunctiva, bacterial properties, transporting metabolic products etc., this film causes that the corneal surface has the optical quality. This smooth surface allows to apply interferometry for measurements. The paper presents tear's layer distribution on the soft contact lens and the cornea in comparison. Tv frame speed registration in the Twyman- Green interferometer was used to observe an unstable biomedical objects like the eye. The proposed method has the advantage of being noncontact and applies the low energy laser beam in interferometric set-up. This provides non- invasive testing of human cornea in vivo and enables observation the kinetics of its tear layer deterioration. The evaluation of non-invasive tear breakup time is possible by use of proposed setup. Further analysis of recorded interferograms helps to examine the matter of the breakup process and can be used for detection of the 'dry eye' symptoms.

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.

PubMed

Maxwell, W M; Johnson, L A

1999-12-01

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.

Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science.

PubMed

Giessen, Tobias W

2016-10-01

Compartmentalization is one of the defining features of life. Cells use protein compartments to exert spatial control over their metabolism, store nutrients and create unique microenvironments needed for essential physiological processes. Encapsulins are a recently discovered class of protein nanocompartments found in bacteria and archaea that naturally encapsulate cargo proteins. A short C-terminal targeting sequence directs the highly specific encapsulation process in vivo. Here, I will initially discuss the properties, diversity and putative function of encapsulins. The unique characteristics and potential uses of the self-sorting cargo-packaging process found in encapsulin systems will then be highlighted. Examples for the application of encapsulins as cell-specific optical nanoprobes and targeted therapeutic delivery systems will be discussed with an emphasis on the ability to integrate multiple functionalities within a single nanodevice. By fusing targeting sequences to non-native proteins, encapsulins can also be used as specific nanocontainers and enzymatic nanoreactors in vivo. I will end by briefly discussing future avenues for encapsulin research related to both basic microbial metabolism and applications in biomedicine, catalysis and materials science. Copyright © 2016 Elsevier Ltd. All rights reserved.

Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function

PubMed Central

Sen, Supriya; Jumaa, Hassan; Webster, Nicholas J.G.

2015-01-01

SR family RNA binding proteins regulate splicing of nascent RNAs in vitro but their physiological role in vivo is largely unexplored, as genetic deletion of many SR protein genes results in embryonic lethality. Here we show that SRSF3HKO mice carrying a hepatocyte-specific deletion of Srsf3 (homologous to human SRSF3/SRp20) have a disrupted hepatic architecture and show pre- and postnatal growth retardation. SRSF3HKO mice exhibit impaired hepatocyte maturation with alterations in glucose and lipid homeostasis characterized by reduced glycogen storage, fasting hypoglycemia, increased insulin sensitivity and reduced cholesterol synthesis. We identify various splicing alterations in the SRSF3HKO liver that explain the in vivo phenotype. In particular, loss of SRSF3 causes aberrant splicing of Hnf1α, Ern1, Hmgcs1, Dhcr7 and Scap genes, which are critical regulators of glucose and lipid metabolism. Our study provides the first evidence for a SRSF3-driven genetic programme required for morphological and functional differentiation of hepatocytes that may have relevance for human liver disease and metabolic dysregulation. PMID:23299886

DJ-1/PARK7, But Not Its L166P Mutant Linked to Autosomal Recessive Parkinsonism, Modulates the Transcriptional Activity of the Orphan Nuclear Receptor Nurr1 In Vitro and In Vivo.

PubMed

Lu, Lingling; Zhao, Shasha; Gao, Ge; Sun, Xiaohong; Zhao, Huanying; Yang, Hui

2016-12-01

Although mutations of DJ-1 have been linked to autosomal recessive Parkinsonism for years, its physiological function and the pathological mechanism of its mutants are not well understood. We report for the first time that exogenous application of DJ-1, but not its L166P mutant, enhances the nuclear translocation and the transcriptional activity of Nurr1, a transcription factor essential for dopaminergic neuron development and maturation, both in vitro and in vivo. Knockdown of DJ-1 attenuates Nurr1 activity. Further investigation showed that signaling of Raf/MEK/ERK MAPKs is involved in this regulatory process and that activation induced by exogenous DJ-1 is antagonized by U0126, an ERK pathway inhibitor, indicating that DJ-1 modulates Nurr1 activity via the Raf/MEK/ERK pathway. Our findings shed light on the novel function of DJ-1 to enhance Nurr1 activity and provide the first insight into the molecular mechanism by which DJ-1 enhances Nurr1 activity.

β2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase

PubMed Central

Chiu, Wen-Chin; Chiou, Tzeon-Jye; Chung, Meng-Ju; Chiang, An-Na

2016-01-01

Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy. PMID:27579889

The effects of acute alcohol exposure on the response properties of neurons in visual cortex area 17 of cats

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

Chen Bo; State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Science, Beijing 100101; Xia Jing

Physiological and behavioral studies have demonstrated that a number of visual functions such as visual acuity, contrast sensitivity, and motion perception can be impaired by acute alcohol exposure. The orientation- and direction-selective responses of cells in primary visual cortex are thought to participate in the perception of form and motion. To investigate how orientation selectivity and direction selectivity of neurons are influenced by acute alcohol exposure in vivo, we used the extracellular single-unit recording technique to examine the response properties of neurons in primary visual cortex (A17) of adult cats. We found that alcohol reduces spontaneous activity, visual evoked unitmore » responses, the signal-to-noise ratio, and orientation selectivity of A17 cells. In addition, small but detectable changes in both the preferred orientation/direction and the bandwidth of the orientation tuning curve of strongly orientation-biased A17 cells were observed after acute alcohol administration. Our findings may provide physiological evidence for some alcohol-related deficits in visual function observed in behavioral studies.« less

Do unliganded thyroid hormone receptors have physiological functions?

PubMed

Chassande, O

2003-08-01

Thyroid hormone (TH) is required for the development of vertebrates and exerts numerous homeostatic functions in adults. TH acts through nuclear receptors which control the transcription of target genes. Unliganded and liganded thyroid hormone receptors (TRs) have been shown to exert opposite effects on the transcription of target genes in vitro. However, the occurance of an aporeceptor activity in vivo and its potential physiological significance has not been clearly addressed. Several data generated using experimental hypothyroidism and thyrotoxicosis in wild type and TR knockout mice support the notion that apoTRs have an intrinsic activity in several tIssues. ApoTRs, and in particular TRalpha1, are predominant during the early stages of vertebrate development and must be turned into holoTRs for post-natal development to proceed normally. However, the absence of striking alterations of embryonic and fetal development in mice devoid of TRs indicates that apoTRs do not play a fundamental role. During development, as well as in adults, apoTRs rather appears as a system which increases the range of transcriptional responses to moderate variations of T3.

History of functional neurosurgery.

PubMed

Iskandar, B J; Nashold, B S

1995-01-01

Whereas in the early days of evil spirits, electric catfish, and phrenology, functional neurosurgery was based on crude observations and dogma, the progress made in neurophysiology at the turn of the century gave the field a strong scientific foundation. Subsequently, the advent of stereotaxis allowed access to deep brain regions and contributed an element of precision. Future directions include the development of frameless stereotaxy; the use of MRI-generated anatomic data, which would circumvent the serious problem of individual variations seen with standard brain atlases; the introduction of various chemicals into brain structures, in an attempt to influence neurochemically mediated disease processes; and finally, the use of the promising techniques of neural transplantation. On hearing of Penfield's intraoperative brain stimulations, Sherrington commented: "It must be great fun to have the physiological preparation speak to you." The idea of therapeutic neurophysiologic interventions is appealing, especially because many disorders show no obvious treatable pathologic cause (e.g., tumor, vascular malformation). As stereotactic technology becomes less cumbersome and more precise, more sophisticated in vivo neurophysiologic preparations become possible. In turn, as our understanding of nervous system physiology grows, our ability to understand pathophysiology and treat disease processes increases.

From functional architecture to functional connectomics.

PubMed

Reid, R Clay

2012-07-26

"Receptive Fields, Binocular Interaction and Functional Architecture in the Cat's Visual Cortex" by Hubel and Wiesel (1962) reported several important discoveries: orientation columns, the distinct structures of simple and complex receptive fields, and binocular integration. But perhaps the paper's greatest influence came from the concept of functional architecture (the complex relationship between in vivo physiology and the spatial arrangement of neurons) and several models of functionally specific connectivity. They thus identified two distinct concepts, topographic specificity and functional specificity, which together with cell-type specificity constitute the major determinants of nonrandom cortical connectivity. Orientation columns are iconic examples of topographic specificity, whereby axons within a column connect with cells of a single orientation preference. Hubel and Wiesel also saw the need for functional specificity at a finer scale in their model of thalamic inputs to simple cells, verified in the 1990s. The difficult but potentially more important question of functional specificity between cortical neurons is only now becoming tractable with new experimental techniques. Copyright © 2012 Elsevier Inc. All rights reserved.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip

PubMed Central

Dauth, Stephanie; Maoz, Ben M.; Sheehy, Sean P.; Hemphill, Matthew A.; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M.; Budnik, Bogdan

2017-01-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. PMID:28031399

Intestinal ABCA1 directly contributes to HDL biogenesis in vivo

PubMed Central

Brunham, Liam R.; Kruit, Janine K.; Iqbal, Jahangir; Fievet, Catherine; Timmins, Jenelle M.; Pape, Terry D.; Coburn, Bryan A.; Bissada, Nagat; Staels, Bart; Groen, Albert K.; Hussain, M. Mahmood; Parks, John S.; Kuipers, Folkert; Hayden, Michael R.

2006-01-01

Plasma HDL cholesterol levels are inversely related to risk for atherosclerosis. The ATP-binding cassette, subfamily A, member 1 (ABCA1) mediates the rate-controlling step in HDL particle formation, the assembly of free cholesterol and phospholipids with apoA-I. ABCA1 is expressed in many tissues; however, the physiological functions of ABCA1 in specific tissues and organs are still elusive. The liver is known to be the major source of plasma HDL, but it is likely that there are other important sites of HDL biogenesis. To assess the contribution of intestinal ABCA1 to plasma HDL levels in vivo, we generated mice that specifically lack ABCA1 in the intestine. Our results indicate that approximately 30% of the steady-state plasma HDL pool is contributed by intestinal ABCA1 in mice. In addition, our data suggest that HDL derived from intestinal ABCA1 is secreted directly into the circulation and that HDL in lymph is predominantly derived from the plasma compartment. These data establish a critical role for intestinal ABCA1 in plasma HDL biogenesis in vivo. PMID:16543947

A quantitative analysis of statistical power identifies obesity endpoints for improved in vivo preclinical study design

PubMed Central

Selimkhanov, Jangir; Thompson, W. Clayton; Guo, Juen; Hall, Kevin D.; Musante, Cynthia J.

2017-01-01

The design of well-powered in vivo preclinical studies is a key element in building knowledge of disease physiology for the purpose of identifying and effectively testing potential anti-obesity drug targets. However, as a result of the complexity of the obese phenotype, there is limited understanding of the variability within and between study animals of macroscopic endpoints such as food intake and body composition. This, combined with limitations inherent in the measurement of certain endpoints, presents challenges to study design that can have significant consequences for an anti-obesity program. Here, we analyze a large, longitudinal study of mouse food intake and body composition during diet perturbation to quantify the variability and interaction of key metabolic endpoints. To demonstrate how conclusions can change as a function of study size, we show that a simulated pre-clinical study properly powered for one endpoint may lead to false conclusions based on secondary endpoints. We then propose guidelines for endpoint selection and study size estimation under different conditions to facilitate proper power calculation for a more successful in vivo study design. PMID:28392555

A simple sensing mechanism for wireless, passive pressure sensors.

PubMed

Drazan, John F; Wassick, Michael T; Dahle, Reena; Beardslee, Luke A; Cady, Nathaniel C; Ledet, Eric H

2016-08-01

We have developed a simple wireless pressure sensor that consists of only three electrically isolated components. Two conductive spirals are separated by a closed cell foam that deforms when exposed to changing pressures. This deformation changes the capacitance and thus the resonant frequency of the sensors. Prototype sensors were submerged and wirelessly interrogated while being exposed to physiologically relevant pressures from 10 to 130 mmHg. Sensors consistently exhibited a sensitivity of 4.35 kHz/mmHg which is sufficient for resolving physiologically relevant pressure changes in vivo. These simple sensors have the potential for in vivo pressure sensing.

Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology

PubMed Central

Kotlikoff, Michael I

2007-01-01

This article reviews genetically encoded Ca2+ indicators (GECIs), with a focus on the use of these novel molecules in the context of understanding complex cell signalling in mammals, in vivo. The review focuses on the advantages and limitations of specific GECI design strategies and the results of experiments in which these molecules have been expressed in transgenic mice, concentrating particularly on recent experiments from our laboratory in which physiological signalling could be monitored in vivo. Finally, newer strategies for effective genetic specification of GECIs are briefly reviewed. PMID:17038427

A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.

PubMed

Koh, James; Hogue, Joyce A; Sosa, Julie A

2016-01-01

The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.

Molecular imaging of photodynamic therapy

NASA Astrophysics Data System (ADS)

Chang, Sung K.; Errabelli, Divya; Rizvi, Imran; Solban, Nicolas; O'Riordan, Katherine; Hasan, Tayyaba

2006-02-01

Recent advances in light sources, detectors and other optical imaging technologies coupled with the development of novel optical contrast agents have enabled real-time, high resolution, in vivo monitoring of molecular targets. Noninvasive monitoring of molecular targets is particularly relevant to photodynamic therapy (PDT), including the delivery of photosensitizer in the treatment site and monitoring of molecular and physiological changes following treatment. Our lab has developed optical imaging technologies to investigate these various aspects of photodynamic therapy (PDT). We used a laser scanning confocal microscope to monitor the pharmacokinetics of various photosensitizers in in vitro as well as ex vivo samples, and developed an intravital fluorescence microscope to monitor photosensitizer delivery in vivo in small animals. A molecular specific contrast agent that targets the vascular endothelial growth factor (VEGF) was developed to monitor the changes in the protein expression following PDT. We were then able to study the physiological changes due to post-treatment VEGF upregulation by quantifying vascular permeability with in vivo imaging.

Developing a Physiologically-Based Pharmacokinetic Model Knowledgebase in Support of Provisional Model Construction - poster

EPA Science Inventory

Building new physiologically based pharmacokinetic (PBPK) models requires a lot data, such as the chemical-specific parameters and in vivo pharmacokinetic data. Previously-developed, well-parameterized, and thoroughly-vetted models can be great resource for supporting the constr...

Unperturbed vs. post-transplantation hematopoiesis: both in vivo but different.

PubMed

Busch, Katrin; Rodewald, Hans-Reimer

2016-07-01

Hematopoietic stem cell (HSC) transplantation has yielded tremendous information on experimental properties of HSCs. Yet, it remains unclear whether transplantation reflects the physiology of hematopoiesis. A limitation is the difficulty in accessing HSC functions without isolation, in-vitro manipulation and readout for potential. New genetic fate mapping and clonal marking techniques now shed light on hematopoiesis under physiological conditions. Transposon-based genetic marks were introduced across the entire hematopoietic system to follow the clonal dynamics of these tags over time. A polyclonal source downstream from stem cells was found responsible for the production of at least granulocytes. In independent experiments, HSCs were genetically marked in adult mice, and the kinetics of label emergence throughout the system was followed over time. These experiments uncovered that during physiological steady-state hematopoiesis large numbers of HSCs yield differentiated progeny. Individual HSCs were active only rarely, indicating their very slow periodicity of differentiation rather than quiescence. Noninvasive genetic experiments in mice have identified a major role of stem and progenitor cells downstream from HSCs as drivers of adult hematopoiesis, and revealed that post-transplantation hematopoiesis differs quantitatively from normal steady-state hematopoiesis.

The physiological rationale of heat and moisture exchangers in post-laryngectomy pulmonary rehabilitation: a review.

PubMed

Zuur, J K; Muller, S H; de Jongh, F H C; van Zandwijk, N; Hilgers, F J M

2006-01-01

Total laryngectomy results in a permanent disconnection of the upper and lower airways and inevitably leads to chronic pulmonary complaints like frequent involuntary coughing, increased sputum production and repeated daily forced expectoration to clean the airway. Heat and moisture exchangers (HMEs), applied in an attempt to compensate for the lost functions of the upper respiratory tract, have been found to diminish these symptoms and improve the quality of life significantly. An HME has three physical properties that might be responsible for these improvements. First, its heat and moisture exchanging capacity improves intra-airway preservation of heat and water. Since the condensation and evaporation of moisture are accompanied by the release and uptake of thermal energy, these parameters are inseparable. Secondly, the HME's resistance may reduce dynamic airway compression, thereby improving ventilation. Thirdly, to some extent, an HME might filter out particles, thereby cleaning inspiratory breathing air. This article summarizes our present knowledge of changes in respiratory physiology after total laryngectomy and the influence of the HME by reviewing the physiological impact of these three physical properties separately for in vitro and in vivo data.

Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Ahmed, Syeed Ehsan

Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

Senescent changes in the ribosomes of animal cells in vivo and in vitro

NASA Technical Reports Server (NTRS)

Miquel, J.; Johnson, J. E., Jr.

1979-01-01

The paper examines RNA-ribosomal changes observed in protozoa and fixed postmitotic cells, as well as the characteristics of intermitotic cells. Attention is given to a discussion of the implications of the reported ribosomal changes as to the senescent deterioration of protein synthesis and physiological functions. A survey of the literature suggests that, while the data on ribosomal change in dividing cells both in vivo and in vitro are inconclusive, there is strong histological and biochemical evidence in favor of some degree of quantitative ribosomal loss in fixed postmitotic cells. Since these decreases in ribosomes are demonstrated in differential cells from nematodes, insects and mammals, they may represent a universal manifestation of cytoplasmic senescence in certain types of fixed postmitotic animal cells. The observed variability in ribosomal loss for cells belonging to the same type suggests that this involution phenomenon is rather related to the wear and tear suffered by a particular cell.

In vivo optical imaging and dynamic contrast methods for biomedical research

PubMed Central

Hillman, Elizabeth M. C.; Amoozegar, Cyrus B.; Wang, Tracy; McCaslin, Addason F. H.; Bouchard, Matthew B.; Mansfield, James; Levenson, Richard M.

2011-01-01

This paper provides an overview of optical imaging methods commonly applied to basic research applications. Optical imaging is well suited for non-clinical use, since it can exploit an enormous range of endogenous and exogenous forms of contrast that provide information about the structure and function of tissues ranging from single cells to entire organisms. An additional benefit of optical imaging that is often under-exploited is its ability to acquire data at high speeds; a feature that enables it to not only observe static distributions of contrast, but to probe and characterize dynamic events related to physiology, disease progression and acute interventions in real time. The benefits and limitations of in vivo optical imaging for biomedical research applications are described, followed by a perspective on future applications of optical imaging for basic research centred on a recently introduced real-time imaging technique called dynamic contrast-enhanced small animal molecular imaging (DyCE). PMID:22006910

Erythrocytes Are Oxygen-Sensing Regulators of the Cerebral Microcirculation.

PubMed

Wei, Helen Shinru; Kang, Hongyi; Rasheed, Izad-Yar Daniel; Zhou, Sitong; Lou, Nanhong; Gershteyn, Anna; McConnell, Evan Daniel; Wang, Yixuan; Richardson, Kristopher Emil; Palmer, Andre Francis; Xu, Chris; Wan, Jiandi; Nedergaard, Maiken

2016-08-17

Energy production in the brain depends almost exclusively on oxidative metabolism. Neurons have small energy reserves and require a continuous supply of oxygen (O2). It is therefore not surprising that one of the hallmarks of normal brain function is the tight coupling between cerebral blood flow and neuronal activity. Since capillaries are embedded in the O2-consuming neuropil, we have here examined whether activity-dependent dips in O2 tension drive capillary hyperemia. In vivo analyses showed that transient dips in tissue O2 tension elicit capillary hyperemia. Ex vivo experiments revealed that red blood cells (RBCs) themselves act as O2 sensors that autonomously regulate their own deformability and thereby flow velocity through capillaries in response to physiological decreases in O2 tension. This observation has broad implications for understanding how local changes in blood flow are coupled to synaptic transmission. Copyright © 2016 Elsevier Inc. All rights reserved.

Metaplasticity of hypothalamic synapses following in vivo challenge.

PubMed

Kuzmiski, J Brent; Pittman, Quentin J; Bains, Jaideep S

2009-06-25

Neural networks that regulate an organism's internal environment must sense perturbations, respond appropriately, and then reset. These adaptations should be reflected as changes in the efficacy of the synapses that drive the final output of these homeostatic networks. Here we show that hemorrhage, an in vivo challenge to fluid homeostasis, induces LTD at glutamate synapses onto hypothalamic magnocellular neurosecretory cells (MNCs). LTD requires the activation of postsynaptic alpha2-adrenoceptors and the production of endocannabinoids that act in a retrograde fashion to inhibit glutamate release. In addition, both hemorrhage and noradrenaline downregulate presynaptic group III mGluRs. This loss of mGluR function allows high-frequency activity to potentiate these synapses from their depressed state. These findings demonstrate that noradrenaline controls a form of metaplasticity that may underlie the resetting of homeostatic networks following a successful response to an acute physiological challenge.

Mapping phosphorylation rate of fluoro-deoxy-glucose in rat brain by 19F chemical shift imaging

PubMed Central

Coman, Daniel; Sanganahalli, Basavaraju G.; Cheng, David; McCarthy, Timothy; Rothman, Douglas L.; Hyder, Fahmeed

2014-01-01

19F magnetic resonance spectroscopy (MRS) studies of 2-fluoro-2-deoxy-D-glucose (FDG) and 2-fluoro-2-deoxy-D-glucose-6-phosphate (FDG-6P) can be used for directly assessing total glucose metabolism in vivo. To date, 19F MRS measurements of FDG phosphorylation in the brain have either been achieved ex vivo from extracted tissue or in vivo by unusually long acquisition times. Electrophysiological and functional magnetic resonance imaging (fMRI) measurements indicate that FDG doses up to 500mg/kg can be tolerated with minimal side effects on cerebral physiology and evoked fMRI-BOLD responses to forepaw stimulation. In halothane-anesthetized rats, we report localized in vivo detection and separation of FDG and FDG-6P MRS signals with 19F 2D chemical shift imaging (CSI) at 11.7T. A metabolic model based on reversible transport between plasma and brain tissue, which included a non-saturable plasma to tissue component, was used to calculate spatial distribution of FDG and FDG-6P concentrations in rat brain. In addition, spatial distribution of rate constants and metabolic fluxes of FDG to FDG-6P conversion were estimated. Mapping the rate of FDG to FDG-6P conversion by 19F CSI provides an MR methodology that could impact other in vivo applications such as characterization of tumor pathophysiology. PMID:24581725

Effects of model traumatic injury on hepatic drug metabolism in the rat. IV. Glucuronidation.

PubMed

Griffeth, L K; Rosen, G M; Rauckman, E J

1985-01-01

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on hepatic glucuronidation activity. As was previously observed with hepatic oxidative drug metabolism, model trauma resulted in a significant decrease in the in vivo glucuronidation of chloramphenicol, with a 23% drop in clearance of this drug. The effect on in vivo pharmacokinetics appeared to result from a complex interaction between trauma's differential influences on conjugating enzyme(s), deconjugating enzyme(s), and hepatic UDP-glucuronic acid levels, as well as the relative physiological importance of these variables. Hepatic UDP-glucuronyltransferase activities towards both p-nitrophenol and chloramphenicol were elevated (44-54%) after model injury when measured in native hepatic microsomes. However, microsomes which had been "activated" by treatment with Triton X-100 showed no significant difference between control and traumatized animals. Serum beta-glucuronidase activities were elevated by 58%, while hepatic beta-glucuronidase rose by about 16%. Nevertheless, in vivo deconjugation showed no significant change. Model trauma also resulted in a 46% decrease in hepatic UDP-glucuronic acid content. Thus, the observed post-traumatic depression of in vivo chloramphenicol glucuronidation could be due either to a diminished availability of a necessary cofactor (UDP-glucuronic acid) or to an alteration in enzyme kinetics or function in vivo.

Laminar shear stress regulates endothelial kinin B1 receptor expression and function: potential implication in atherogenesis

PubMed Central

Duchene, Johan; Cayla, Cécile; Vessillier, Sandrine; Scotland, Ramona; Yamashiro, Kazuo; Lecomte, Florence; Syed, Irfan; Vo, Phuong; Marrelli, Alessandra; Pitzalis, Costantino; Cipollone, Francesco; Schanstra, Joost; Bascands, Jean-Loup; Hobbs, Adrian J; Perretti, Mauro; Ahluwalia, Amrita

2009-01-01

OBJECTIVE The pro-inflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many pro-inflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. METHODS AND RESULTS Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE-/- mice fed a high fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE-/- mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVEC) exposed to low LSS (0-2dyn/cm2) compared to physiological LSS (6-10dyn/cm2): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFκB activation. CONCLUSION Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whilst physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma are associated with substantial upregulation, identifying this receptor as a potential therapeutic target. CONDENSED ABSTRACT Low laminar shear stress (LSS) underlies the pro-inflammatory processes in atherogenesis. Herein, we demonstrate that whilst physiological LSS represses inflammatory kinin B1 receptor (B1R) expression/function, low atherogenic LSS is associated with profound upregulation of both in atherosclerosis in both humans and animal models, highlighting B1R as an exciting potential therapeutic target. PMID:19661485

Mesenchymal Stem Cells for Osteochondral Tissue Engineering

PubMed Central

Ng, Johnathan; Bernhard, Jonathan; Vunjak-Novakovic, Gordana

2017-01-01

Summary Mesenchymal stem cells (MSC) are of major interest to regenerative medicine, because of the ease of harvesting from a variety of sources (including bone marrow and fat aspirates) and ability to form a range of mesenchymal tissues, in vitro and in vivo. We focus here on the use of MSCs for engineering of cartilage, bone, and complex osteochondral tissue constructs, using protocols that replicate some aspects of the natural mesodermal development. For engineering of human bone, we discuss some of the current advances, and highlight the use of perfusion bioreactors for supporting anatomically exact human bone grafts. For engineering of human cartilage, we discuss limitations of current approaches, and highlight engineering of stratified, mechanically functional human cartilage interfaced with bone by mesenchymal condensation of MSCs. Taken together, the current advances enable engineering physiologically relevant bone, cartilage and osteochondral composites, and physiologically relevant studies of osteochondral development and disease. PMID:27236665

Mechanical deformation induces depolarization of neutrophils.

PubMed

Ekpenyong, Andrew E; Toepfner, Nicole; Fiddler, Christine; Herbig, Maik; Li, Wenhong; Cojoc, Gheorghe; Summers, Charlotte; Guck, Jochen; Chilvers, Edwin R

2017-06-01

The transition of neutrophils from a resting state to a primed state is an essential requirement for their function as competent immune cells. This transition can be caused not only by chemical signals but also by mechanical perturbation. After cessation of either, these cells gradually revert to a quiescent state over 40 to 120 min. We use two biophysical tools, an optical stretcher and a novel microcirculation mimetic, to effect physiologically relevant mechanical deformations of single nonadherent human neutrophils. We establish quantitative morphological analysis and mechanical phenotyping as label-free markers of neutrophil priming. We show that continued mechanical deformation of primed cells can cause active depolarization, which occurs two orders of magnitude faster than by spontaneous depriming. This work provides a cellular-level mechanism that potentially explains recent clinical studies demonstrating the potential importance, and physiological role, of neutrophil depriming in vivo and the pathophysiological implications when this deactivation is impaired, especially in disorders such as acute lung injury.

Biochemical and functional characterization of the periplasmic domain of the outer membrane protein A from enterohemorrhagic Escherichia coli.

PubMed

Wang, Haiguang; Li, Qian; Fang, Yao; Yu, Shu; Tang, Bin; Na, Li; Yu, Bo; Zou, Quanming; Mao, Xuhu; Gu, Jiang

2016-01-01

Outer membrane protein A (OmpA) plays multiple roles in the physiology and pathogenesis of the zoonotic pathogen enterohemorrhagic Escherichia coli (EHEC). The N-terminus of OmpA forms a transmembrane domain (OmpA™), and the roles of this domain in bacterial pathogenesis have been well studied. However, how its C-terminal domain (OmpAper), which is located at the periplasmic space in the bacterial membrane, contributes to virulence remains unclear. Herein, we report that OmpAper forms a dimer and binds to peptidoglycan in vitro. Furthermore, OmpAper is responsible for bacterial resistance to acidic conditions, high osmotic pressure and high SDS environments. In addition, OmpAper contributes to the adhesion of bacteria to HeLa cells in vitro and ex vivo. These results provide an additional understanding of the role of OmpA in EHEC physiology and pathogenesis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Brown fat in a protoendothermic mammal fuels eutherian evolution.

PubMed

Oelkrug, Rebecca; Goetze, Nadja; Exner, Cornelia; Lee, Yang; Ganjam, Goutham K; Kutschke, Maria; Müller, Saskia; Stöhr, Sigrid; Tschöp, Matthias H; Crichton, Paul G; Heldmaier, Gerhard; Jastroch, Martin; Meyer, Carola W

2013-01-01

Endothermy has facilitated mammalian species radiation, but the sequence of events leading to sustained thermogenesis is debated in multiple evolutionary models. Here we study the Lesser hedgehog tenrec (Echinops telfairi), a phylogenetically ancient, 'protoendothermic' eutherian mammal, in which constantly high body temperatures are reported only during reproduction. Evidence for nonshivering thermogenesis is found in vivo during periodic ectothermic-endothermic transitions. Anatomical studies reveal large brown fat-like structures in the proximity of the reproductive organs, suggesting physiological significance for parental care. Biochemical analysis demonstrates high mitochondrial proton leak catalysed by an uncoupling protein 1 ortholog. Strikingly, bioenergetic profiling of tenrec uncoupling protein 1 reveals similar thermogenic potency as modern mouse uncoupling protein 1, despite the large phylogenetic distance. The discovery of functional brown adipose tissue in this 'protoendothermic' mammal links nonshivering thermogenesis directly to the roots of eutherian evolution, suggesting physiological importance prior to sustained body temperatures and migration to the cold.

Brown fat in a protoendothermic mammal fuels eutherian evolution

PubMed Central

Oelkrug, Rebecca; Goetze, Nadja; Exner, Cornelia; Lee, Yang; Ganjam, Goutham K.; Kutschke, Maria; Müller, Saskia; Stöhr, Sigrid; Tschöp, Matthias H.; Crichton, Paul G.; Heldmaier, Gerhard; Jastroch, Martin; Meyer, Carola W.

2013-01-01

Endothermy has facilitated mammalian species radiation, but the sequence of events leading to sustained thermogenesis is debated in multiple evolutionary models. Here we study the Lesser hedgehog tenrec (Echinops telfairi), a phylogenetically ancient, ‘protoendothermic’ eutherian mammal, in which constantly high body temperatures are reported only during reproduction. Evidence for nonshivering thermogenesis is found in vivo during periodic ectothermic–endothermic transitions. Anatomical studies reveal large brown fat-like structures in the proximity of the reproductive organs, suggesting physiological significance for parental care. Biochemical analysis demonstrates high mitochondrial proton leak catalysed by an uncoupling protein 1 ortholog. Strikingly, bioenergetic profiling of tenrec uncoupling protein 1 reveals similar thermogenic potency as modern mouse uncoupling protein 1, despite the large phylogenetic distance. The discovery of functional brown adipose tissue in this ‘protoendothermic’ mammal links nonshivering thermogenesis directly to the roots of eutherian evolution, suggesting physiological importance prior to sustained body temperatures and migration to the cold. PMID:23860571

Insights into the selective binding and toxic mechanism of microcystin to catalase

NASA Astrophysics Data System (ADS)

Hu, Yuandong; Da, Liangjun

2014-03-01

Microcystin is a sort of cyclic nonribosomal peptides produced by cyanobacteria. It is cyanotoxin, which can be very toxic for plants and animals including humans. The present study evaluated the interaction of microcystin and catalase, under physiological conditions by means of fluorescence, three-dimensional (3D) fluorescence, circular dichroism (CD), Fourier Transform infrared (FT-IR) spectroscopy, and enzymatic reactionkinetic techniques. The fluorescence data showed that microcystin could bind to catalase to form a complex. The binding process was a spontaneous molecular interaction procedure, in which electrostatic interactions played a major role. Energy transfer and fluorescence studies proved the existence of a static binding process. Additionally, as shown by the three-dimensional fluorescence, CD and FT-IR results, microcystin could lead to conformational and microenvironmental changes of the protein, which may affect the physiological functions of catalase. The work provides important insights into the toxicity mechanism of microcystin in vivo.

Local pruning of dendrites and spines by caspase-3-dependent and proteasome-limited mechanisms.

PubMed

Ertürk, Ali; Wang, Yuanyuan; Sheng, Morgan

2014-01-29

Synapse loss occurs normally during development and pathologically during neurodegenerative disease. Long-term depression, a proposed physiological correlate of synapse elimination, requires caspase-3 and the mitochondrial pathway of apoptosis. Here, we show that caspase-3 activity is essential--and can act locally within neurons--for regulation of spine density and dendrite morphology. By photostimulation of Mito-KillerRed, we induced caspase-3 activity in defined dendritic regions of cultured neurons. Within the photostimulated region, local elimination of dendritic spines and dendrite retraction occurred in a caspase-3-dependent manner without inducing cell death. However, pharmacological inhibition of inhibitor of apoptosis proteins or proteasome function led to neuronal death, suggesting that caspase activation is spatially restricted by these "molecular brakes" on apoptosis. Caspase-3 knock-out mice have increased spine density and altered miniature EPSCs, confirming a physiological involvement of caspase-3 in the regulation of spines in vivo.

Biophysical characterization of α-synuclein and its controversial structure

PubMed Central

Alderson, T Reid; Markley, John L

2013-01-01

α-synuclein, a presynaptic protein of poorly defined function, constitutes the main component of Parkinson disease-associated Lewy bodies. Extensive biophysical investigations have provided evidence that isolated α-synuclein is an intrinsically disordered protein (IDP) in vitro. Subsequently serving as a model IDP in numerous studies, α-synuclein has aided in the development of many technologies used to characterize IDPs and arguably represents the most thoroughly analyzed IDP to date. Recent reports, however, have challenged the disordered nature of α-synuclein inside cells and have instead proposed a physiologically relevant helical tetramer. Despite α-synuclein’s rich biophysical history, a single coherent picture has not yet emerged concerning its in vivo structure, dynamics, and physiological role(s). We present herein a review of the biophysical discoveries, developments, and models pertinent to the characterization of α-synuclein’s structure and analysis of the native tetramer controversy. PMID:24634806

Polyamine oxidase activity in rats treated with mitoguazone: specific and permanent decrease in thymus.

PubMed

Ferioli, M E; Armanni, A

2003-01-01

To extend the knowledge on the role of polyamine oxidase in thymus physiology, we evaluated the in vivo effect of the polyamine biosynthetic pathway inhibitor mitoguazone. The drug markedly and permanently decreased the enzyme activity in the organ, in which the level of putrescine also decreased at the later times observed. A byproduct of the reaction catalyzed by polyamine oxidase is hydrogen peroxide, a well known inducer of apoptosis. The decrease in polyamine oxidase activity, with the consequent decrease in hydrogen peroxide production, is correlated with a positive effect on thymus physiology. Since mitoguazone has been successfully employed in patients with AIDS-related diseases, in which the reconstitution of the immune function is a favorable prognostic index, we hypothesized that mitoguazone may have the thymus as target organ, and that the decrease in polyamine oxidase activity may have a role in the positive effect of the drug.

Expression of human olfactory receptor 10J5 in heart aorta, coronary artery, and endothelial cells and its functional role in angiogenesis.

PubMed

Kim, Sung-Hee; Yoon, Yeo Cho; Lee, Ae Sin; Kang, NaNa; Koo, JaeHyung; Rhyu, Mee-Ra; Park, Jae-Ho

2015-05-01

ORs are ectopically expressed in non-chemosensory tissues including muscle, kidney, and keratinocytes; however, their physiological roles are largely unknown. We found that human olfactory receptor 10J5 (OR10J5) is expressed in the human aorta, coronary artery, and umbilical vein endothelial cells (HUVEC). Lyral induces Ca(2+) and phosphorylation of AKT in HUVEC. A knockdown study showed the inhibition of the lyral-induced Ca(2+) and the phosphorylation AKT and implied that these processes are mediated by OR10J5. In addition, lyral enhanced migration of HUVEC, which were also inhibited by RNAi in a migration assay. In addition, matrigel plug assay showed that lyral enhanced angiogenesis in vivo. Together these data demonstrate the physiological role of OR10J5 in angiogenesis and represent roles of ORs in HUVEC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Cadherins and Their Partners in the Nematode Worm Caenorhabditis elegans

PubMed Central

Hardin, Jeff; Lynch, Allison; Loveless, Timothy; Pettitt, Jonathan

2018-01-01

The extreme simplicity of Caenorhabditis elegans makes it an ideal system to study the basic principles of cadherin function at the level of single cells within the physiologically relevant context of a developing animal. The genetic tractability of C. elegans also means that components of cadherin complexes can be identified through genetic modifier screens, allowing a comprehensive in vivo characterization of the macromolecular assemblies involved in cadherin function during tissue formation and maintenance in C. elegans. This work shows that a single cadherin system, the classical cadherin–catenin complex, is essential for diverse morphogenetic events during embryogenesis through its interactions with a range of mostly conserved proteins that act to modulate its function. The role of other members of the cadherin family in C. elegans, including members of the Fat-like, Flamingo/CELSR and calsyntenin families is less well characterized, but they have clear roles in neuronal development and function. PMID:23481198

Extracellular matrix and its receptors in Drosophila neural development

PubMed Central

Broadie, Kendal; Baumgartner, Stefan; Prokop, Andreas

2011-01-01

Extracellular matrix (ECM) and matrix receptors are intimately involved in most biological processes. The ECM plays fundamental developmental and physiological roles in health and disease, including processes underlying the development, maintenance and regeneration of the nervous system. To understand the principles of ECM-mediated functions in the nervous system, genetic model organisms like Drosophila provide simple, malleable and powerful experimental platforms. This article provides an overview of ECM proteins and receptors in Drosophila. It then focuses on their roles during three progressive phases of neural development: 1) neural progenitor proliferation, 2) axonal growth and pathfinding and 3) synapse formation and function. Each section highlights known ECM and ECM-receptor components and recent studies done in mutant conditions to reveal their in vivo functions, all illustrating the enormous opportunities provided when merging work on the nervous system with systematic research into ECM-related gene functions. PMID:21688401

GRASP1 regulates synaptic plasticity and learning through endosomal recycling of AMPA receptors

PubMed Central

Chiu, Shu-Ling; Diering, Graham Hugh; Ye, Bing; Takamiya, Kogo; Chen, Chih-Ming; Jiang, Yuwu; Niranjan, Tejasvi; Schwartz, Charles E.; Wang, Tao; Huganir, Richard L.

2017-01-01

Summary Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects on AMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescues spine loss in hippocampal CA1 neurons of Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity in vivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders. PMID:28285821

Relevance of deprivation studies in understanding rapid eye movement sleep

PubMed Central

Mehta, Rachna; Khan, Shafa; Mallick, Birendra N

2018-01-01

Rapid eye movement sleep (REMS) is a unique phenomenon essential for maintaining normal physiological processes and is expressed at least in species higher in the evolution. The basic scaffold of the neuronal network responsible for REMS regulation is present in the brainstem, which may be directly or indirectly influenced by most other physiological processes. It is regulated by the neurons in the brainstem. Various manipulations including chemical, elec-trophysiological, lesion, stimulation, behavioral, ontogenic and deprivation studies have been designed to understand REMS genesis, maintenance, physiology and functional significance. Although each of these methods has its significance and limitations, deprivation studies have contributed significantly to the overall understanding of REMS. In this review, we discuss the advantages and limitations of various methods used for REMS deprivation (REMSD) to understand neural regulation and physiological significance of REMS. Among the deprivation strategies, the flowerpot method is by far the method of choice because it is simple and convenient, exploits physiological parameter (muscle atonia) for REMSD and allows conducting adequate controls to overcome experimental limitations as well as to rule out nonspecific effects. Notwithstanding, a major criticism that the flowerpot method faces is that of perceived stress experienced by the experimental animals. Nevertheless, we conclude that like most methods, particularly for in vivo behavioral studies, in spite of a few limitations, given the advantages described above, the flowerpot method is the best method of choice for REMSD studies. PMID:29881316

Relevance of deprivation studies in understanding rapid eye movement sleep.

PubMed

Mehta, Rachna; Khan, Shafa; Mallick, Birendra N

2018-01-01

Rapid eye movement sleep (REMS) is a unique phenomenon essential for maintaining normal physiological processes and is expressed at least in species higher in the evolution. The basic scaffold of the neuronal network responsible for REMS regulation is present in the brainstem, which may be directly or indirectly influenced by most other physiological processes. It is regulated by the neurons in the brainstem. Various manipulations including chemical, elec-trophysiological, lesion, stimulation, behavioral, ontogenic and deprivation studies have been designed to understand REMS genesis, maintenance, physiology and functional significance. Although each of these methods has its significance and limitations, deprivation studies have contributed significantly to the overall understanding of REMS. In this review, we discuss the advantages and limitations of various methods used for REMS deprivation (REMSD) to understand neural regulation and physiological significance of REMS. Among the deprivation strategies, the flowerpot method is by far the method of choice because it is simple and convenient, exploits physiological parameter (muscle atonia) for REMSD and allows conducting adequate controls to overcome experimental limitations as well as to rule out nonspecific effects. Notwithstanding, a major criticism that the flowerpot method faces is that of perceived stress experienced by the experimental animals. Nevertheless, we conclude that like most methods, particularly for in vivo behavioral studies, in spite of a few limitations, given the advantages described above, the flowerpot method is the best method of choice for REMSD studies.

Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins.

PubMed

Tiede, L M; Cook, E A; Morsey, B; Fox, H S

2011-12-22

Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1-11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons.

Three-Dimensional Cell Culture Models for Infectious Disease and Drug Development

NASA Technical Reports Server (NTRS)

Nickerson, Cheryl A.; Honer zu Bentrup, Kerstin; Ott, C. Mark

2005-01-01

Three-dimensional (3-D) cell cultures hold enormous potential to advance our understanding of infectious disease and to effectively translate basic cellular research into clinical applications. Using novel NASA bioreactor technology, the rotating wall vessel (RWV), we have engineered physiologically relevant 3-D human tissue culture models for infectious disease studies. The design of the RWV is based on the understanding that organs and tissues function in a 3-D environment, and that this 3-D architecture is critical for the differentiated form and function of tissues in vivo. The RWV provides large numbers of cells which are amenable to a wide variety of experimental manipulations and provides an easy, reproducible, and cost-effective approach to enhance differentiated features of cell culture models.

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

PubMed Central

Bazzini, Ariel A; Johnstone, Timothy G; Christiano, Romain; Mackowiak, Sebastian D; Obermayer, Benedikt; Fleming, Elizabeth S; Vejnar, Charles E; Lee, Miler T; Rajewsky, Nikolaus; Walther, Tobias C; Giraldez, Antonio J

2014-01-01

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo. PMID:24705786

Metals and Breast Cancer

PubMed Central

Byrne, Celia; Divekar, Shailaja D.; Storchan, Geoffrey B.; Parodi, Daniela A.; Martin, Mary Beth

2014-01-01

Metalloestrogens are metals that activate the estrogen receptor in the absence of estradiol. The metalloestrogens fall into two subclasses: metal/metalloid anions and bivalent cationic metals. The metal/metalloid anions include compounds such as arsenite, nitrite, selenite, and vanadate while the bivalent cations include metals such as cadmium, calcium, cobalt, copper, nickel, chromium, lead, mercury, and tin. The best studied metalloestrogen is cadmium. It is a heavy metal and a prevalent environmental contaminant with no known physiological function. This review addresses our current understanding of the mechanism by which cadmium and the bivalent cationic metals activate estrogen receptor-α. The review also summarizes the in vitro and in vivo evidence that cadmium functions as an estrogen and the potential role of cadmium in breast cancer. PMID:23338949

Neuroscience and education.

PubMed

Goswami, Usha

2004-03-01

Neuroscience is a relatively new discipline encompassing neurology, psychology and biology. It has made great strides in the last 100 years, during which many aspects of the physiology, biochemistry, pharmacology and structure of the vertebrate brain have been understood. Understanding of some of the basic perceptual, cognitive, attentional, emotional and mnemonic functions is also making progress, particularly since the advent of the cognitive neurosciences, which focus specifically on understanding higher level processes of cognition via imaging technology. Neuroimaging has enabled scientists to study the human brain at work in vivo, deepening our understanding of the very complex processes underpinning speech and language, thinking and reasoning, reading and mathematics. It seems timely, therefore, to consider how we might implement our increased understanding of brain development and brain function to explore educational questions.

Pathways of the Maillard reaction under physiological conditions.

PubMed

Henning, Christian; Glomb, Marcus A

2016-08-01

Initially investigated as a color formation process in thermally treated foods, nowadays, the relevance of the Maillard reaction in vivo is generally accepted. Many chronic and age-related diseases such as diabetes, uremia, atherosclerosis, cataractogenesis and Alzheimer's disease are associated with Maillard derived advanced glycation endproducts (AGEs) and α-dicarbonyl compounds as their most important precursors in terms of reactivity and abundance. However, the situation in vivo is very challenging, because Maillard chemistry is paralleled by enzymatic reactions which can lead to both, increases and decreases in certain AGEs. In addition, mechanistic findings established under the harsh conditions of food processing might not be valid under physiological conditions. The present review critically discusses the relevant α-dicarbonyl compounds as central intermediates of AGE formation in vivo with a special focus on fragmentation pathways leading to formation of amide-AGEs.

Optical Drug Monitoring: Photoacoustic Imaging of Nanosensors to Monitor Therapeutic Lithium In Vivo

PubMed Central

Cash, Kevin J.; Li, Chiye; Xia, Jun; Wang, Lihong V.; Clark, Heather A.

2015-01-01

Personalized medicine could revolutionize how primary care physicians treat chronic disease and how researchers study fundamental biological questions. To realize this goal we need to develop more robust, modular tools and imaging approaches for in vivo monitoring of analytes. In this report, we demonstrate that synthetic nanosensors can measure physiologic parameters with photoacoustic contrast, and we apply that platform to continuously track lithium levels in vivo. Photoacoustic imaging achieves imaging depths that are unattainable with fluorescence or multiphoton microscopy. We validated the photoacoustic results that illustrate the superior imaging depth and quality of photoacoustic imaging with optical measurements. This powerful combination of techniques will unlock the ability to measure analyte changes in deep tissue and will open up photoacoustic imaging as a diagnostic tool for continuous physiological tracking of a wide range of analytes. PMID:25588028

Optical drug monitoring: photoacoustic imaging of nanosensors to monitor therapeutic lithium in vivo.

PubMed

Cash, Kevin J; Li, Chiye; Xia, Jun; Wang, Lihong V; Clark, Heather A

2015-02-24

Personalized medicine could revolutionize how primary care physicians treat chronic disease and how researchers study fundamental biological questions. To realize this goal, we need to develop more robust, modular tools and imaging approaches for in vivo monitoring of analytes. In this report, we demonstrate that synthetic nanosensors can measure physiologic parameters with photoacoustic contrast, and we apply that platform to continuously track lithium levels in vivo. Photoacoustic imaging achieves imaging depths that are unattainable with fluorescence or multiphoton microscopy. We validated the photoacoustic results that illustrate the superior imaging depth and quality of photoacoustic imaging with optical measurements. This powerful combination of techniques will unlock the ability to measure analyte changes in deep tissue and will open up photoacoustic imaging as a diagnostic tool for continuous physiological tracking of a wide range of analytes.

Developmental and Life-Stage Physiologically-Based Pharmacokinetic (PBPK) Models in Humans and Animal Models.

EPA Science Inventory

PBPK models provide a computational framework for incorporating pertinent physiological and biochemical information to estimate in vivo levels of xenobiotics in biological tissues. In general, PBPK models are used to correlate exposures to target tissue levels of chemicals and th...

Dose-dependent DNA adduct formation by cinnamaldehyde and other food-borne α,β-unsaturated aldehydes predicted by physiologically based in silico modelling.

PubMed

Kiwamoto, R; Ploeg, D; Rietjens, I M C M; Punt, A

2016-03-01

Genotoxicity of α,β-unsaturated aldehydes shown in vitro raises a concern for the use of the aldehydes as food flavourings, while at low dose exposures the formation of DNA adducts may be prevented by detoxification. Unlike many α,β-unsaturated aldehydes for which in vivo data are absent, cinnamaldehyde was shown to be not genotoxic or carcinogenic in vivo. The present study aimed at comparing dose-dependent DNA adduct formation by cinnamaldehyde and 18 acyclic food-borne α,β-unsaturated aldehydes using physiologically based kinetic/dynamic (PBK/D) modelling. In rats, cinnamaldehyde was predicted to induce higher DNA adducts levels than 6 out of the 18 α,β-unsaturated aldehydes, indicating that these 6 aldehydes may also test negative in vivo. At the highest cinnamaldehyde dose that tested negative in vivo, cinnamaldehyde was predicted to form at least three orders of magnitude higher levels of DNA adducts than the 18 aldehydes at their respective estimated daily intake. These results suggest that for all the 18 α,β-unsaturated aldehydes DNA adduct formation at doses relevant for human dietary exposure may not raise a concern. The present study illustrates a possible use of physiologically based in silico modelling to facilitate a science-based comparison and read-across on the possible risks posed by DNA reactive agents. Copyright © 2015 Elsevier B.V. All rights reserved.

Chitosan coated carbon fiber microelectrode for selective in vivo detection of neurotransmitters in live zebrafish embryos

PubMed Central

Özel, Rıfat Emrah; Wallace, Kenneth N.; Andreescu, Silvana

2011-01-01

We report the development of a chitosan modified carbon fiber microelectrode for in vivo detection of serotonin. We find that chitosan has the ability to reject physiological levels of ascorbic acid interferences and facilitate selective and sensitive detection of in vivo levels of serotonin, a common catecholamine neurotransmitter. Presence of chitosan on the microelectrode surface was investigated using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The electrode was characterized using differential pulse voltammetry (DPV). A detection limit of 1.6 nM serotonin with a sensitivity of 5.12 nA/µM, a linear range from 2 to 100 nM and a reproducibility of 6.5 % for n=6 electrodes were obtained. Chitosan modified microelectrodes selectively measure serotonin in presence of physiological levels of ascorbic acid. In vivo measurements were performed to measure concentration of serotonin in the live embryonic zebrafish intestine. The sensor quantifies in vivo intestinal levels of serotonin while successfully rejecting ascorbic acid interferences. We demonstrate that chitosan can be used as an effective coating to reject ascorbic acid interferences at carbon fiber microelectrodes, as an alternative to Nafion, and that chitosan modified microelectrodes are reliable tools for in vivo monitoring of changes in neurotransmitter levels. PMID:21601035

Physical limits to autofluorescence signals in vivo recordings in the rat olfactory bulb: a Monte Carlo study

NASA Astrophysics Data System (ADS)

L'Heureux, B.; Gurden, H.; Pinot, L.; Mastrippolito, R.; Lefebvre, F.; Lanièce, P.; Pain, F.

2007-07-01

Understanding the cellular mechanisms of energy supply to neurons following physiological activation is still challenging and has strong implications to the interpretation of clinical functional images based on metabolic signals such as Blood Oxygen Level Dependent Magnetic Resonance Imaging or 18F-Fluorodexoy-Glucose Positron Emission Tomography. Intrinsic Optical Signal Imaging provides with high spatio temporal resolution in vivo imaging in the anaesthetized rat. In that context, intrinsic signals are mainly related to changes in the optical absorption of haemoglobin depending on its oxygenation state. This technique has been validated for imaging of the rat olfactory bulb, providing with maps of the actived olfactory glomeruli, the functional modules involved in the first step of olfactory coding. A complementary approach would be autofluorescence imaging relying on the fluorescence properties of endogenous Flavin Adenine Dinucleotide (FAD) or Nicotinamide Adenine Dinucleotide (NADH) both involved in intracellular metabolic pathways. The purpose of the present study was to investigate the feasibility of in vivo autofluorescence imaging in the rat olfactory bulb. We performed standard Monte Carlo simulations of photons scattering and absorption at the excitation and emission wavelengths of FAD and NADH fluorescence. Characterization of the fluorescence distribution in the glomerulus, effect of hemoglobin absorption at the excitation and absorption wavelengths as well as the effect of the blurring due to photon scattering and the depth of focus of the optical apparatus have been studied. Finally, optimal experimental parameters are proposed to achieve in vivo validation of the technique in the rat olfactory bulb.

Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo.

PubMed

Hirai, Yasuharu; Nishino, Eri; Ohmori, Harunori

2015-06-01

Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices. Copyright © 2015 the American Physiological Society.

Neutralization of Nogo-A Enhances Synaptic Plasticity in the Rodent Motor Cortex and Improves Motor Learning in Vivo

PubMed Central

Weinmann, Oliver; Kellner, Yves; Yu, Xinzhu; Vicente, Raul; Gullo, Miriam; Kasper, Hansjörg; Lussi, Karin; Ristic, Zorica; Luft, Andreas R.; Rioult-Pedotti, Mengia; Zuo, Yi; Zagrebelsky, Marta; Schwab, Martin E.

2014-01-01

The membrane protein Nogo-A is known as an inhibitor of axonal outgrowth and regeneration in the CNS. However, its physiological functions in the normal adult CNS remain incompletely understood. Here, we investigated the role of Nogo-A in cortical synaptic plasticity and motor learning in the uninjured adult rodent motor cortex. Nogo-A and its receptor NgR1 are present at cortical synapses. Acute treatment of slices with function-blocking antibodies (Abs) against Nogo-A or against NgR1 increased long-term potentiation (LTP) induced by stimulation of layer 2/3 horizontal fibers. Furthermore, anti-Nogo-A Ab treatment increased LTP saturation levels, whereas long-term depression remained unchanged, thus leading to an enlarged synaptic modification range. In vivo, intrathecal application of Nogo-A-blocking Abs resulted in a higher dendritic spine density at cortical pyramidal neurons due to an increase in spine formation as revealed by in vivo two-photon microscopy. To investigate whether these changes in synaptic plasticity correlate with motor learning, we trained rats to learn a skilled forelimb-reaching task while receiving anti-Nogo-A Abs. Learning of this cortically controlled precision movement was improved upon anti-Nogo-A Ab treatment. Our results identify Nogo-A as an influential molecular modulator of synaptic plasticity and as a regulator for learning of skilled movements in the motor cortex. PMID:24966370

In Vivo Evaluation and Proof of Radiofrequency Safety of a Novel Diagnostic MR-Electrophysiology Catheter

PubMed Central

Weiss, Steffen; Wirtz, Daniel; David, Bernd; Krueger, Sascha; Lips, Oliver; Caulfield, Dennis; Pedersen, Steen Fjord; Bostock, Julian; Razavi, Reza; Schaeffter, Tobias

2013-01-01

An MR-electrophysiology (EP) catheter is presented that provides full diagnostic EP functionality and a high level of radiofrequency safety achieved by custom-designed transmission lines. Highly resistive wires transmit intracardiac electrograms and currents for intracardiac pacing. A transformer cable transmits the localization signal of a tip coil. Specific absorption rate simulations and temperature measurements at 1.5 T demonstrate that a wire resistance > 3 kΩ/m limits dielectric heating to a physiologically irrelevant level. Additional wires do not increase tip specific absorption rate significantly, which is important because some clinical catheters require up to 20 electrodes. It is further demonstrated that radiofrequency-induced and pacing-induced resistive heating of the wires is negligible under clinical conditions. The MR-EP catheters provided uncompromised recording of electrograms and cardiac pacing in combination with a standard EP recorder in MR-guided in vivo EP studies, and the tip coil enabled fast and robust catheter localization. In vivo temperature measurements during such a study did not detect any device-related heating, which confirms the high level of safety of the catheter, whereas unacceptable heating was found with a standard EP catheter. The presented concept for the first time enables catheters with full diagnostic EP functionality and active tracking and at the same time a sufficient level of radiofrequency safety for MRI without specific absorption rate-related limitations. PMID:21337409

HIF2α reduces growth rate but promotes angiogenesis in a mouse model of neuroblastoma

PubMed Central

Favier, Judith; Lapointe, Stéphanie; Maliba, Ricardo; Sirois, Martin G

2007-01-01

Background HIF2α/EPAS1 is a hypoxia-inducible transcription factor involved in catecholamine homeostasis, vascular remodelling, physiological angiogenesis and adipogenesis. It is overexpressed in many cancerous tissues, but its exact role in tumour progression remains to be clarified. Methods In order to better establish its function in tumourigenesis and tumour angiogenesis, we have stably transfected mouse neuroblastoma N1E-115 cells with the native form of HIF2α or with its dominant negative mutant, HIF2α (1–485) and studied their phenotype in vitro and in vivo. Results In vitro studies reveal that HIF2α induces neuroblastoma cells hypertrophy and decreases their proliferation rate, while its inactivation by the HIF2α (1–485) mutant leads to a reduced cell size, associated with an accelerated proliferation. However, our in vivo experiments show that subcutaneous injection of cells overexpressing HIF2α into syngenic mice, leads to the formation of tumour nodules that grow slower than controls, but that are well structured and highly vascularized. In contrast, HIF2α (1–485)-expressing neuroblastomas grow fast, but are poorly vascularized and quickly tend to extended necrosis. Conclusion Together, our data reveal an unexpected combination between an antiproliferative and a pro-angiogenic function of HIF2α that actually seems to be favourable to the establishment of neuroblastomas in vivo. PMID:17655754

Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo.

PubMed

Schnoor, Michael; Lai, Frank P L; Zarbock, Alexander; Kläver, Ruth; Polaschegg, Christian; Schulte, Dörte; Weich, Herbert A; Oelkers, J Margit; Rottner, Klemens; Vestweber, Dietmar

2011-08-01

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of β(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

In vivo evaluation and proof of radiofrequency safety of a novel diagnostic MR-electrophysiology catheter.

PubMed

Weiss, Steffen; Wirtz, Daniel; David, Bernd; Krueger, Sascha; Lips, Oliver; Caulfield, Dennis; Pedersen, Steen Fjord; Bostock, Julian; Razavi, Reza; Schaeffter, Tobias

2011-03-01

An MR-electrophysiology (EP) catheter is presented that provides full diagnostic EP functionality and a high level of radiofrequency safety achieved by custom-designed transmission lines. Highly resistive wires transmit intracardiac electrograms and currents for intracardiac pacing. A transformer cable transmits the localization signal of a tip coil. Specific absorption rate simulations and temperature measurements at 1.5 T demonstrate that a wire resistance > 3 kΩ/m limits dielectric heating to a physiologically irrelevant level. Additional wires do not increase tip specific absorption rate significantly, which is important because some clinical catheters require up to 20 electrodes. It is further demonstrated that radiofrequency-induced and pacing-induced resistive heating of the wires is negligible under clinical conditions. The MR-EP catheters provided uncompromised recording of electrograms and cardiac pacing in combination with a standard EP recorder in MR-guided in vivo EP studies, and the tip coil enabled fast and robust catheter localization. In vivo temperature measurements during such a study did not detect any device-related heating, which confirms the high level of safety of the catheter, whereas unacceptable heating was found with a standard EP catheter. The presented concept for the first time enables catheters with full diagnostic EP functionality and active tracking and at the same time a sufficient level of radiofrequency safety for MRI without specific absorption rate-related limitations. Copyright © 2010 Wiley-Liss, Inc.

Resistance of chemokine receptor 6-deficient mice to Yersinia enterocolitica infection: evidence of defective M-cell formation in vivo.

PubMed

Westphal, Sabine; Lügering, Andreas; von Wedel, Julia; von Eiff, Christof; Maaser, Christian; Spahn, Thomas; Heusipp, Gerhard; Schmidt, M Alexander; Herbst, Hermann; Williams, Ifor R; Domschke, Wolfram; Kucharzik, Torsten

2008-03-01

M cells, specialized cells within Peyer's patches (PPs), are reduced in number in chemokine receptor 6 (CCR6)-deficient mice. The pathogenic microorganism Yersinia enterocolitica exploits M cells for the purpose of mucosal tissue invasion exclusively through PPs. The aim of this study was to evaluate the course of yersiniosis in CCR6-deficient mice and to investigate whether these mice might be used as an in vivo model to determine M-cell function. After oral challenge with Y. enterocolitica, control mice suffered from lethal septic infection whereas CCR6-deficient mice showed very limited symptoms of infection. Immunohistochemical analysis demonstrated PP invasion by Y. enterocolitica in control mice whereas no bacteria could be found in CCR6-deficient mice. In addition, a significant induction of proinflammatory cytokines could be found in control mice whereas proinflammatory cytokine levels in CCR6-deficient mice remained unchanged. In contrast, intraperitoneal infection resulted in severe systemic yersiniosis in both mouse groups. Abrogated oral Y. enterocolitica infection in CCR6-deficient mice demonstrates the importance of CCR6 expression in the physiological and pathological immune responses generated within PPs by influencing M-cell differentiation, underscoring the important role of M cells in the process of microbial uptake. CCR6-deficient mice may therefore represent a suitable model for the study of M-cell function in vivo.

An In Vivo Photo-Cross-Linking Approach Reveals a Homodimerization Domain of Aha1 in S. cerevisiae

PubMed Central

Berg, Michael; Michalowski, Annette; Palzer, Silke; Rupp, Steffen; Sohn, Kai

2014-01-01

Protein-protein interactions play an essential role in almost any biological processes. Therefore, there is a particular need for methods which describe the interactions of a defined target protein in its physiological context. Here we report a method to photo-cross-link interacting proteins in S. cerevisiae by using the non-canonical amino acid p-azido-L-phenylalanine (pAzpa). Based on the expanded genetic code the photoreactive non-canonical amino acid pAzpa was site-specifically incorporated at eight positions into a domain of Aha1 that was previously described to bind Hsp90 in vitro to function as a cochaperone of Hsp90 and activates its ATPase activity. In vivo photo-cross-linking to the cognate binding partner of Aha1 was carried out by irradiation of mutant strains with UV light (365 nm) to induce covalent intermolecular bonds. Surprisingly, an interaction between Aha1 and Hsp90 was not detected, although, we could confirm binding of suppressed pAzpa containing Aha1 to Hsp90 by native co-immunoprecipitation. However, a homodimer consisting of two covalently crosslinked Aha1 monomers was identified by mass spectrometry. This homodimer could also be confirmed using p-benzoyl-L-phenylalanine, another photoreactive non-canonical amino acid. Crosslinking was highly specific as it was dependent on irradiation using UV light, the exact position of the non-canonical amino acid in the protein sequence as well as on the addition of the non-canonical amino acid to the growth medium. Therefore it seems possible that an interaction of Aha1 with Hsp90 takes place at different positions than previously described in vitro highlighting the importance of in vivo techniques to study protein-protein interactions. Accordingly, the expanded genetic code can easily be applied to other S. cerevisiae proteins to study their interaction under physiological relevant conditions in vivo. PMID:24614167

Mechanisms of EHD/RME-1 Protein Function in Endocytic Transport

PubMed Central

Grant, Barth D.; Caplan, Steve

2009-01-01

The evolutionarily conserved Eps15 homology domain (EHD)/receptor-mediated endocytosis (RME)-1 family of C-terminal EH domain proteins has recently come under intense scrutiny because of its importance in intracellular membrane transport, especially with regard to the recycling of receptors from endosomes to the plasma membrane. Recent studies have shed new light on the mode by which these adenosine triphosphatases function on endosomal membranes in mammals and Caenorhabditis elegans. This review highlights our current understanding of the physiological roles of these proteins in vivo, discussing conserved features as well as emerging functional differences between individual mammalian paralogs. In addition, these findings are discussed in light of the identification of novel EHD/RME-1 protein and lipid interactions and new structural data for proteins in this family, indicating intriguing similarities to the Dynamin superfamily of large guanosine triphosphatases. PMID:18801062

Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ

PubMed Central

Hirayama, Masatoshi; Ogawa, Miho; Oshima, Masamitsu; Sekine, Yurie; Ishida, Kentaro; Yamashita, Kentaro; Ikeda, Kazutaka; Shimmura, Shigeto; Kawakita, Tetsuya; Tsubota, Kazuo; Tsuji, Takashi

2013-01-01

The lacrimal gland has a multifaceted role in maintaining a homeostatic microenvironment for a healthy ocular surface via tear secretion. Dry-eye disease, which is caused by lacrimal gland dysfunction, is one of the most prevalent eye diseases that cause corneal epithelial damage and results in significant loss of vision and a reduction in the quality of life. Here we demonstrate orthotopic transplantation of bioengineered lacrimal gland germs into adult mice with an extra-orbital lacrimal gland defect, a mouse model that mimics the corneal epithelial damage caused by lacrimal gland dysfunction. The bioengineered lacrimal gland germs and harderian gland germs both develop in vivo and achieve sufficient physiological functionality, including tear production in response to nervous stimulation and ocular surface protection. This study demonstrates the potential for bioengineered organ replacement to functionally restore the lacrimal gland. PMID:24084941

In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease

PubMed Central

Cruzat, Andrea; Qazi, Yureeda; Hamrah, Pedram

2016-01-01

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to that of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, and proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases. PMID:27771327

A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

PubMed

Lechuga, Susana; Naydenov, Nayden G; Feygin, Alex; Jimenez, Antonio J; Ivanov, Andrei I

2017-05-13

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

A VESICLE TRAFFICKING PROTEIN αSNAP REGULATES PANETH CELL DIFFERENTIATION IN VIVO

PubMed Central

Lechuga, Susana; Naydenov, Nayden G.; Feygin, Alex; Jimenez, Antonio J.; Ivanov, Andrei I.

2017-01-01

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. PMID:28359759

On being the right size: scaling effects in designing a human-on-a-chip

PubMed Central

Moraes, Christopher; Labuz, Joseph M.; Leung, Brendan M.; Inoue, Mayumi; Chun, Tae-Hwa; Takayama, Shuichi

2013-01-01

Developing a human-on-a-chip by connecting multiple model organ systems would provide an intermediate screen for therapeutic efficacy and toxic side effects of drugs prior to conducting expensive clinical trials. However, correctly designing individual organs and scaling them relative to each other to make a functional microscale human analog is challenging, and a generalized approach has yet to be identified. In this work, we demonstrate the importance of rational design of both the individual organ and its relationship with other organs, using a simple two-compartment system simulating insulin-dependent glucose uptake in adipose tissues. We demonstrate that inter-organ scaling laws depend on both the number of cells, and on the spatial arrangement of those cells within the microfabricated construct. We then propose a simple and novel inter-organ ‘metabolically-supported functional scaling’ approach predicated on maintaining in vivo cellular basal metabolic rates, by limiting resources available to cells on the chip. This approach leverages findings from allometric scaling models in mammals that limited resources in vivo prompts cells to behave differently than in resource-rich in vitro cultures. Although applying scaling laws directly to tissues can result in systems that would be quite challenging to implement, engineering workarounds may be used to circumvent these scaling issues. Specific workarounds discussed include the limited oxygen carrying capacity of cell culture media when used as a blood substitute and the ability to engineer non-physiological structures to augment organ function, to create the transport-accessible, yet resource-limited environment necessary for cells to mimic in vivo functionality. Furthermore, designing the structure of individual tissues in each organ compartment may be a useful strategy to bypass scaling concerns at the inter-organ level. PMID:23925524

Effect of Different Formulations of Magnesium Chloride Used As Anesthetic Agents on the Performance of the Isolated Heart of Octopus vulgaris.

PubMed

Pugliese, Chiara; Mazza, Rosa; Andrews, Paul L R; Cerra, Maria C; Fiorito, Graziano; Gattuso, Alfonsina

2016-01-01

Magnesium chloride (MgCl 2 ) is commonly used as a general anesthetic in cephalopods, but its physiological effects including those at cardiac level are not well-characterized. We used an in vitro isolated perfused systemic heart preparation from the common octopus, Octopus vulgaris , to investigate: (a) if in vivo exposure to MgCl 2 formulations had an effect on cardiac function in vitro and, if so, could this impact recovery and (b) direct effects of MgCl 2 formulations on cardiac function. In vitro hearts removed from animals exposed in vivo to 3.5% MgCl 2 in sea water (20 min) or to a mixture of MgCl 2 + ethanol (1.12/1%; 20 min) showed cardiac function (heart rate, stroke volume, cardiac output) comparable to hearts removed from animals killed under hypothermia. However, 3.5% MgCl 2 (1:1, sea water: distilled water, 20 min) produced a significant impairment of the Frank-Starling response as did 45 min exposure to the MgCl 2 + ethanol mixture. Perfusion of the isolated heart with MgCl 2 ± ethanol formulations produced a concentration-related bradycardia (and arrest), a decreased stroke volume and cardiac output indicating a direct effect on the heart. The cardiac effects of MgCl 2 are discussed in relation to the involvement of magnesium, sodium, chloride, and calcium ions, exposure time and osmolality of the formulations and the implications for the use of various formulations of MgCl 2 as anesthetics in octopus. Overall, provided that the in vivo exposure to 3.5% MgCl 2 in sea water or to a mixture of MgCl 2 + ethanol is limited to ~20 min, residual effects on cardiac function are unlikely to impact post-anesthetic recovery.

Life-Stage Physiologically-Based Pharmacokinetic (PBPK) Model Applications to Screen Environmental Hazards.

EPA Science Inventory

This presentation discusses methods used to extrapolate from in vitro high-throughput screening (HTS) toxicity data for an endocrine pathway to in vivo for early life stages in humans, and the use of a life stage PBPK model to address rapidly changing physiological parameters. A...

Physiologic Levels of Endogenous Hydrogen Sulfide Maintain the Proliferation and Differentiation Capacity of Periodontal Ligament Stem Cells.

PubMed

Su, Yingying; Liu, Dayong; Liu, Yi; Zhang, Chunmei; Wang, Jinsong; Wang, Songlin

2015-11-01

Many invading oral bacteria are known to produce considerable amounts of hydrogen sulfide (H2S). The toxic activity of exogenous H2S in periodontal tissue has been demonstrated, but the role of endogenous H2S in the physiologic function of periodontal tissue remains poorly understood. The purpose of the present study is to investigate the biologic functions of H2S in the proliferation and differentiation of human periodontal ligament stem cells (PDLSCs). PDLSCs were isolated from periodontal ligament tissues of periodontally healthy volunteers or patients with periodontitis. Immunocytochemical staining, flow cytometry, and Western blot analysis were used to examine the expression of H2S-synthesizing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). The proliferation capacity of PDLSCs was determined by cell counting kit-8 assay, carboxyfluorescein succinimidyl ester analysis, and 5-ethynyl-2'-deoxyuridine assay. The osteogenic potential of PDLSCs was tested using alkaline phosphatase staining, Alizarin Red staining, and in vivo transplantation experiments. Oil Red O staining was used to analyze adipogenic ability. The results show that human PDLSCs express both CBS and CSE and produce H2S. Blocking the generation of endogenous H2S with CBS inhibitor hydroxylamine significantly attenuated PDLSC proliferation and reduced the osteogenic and adipogenic differentiation capacity of PDLSCs. In contrast, CSE inhibitor DL-propargylglycine had no effect on PDLSC function. Exogenous H2S could inhibit the production of endogenous H2S and impair PDLSC function in a dose-dependent manner. Physiologic levels of endogenous H2S maintain the proliferation and differentiation capacity of PDLSCs, and CBS may be the main source of endogenous H2S in PDLSCs.

Stimulatory effects of calcium on respiration and NAD(P)H synthesis in intact rat heart mitochondria utilizing physiological substrates cannot explain respiratory control in vivo.

PubMed

Vinnakota, Kalyan C; Dash, Ranjan K; Beard, Daniel A

2011-09-02

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca(2+) under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca(2+) in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca(2+) can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca(2+) concentration averaged per beat, Ca(2+) had no observable stimulatory effect. A modest apparent stimulatory effect (22-27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mM initial phosphate. The stimulatory effects observed over the physiological Ca(2+) range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.

Stimulatory Effects of Calcium on Respiration and NAD(P)H Synthesis in Intact Rat Heart Mitochondria Utilizing Physiological Substrates Cannot Explain Respiratory Control in Vivo*

PubMed Central

Vinnakota, Kalyan C.; Dash, Ranjan K.; Beard, Daniel A.

2011-01-01

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca2+ under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca2+ in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mm extramitochondrial free magnesium, Ca2+ can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca2+ concentration averaged per beat, Ca2+ had no observable stimulatory effect. A modest apparent stimulatory effect (22–27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mm initial phosphate. The stimulatory effects observed over the physiological Ca2+ range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo. PMID:21757763

[Study on material basis of essential oil from Yin Teng Gu Bi Kang prescription on activating blood circulation].

PubMed

Wang, Yuan-Qing; Yan, Jian-Ye; Gong, Li-Min; Luo, Kun; Li, Shun-Xiang; Yang, Yan-Tao; Xie, Yu

2014-08-01

To explore the component difference of the serum containing essential oil from Yin Teng Gu Bi Kang prescription in pathologic and physiologic rat models, and to reveal the material basis of its efficacy of activating blood circulation. The essential oils were obtained by CO2 supercritical fluid extraction and the ingredients of the essential oils in vitro and in vivo (under physiological and pathological status) were analyzed by GC-MS to compare differences of the essential oil under physiological and pathological status in rats. 32 components were identified with the main components of Z-ligustilide (39.23%) and d-limonene (21.7%) in the essential oil. In vivo analysis on the essential oil indicated that 16 components were identified, 7 existed originally in essential oil and 9 were metabolites under physiological status; while 22 components were identified, 10 existed originally in essential oil and 12 were metabolites under pathological status (acute blood stasis). There were 7 common prototypes and 8 common metabolites under different physiological status. The absorption and metabolism of essential oils were affected by blood stasis and the compounds migrating to blood may be the effective substance in activating blood circulation.

The impact of supersaturation level for oral absorption of BCS class IIb drugs, dipyridamole and ketoconazole, using in vivo predictive dissolution system: Gastrointestinal Simulator (GIS).

PubMed

Tsume, Yasuhiro; Matsui, Kazuki; Searls, Amanda L; Takeuchi, Susumu; Amidon, Gregory E; Sun, Duxin; Amidon, Gordon L

2017-05-01

The development of formulations and the assessment of oral drug absorption for Biopharmaceutical Classification System (BCS) class IIb drugs is often a difficult issue due to the potential for supersaturation and precipitation in the gastrointestinal (GI) tract. The physiological environment in the GI tract largely influences in vivo drug dissolution rates of those drugs. Thus, those physiological factors should be incorporated into the in vitro system to better assess in vivo performance of BCS class IIb drugs. In order to predict oral bioperformance, an in vitro dissolution system with multiple compartments incorporating physiologically relevant factors would be expected to more accurately predict in vivo phenomena than a one-compartment dissolution system like USP Apparatus 2 because, for example, the pH change occurring in the human GI tract can be better replicated in a multi-compartmental platform. The Gastrointestinal Simulator (GIS) consists of three compartments, the gastric, duodenal and jejunal chambers, and is a practical in vitro dissolution apparatus to predict in vivo dissolution for oral dosage forms. This system can demonstrate supersaturation and precipitation and, therefore, has the potential to predict in vivo bioperformance of oral dosage forms where this phenomenon may occur. In this report, in vitro studies were performed with dipyridamole and ketoconazole to evaluate the precipitation rates and the relationship between the supersaturation levels and oral absorption of BCS class II weak base drugs. To evaluate the impact of observed supersaturation levels on oral absorption, a study utilizing the GIS in combination with mouse intestinal infusion was conducted. Supersaturation levels observed in the GIS enhanced dipyridamole and ketoconazole absorption in mouse, and a good correlation between their supersaturation levels and their concentration in plasma was observed. The GIS, therefore, appears to represent in vivo dissolution phenomena and demonstrate supersaturation and precipitation of dipyridamole and ketoconazole. We therefore conclude that the GIS has been shown to be a good biopredictive tool to predict in vivo bioperformance of BCS class IIb drugs that can be used to optimize oral formulations. Copyright © 2017. Published by Elsevier B.V.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

PubMed Central

Perrin-Terrin, Anne-Sophie; Jeton, Florine; Pichon, Aurelien; Frugière, Alain; Richalet, Jean-Paul; Bodineau, Laurence; Voituron, Nicolas

2016-01-01

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive. PMID:27167092

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

PubMed Central

Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

2015-01-01

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

In vivo speciation studies and antioxidant properties of bromine in Laminaria digitata reinforce the significance of iodine accumulation for kelps

PubMed Central

Küpper, Frithjof C.; Carpenter, Lucy J.; Leblanc, Catherine; Toyama, Chiaki; Uchida, Yuka; Maskrey, Benjamin H.; Robinson, Joanne; Verhaeghe, Elodie F.; Malin, Gill; Luther, George W.; Kroneck, Peter M. H.; Kloareg, Bernard; Meyer-Klaucke, Wolfram; Muramatsu, Yasuyuki; Megson, Ian L.; Potin, Philippe; Feiters, Martin C.

2013-01-01

The metabolism of bromine in marine brown algae remains poorly understood. This contrasts with the recent finding that the accumulation of iodide in the brown alga Laminaria serves the provision of an inorganic antioxidant – the first case documented from a living system. The aim of this study was to use an interdisciplinary array of techniques to study the chemical speciation, transformation, and function of bromine in Laminaria and to investigate the link between bromine and iodine metabolism, in particular in the antioxidant context. First, bromine and iodine levels in different Laminaria tissues were compared by inductively coupled plasma MS. Using in vivo X-ray absorption spectroscopy, it was found that, similarly to iodine, bromine is predominantly present in this alga in the form of bromide, albeit at lower concentrations, and that it shows similar behaviour upon oxidative stress. However, from a thermodynamic and kinetic standpoint, supported by in vitro and reconstituted in vivo assays, bromide is less suitable than iodide as an antioxidant against most reactive oxygen species except superoxide, possibly explaining why kelps prefer to accumulate iodide. This constitutes the first-ever study exploring the potential antioxidant function of bromide in a living system and other potential physiological roles. Given the tissue-specific differences observed in the content and speciation of bromine, it is concluded that the bromide uptake mechanism is different from the vanadium iodoperoxidase-mediated uptake of iodide in L. digitata and that its function is likely to be complementary to the iodide antioxidant system for detoxifying superoxide. PMID:23606364

Liver Cytochrome P450 3A Ubiquitination in Vivo by gp78/Autocrine Motility Factor Receptor and C Terminus of Hsp70-interacting Protein (CHIP) E3 Ubiquitin Ligases

PubMed Central

Kim, Sung-Mi; Acharya, Poulomi; Engel, Juan C.; Correia, Maria Almira

2010-01-01

CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drug-drug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function. PMID:20819951

Bioengineered transplantable porcine livers with re-endothelialized vasculature.

PubMed

Ko, In Kap; Peng, Li; Peloso, Andrea; Smith, Charesa J; Dhal, Abritee; Deegan, Daniel B; Zimmerman, Cindy; Clouse, Cara; Zhao, Weixin; Shupe, Thomas D; Soker, Shay; Yoo, James J; Atala, Anthony

2015-02-01

Donor shortage remains a continued challenge in liver transplantation. Recent advances in tissue engineering have provided the possibility of creating functional liver tissues as an alternative to donor organ transplantation. Small bioengineered liver constructs have been developed, however a major challenge in achieving functional bioengineered liver in vivo is the establishment of a functional vasculature within the scaffolds. Our overall goal is to bioengineer intact livers, suitable for transplantation, using acellular porcine liver scaffolds. We developed an effective method for reestablishing the vascular network within decellularized liver scaffolds by conjugating anti-endothelial cell antibodies to maximize coverage of the vessel walls with endothelial cells. This procedure resulted in uniform endothelial attachment throughout the liver vasculature extending to the capillary bed of the liver scaffold and greatly reduced platelet adhesion upon blood perfusion in vitro. The re-endothelialized livers, when transplanted to recipient pigs, were able to withstand physiological blood flow and maintained for up to 24 h. This study demonstrates, for the first time, that vascularized bioengineered livers, of clinically relevant size, can be transplanted and maintained in vivo, and represents the first step towards generating engineered livers for transplantation to patients with end-stage liver failure. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vivo measurement of blood flow in the vitelline network

NASA Astrophysics Data System (ADS)

Poelma, Christian; Vennemann, Peter; Lindken, Ralph; Westerweel, Jerry

2007-11-01

The growth and adaptation of blood vessels is studied in vivo in the so-called vitelline network of a chick embryo. The vitelline network, a system of extra-embryonic blood vessels that transports nutrients from the yolk sac to the chick embryo, is an easily accessible model system for the study of human cardiovascular development and functioning. We present measurements obtained by means of scanning microscopic Particle Image Velocimetry. Using phase-locking, we can reconstruct the full three-dimensional flow as a function of the cardiac cycle. Typical reconstructed volumes are 0.4x0.5x0.2 mm^3 with a spatial resolution (i.e. vector spacing) of 6 μm. These hemodynamic measurements allow a study of the coupling between form and functioning of the blood vessels. Special attention is given to the local wall shear stress (WSS), an important physiological parameter that is thought to determine - to great extent - the adaptation of the vessels to changing conditions. The WSS can be estimated directly from the velocity gradient at the wall or from a fit to the blood velocity profile. The former method slightly underestimates the WSS (most likely due to lack of resolution) but is significantly easier to apply in the complex geometries under consideration.

Structure-function analysis of genetically defined neuronal populations.

PubMed

Groh, Alexander; Krieger, Patrik

2013-10-01

Morphological and functional classification of individual neurons is a crucial aspect of the characterization of neuronal networks. Systematic structural and functional analysis of individual neurons is now possible using transgenic mice with genetically defined neurons that can be visualized in vivo or in brain slice preparations. Genetically defined neurons are useful for studying a particular class of neurons and also for more comprehensive studies of the neuronal content of a network. Specific subsets of neurons can be identified by fluorescence imaging of enhanced green fluorescent protein (eGFP) or another fluorophore expressed under the control of a cell-type-specific promoter. The advantages of such genetically defined neurons are not only their homogeneity and suitability for systematic descriptions of networks, but also their tremendous potential for cell-type-specific manipulation of neuronal networks in vivo. This article describes a selection of procedures for visualizing and studying the anatomy and physiology of genetically defined neurons in transgenic mice. We provide information about basic equipment, reagents, procedures, and analytical approaches for obtaining three-dimensional (3D) cell morphologies and determining the axonal input and output of genetically defined neurons. We exemplify with genetically labeled cortical neurons, but the procedures are applicable to other brain regions with little or no alterations.

Ex vivo perfusion of human spleens maintains clearing and processing functions.

PubMed

Buffet, Pierre A; Milon, Geneviève; Brousse, Valentine; Correas, Jean-Michel; Dousset, Bertrand; Couvelard, Anne; Kianmanesh, Reza; Farges, Olivier; Sauvanet, Alain; Paye, François; Ungeheuer, Marie-Noëlle; Ottone, Catherine; Khun, Huot; Fiette, Laurence; Guigon, Ghislaine; Huerre, Michel; Mercereau-Puijalon, Odile; David, Peter H

2006-05-01

The spleen plays a central role in the pathophysiology of several potentially severe diseases such as inherited red cell membrane disorders, hemolytic anemias, and malaria. Research on these diseases is hampered by ethical constraints that limit human spleen tissue explorations. We identified a surgical situation--left splenopancreatectomy for benign pancreas tumors--allowing spleen retrieval at no risk for patients. Ex vivo perfusion of retrieved intact spleens for 4 to 6 hours maintained a preserved parenchymal structure, vascular flow, and metabolic activity. Function preservation was assessed by testing the ability of isolated-perfused spleens to retain Plasmodium falciparum-infected erythrocytes preexposed to the antimalarial drug artesunate (Art-iRBCs). More than 95% of Art-iRBCs were cleared from the perfusate in 2 hours. At each transit through isolated-perfused spleens, parasite remnants were removed from 0.2% to 0.23% of Art-iRBCs, a proportion consistent with the 0.02% to 1% pitting rate previously established in artesunate-treated patients. Histologic analysis showed that more than 90% of Art-iRBCs were retained and processed in the red pulp, providing the first direct evidence of a zone-dependent parasite clearance by the human spleen. Human-specific physiologic or pathophysiologic mechanisms involving clearing or processing functions of the spleen can now be experimentally explored in a human tissue context.

Obesity-induced oxidative stress, accelerated functional decline with age and increased mortality in mice

PubMed Central

Zhang, Yiqiang; Fischer, Kathleen E.; Soto, Vanessa; Liu, Yuhong; Sosnowska, Danuta; Richardson, Arlan; Salmon, Adam B.

2015-01-01

Obesity is a serious chronic disease that increases the risk of numerous co-morbidities including metabolic syndrome, cardiovascular disease and cancer as well as increases risk of mortality leading some to suggest this represents accelerated aging. Obesity is associated with significant increases in oxidative stress in vivo and, despite the well-explored relationship between oxidative stress and aging, the role this plays in the increased mortality of obese subjects remains an unanswered question. Here, we addressed this by undertaking a comprehensive, longitudinal study of a group of high fat-fed obese mice and assessed both their changes in oxidative stress and in their performance in physiological assays known to decline with aging. In female C57BL/6J mice fed a high-fat diet starting in adulthood, mortality was significantly increased in high fat-fed mice as was oxidative damage in vivo. High fat-feeding significantly accelerated the decline in performance in several assays, including activity, gait, and rotarod. However, we also found that obesity had little effect on other markers and actually improved performance in grip strength, a marker of muscular function. Together, this first comprehensive assessment of longitudinal functional changes in high fat-fed mice suggests that obesity may induce segmental acceleration of some of the aging process. PMID:25558793

Novel functional Renilla luciferase mutant provides long-term serum stability and high luminescence activity.

PubMed

Song, Woo Chul; Sung, Hye-Jin; Park, Kyung Soo; Choi, Jeong-Woo; Cho, Je-Yeol; Um, Soong Ho

2013-10-01

Fluorescent and luminescent chemical probes are essential in recent medical diagnostics. However, the use of these probes in vivo has raised concerns due to their low sensitivity, background signal interference, and non-biocompatibility. Therefore, biological chromophores have received much attention as new alternatives. In particular, luciferase, a class of oxidative enzyme with bioluminescence, has emerged as a promising fluorophore due to its improved biocompatibility. However, the enzyme usually possesses weaker luminescence and stability relative to its chemically-based competitors. Here, we report a novel functional mutant luciferase with both enhanced luminescence and long-term serum stability. For the preparation of the modified Renilla luciferase, a new bacterial subcloning design was established. The luciferase coding DNA sequence was redesigned so that mutant luciferase could be easily expressed in an Escherichia coli system. The mutant Renilla luciferase, which we called "m-Rluc," demonstrated characteristic enzymatic functions and showed a 5.6-fold increase in luminescence activity. In addition, the enzyme's physiological stability remained >80% for more than 5days, in contrast to conventional luciferase, termed "hrluc," which disappeared within a few hours. We suggest that this novel biological luciferase probe may be a great tool for both in vitro and in vivo medical diagnostics. Copyright © 2013 Elsevier Inc. All rights reserved.

In vivo observation of transient photoreceptor movement correlated with oblique light stimulation

NASA Astrophysics Data System (ADS)

Lu, Yiming; Liu, Changgeng; Yao, Xincheng

2018-02-01

Rod-dominated transient retinal phototropism (TRP) has been observed in freshly isolated retinas, promising a noninvasive biomarker for high resolution assessment of retinal physiology. However, in vivo mapping of TRP is challenging due to its fast time course and sub-cellular signal magnitude. By developing a line-scanning and virtually structured detection based super-resolution ophthalmoscope, we report here in vivo observation of TRP in frog retina. In vivo characterization of TRP time course and magnitude were implemented by using variable light stimulus intensities.

Assessment of the Role of MAP Kinase in Mediating Activity-Dependent Transcriptional Activation of the Immediate Early Gene "Arc/Arg3.1" in the Dentate Gyrus in Vivo

ERIC Educational Resources Information Center

Chotiner, Jennifer K.; Nielson, Jessica; Farris, Shannon; Lewandowski, Gail; Huang, Fen; Banos, Karla; de Leon, Ray; Steward, Oswald

2010-01-01

Different physiological and behavioral events activate transcription of "Arc/Arg3.1" in neurons in vivo, but the signal transduction pathways that mediate induction in particular situations remain to be defined. Here, we explore the relationships between induction of "Arc/Arg3.1" transcription in dentate granule cells in vivo and activation of…

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

PubMed Central

Naik, Meghna U.; Stalker, Timothy J.; Brass, Lawrence F.

2012-01-01

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation. PMID:22271446

Microfluidic cardiac cell culture model (μCCCM).

PubMed

Giridharan, Guruprasad A; Nguyen, Mai-Dung; Estrada, Rosendo; Parichehreh, Vahidreza; Hamid, Tariq; Ismahil, Mohamed Ameen; Prabhu, Sumanth D; Sethu, Palaniappan

2010-09-15

Physiological heart development and cardiac function rely on the response of cardiac cells to mechanical stress during hemodynamic loading and unloading. These stresses, especially if sustained, can induce changes in cell structure, contractile function, and gene expression. Current cell culture techniques commonly fail to adequately replicate physical loading observed in the native heart. Therefore, there is a need for physiologically relevant in vitro models that recreate mechanical loading conditions seen in both normal and pathological conditions. To fulfill this need, we have developed a microfluidic cardiac cell culture model (μCCCM) that for the first time allows in vitro hemodynamic stimulation of cardiomyocytes by directly coupling cell structure and function with fluid induced loading. Cells are cultured in a small (1 cm diameter) cell culture chamber on a thin flexible silicone membrane. Integrating the cell culture chamber with a pump, collapsible pulsatile valve and an adjustable resistance element (hemostatic valve) in series allow replication of various loading conditions experienced in the heart. This paper details the design, modeling, fabrication and characterization of fluid flow, pressure and stretch generated at various frequencies to mimic hemodynamic conditions associated with the normal and failing heart. Proof-of-concept studies demonstrate successful culture of an embryonic cardiomyoblast line (H9c2 cells) and establishment of an in vivo like phenotype within this system.

Crystal structure and potential physiological role of zebra fish thioesterase superfamily member 2 (fTHEM2)

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

Yu, Shanshan; Li, Han; Gao, Feng

2015-08-07

Thioesterase superfamily member 2 (THEM2) is an essential protein for mammalian cell proliferation. It belongs to the hotdog-fold thioesterase superfamily and catalyzes hydrolysis of thioester bonds of acyl-CoA in vitro, while its in vivo function remains unrevealed. In this study, Zebra fish was selected as a model organism to facilitate the investigations on THEM2. First, we solved the crystal structure of recombinant fTHEM2 at the resolution of 1.80 Å, which displayed a similar scaffolding as hTHEM2. Second, functional studies demonstrated that fTHEM2 is capable of hydrolyzing palmitoyl-CoA in vitro. In addition, injection of morpholino against fTHEM2 at one-cell stage resulted in distorted early embryomore » development, including delayed cell division, retarded development and increased death rate. The above findings validated our hypothesis that fTHEM2 could serve as an ideal surrogate for studying the physiological functions of THEM2. - Highlights: • The crystal structure of recombinant fTHEM2 is presented. • fTHEM2 is capable of hydrolyzing palmitoyl-CoA. • The influence of fTHEM2 on early embryo development is demonstrated.« less

Revascularization of decellularized lung scaffolds: principles and progress

PubMed Central

Stabler, Collin T.; Lecht, Shimon; Mondrinos, Mark J.; Goulart, Ernesto; Lazarovici, Philip

2015-01-01

There is a clear unmet clinical need for novel biotechnology-based therapeutic approaches to lung repair and/or replacement, such as tissue engineering of whole bioengineered lungs. Recent studies have demonstrated the feasibility of decellularizing the whole organ by removal of all its cellular components, thus leaving behind the extracellular matrix as a complex three-dimensional (3D) biomimetic scaffold. Implantation of decellularized lung scaffolds (DLS), which were recellularized with patient-specific lung (progenitor) cells, is deemed the ultimate alternative to lung transplantation. Preclinical studies demonstrated that, upon implantation in rodent models, bioengineered lungs that were recellularized with airway and vascular cells were capable of gas exchange for up to 14 days. However, the long-term applicability of this concept is thwarted in part by the failure of current approaches to reconstruct a physiologically functional, quiescent endothelium lining the entire vascular tree of reseeded lung scaffolds, as inferred from the occurrence of hemorrhage into the airway compartment and thrombosis in the vasculature in vivo. In this review, we explore the idea that successful whole lung bioengineering will critically depend on 1) preserving and/or reestablishing the integrity of the subendothelial basement membrane, especially of the ultrathin respiratory membrane separating airways and capillaries, during and following decellularization and 2) restoring vascular physiological functionality including the barrier function and quiescence of the endothelial lining following reseeding of the vascular compartment. We posit that physiological reconstitution of the pulmonary vascular tree in its entirety will significantly promote the clinical translation of the next generation of bioengineered whole lungs. PMID:26408553

Development of a Rat Model to Investigate Contributions of Anatomic and Physiologic Determinants of in Vivo Skin Permeation

DTIC Science & Technology

1991-08-30

physiologic states. Physiologic perturbations were performed to test the sensitivity of the model system to detect effects of minoxidil -mediated... Minoxidil , leukotriene D̂ , tape stripping of stratum corneum, and topical ether-ethanol experiments produced statistically significant increases (52 to... Minoxidil 68 B. Phenylephrine 69 C. Leukotriene D̂ 69 D. Ether-Ethanol 70 E. Tape stripping 76 8. Data analysis 77 Vlll RESULTS 80 1

Therapeutic perspectives for melatonin agonists and antagonists.

PubMed

Delagrange, P; Atkinson, J; Boutin, J A; Casteilla, L; Lesieur, D; Misslin, R; Pellissier, S; Pénicaud, L; Renard, P

2003-04-01

Melatonin is a neurohormone synthesized in the pineal gland during the dark period in all species, including humans. The diversity and differences in melatonin receptor distribution in the brain and extracerebral organs suggest multiple functional roles for melatonin. Administration of melatonin agonists reduces neophobia and treatment with a melatonin antagonist during the dark period reverses the anxiolytic-like effect of endogenous melatonin. Chronic treatment with agonists prevents various perturbations induced by chronic mild stress. Melatonin in vivo directly constricts cerebral arterioles in rats and decreases the lower limit of cerebral blood flow autoregulation, suggesting that melatonin may diminish the risk of hypoperfusion-induced cerebral ischemia. At the extracerebral level, melatonin regulates intestinal motility in rats. The intestinal postprandial motor response is shorter in the dark phase than in the light phase and this reduction is reversed in animals pretreated with a melatonin antagonist. Moreover, melatonin reduces the duration of cholecystokinin excitomotor effect. Endogenous melatonin may modulate intestinal motility to coordinate intestinal functions such as digestion and transit and control the metabolism of the animal. An adipocyte melatonin binding site may also participate in this control. Melatonin is involved in a wide range of physiological functions. The question remains as to whether evolution, adaptation and diurnal life have modified the physiological role of melatonin in humans. Moreover, the functional role of each of the receptor subtypes has to be characterized to design selective ligands to treat specific diseases.

Evolution of a detailed physiological model to simulate the gastrointestinal transit and absorption process in humans, part II: extension to describe performance of solid dosage forms.

PubMed

Thelen, Kirstin; Coboeken, Katrin; Willmann, Stefan; Dressman, Jennifer B; Lippert, Jörg

2012-03-01

The physiological absorption model presented in part I of this work is now extended to account for dosage-form-dependent gastrointestinal (GI) transit as well as disintegration and dissolution processes of various immediate-release and modified-release dosage forms. Empirical functions of the Weibull type were fitted to experimental in vitro dissolution profiles of solid dosage forms for eight test compounds (aciclovir, caffeine, cimetidine, diclofenac, furosemide, paracetamol, phenobarbital, and theophylline). The Weibull functions were then implemented into the model to predict mean plasma concentration-time profiles of the various dosage forms. On the basis of these dissolution functions, pharmacokinetics (PK) of six model drugs was predicted well. In the case of diclofenac, deviations between predicted and observed plasma concentrations were attributable to the large variability in gastric emptying time of the enteric-coated tablets. Likewise, oral PK of furosemide was found to be predominantly governed by the gastric emptying patterns. It is concluded that the revised model for GI transit and absorption was successfully integrated with dissolution functions of the Weibull type, enabling prediction of in vivo PK profiles from in vitro dissolution data. It facilitates a comparative analysis of the parameters contributing to oral drug absorption and is thus a powerful tool for formulation design. Copyright © 2011 Wiley Periodicals, Inc.

In vivo monitoring of Ca2+ uptake into mitochondria of mouse skeletal muscle during contraction

PubMed Central

Rudolf, Rüdiger; Mongillo, Marco; Magalhães, Paulo J.; Pozzan, Tullio

2004-01-01

Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca2+ handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca2+ transients reliably during the contraction–relaxation cycle. Using two-photon microscopy and genetically expressed “cameleon” Ca2+ sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca2+ transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca2+ during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca2+ increase is delayed by a few milliseconds compared with the cytosolic Ca2+ rise and occurs both during a single twitch and upon tetanic contraction. PMID:15314066

A novel bronchial ring bioassay for the evaluation of small airway smooth muscle function in mice.

PubMed

Liu, John Q; Yang, Dennis; Folz, Rodney J

2006-08-01

Advances in our understanding of murine airway physiology have been hindered by the lack of suitable, ex vivo, small airway bioassay systems. In this study, we introduce a novel small murine airway bioassay system that permits the physiological and pharmacological study of intrapulmonary bronchial smooth muscle via a bronchial ring (BR) preparation utilizing BR segments as small as 200 microm in diameter. Using this ex vivo BR bioassay, we characterized small airway smooth muscle contraction and relaxation in the presence and absence of bronchial epithelium. In control BRs, the application of mechanical stretch is followed by spontaneous bronchial smooth muscle relaxation. BRs pretreated with methacholine (MCh) partially attenuate this stretch-induced relaxation by as much as 42% compared with control. MCh elicited a dose-dependent bronchial constriction with a maximal tension (E(max)) of 8.7 +/- 0.2 mN at an EC(50) of 0.33 +/- 0.02 microM. In the presence of nifedipine, ryanodine, 2-aminoethoxydiphenyl borate, and SKF-96365, E(max) to MCh was significantly reduced. In epithelium-denuded BRs, MCh-induced contraction was significantly enhanced to 11.4 +/- 1.0 mN with an EC(50) of 0.16 +/- 0.04 microM (P < 0.01). Substance P relaxed MCh-precontracted BR by 62.1%; however, this bronchial relaxation effect was completely lost in epithelium-denuded BRs. Papaverine virtually abolished MCh-induced constriction in both epithelium-intact and epithelium-denuded bronchial smooth muscle. In conclusion, this study introduces a novel murine small airway BR bioassay that allows for the physiological study of smooth muscle airway contractile responses that may aid in our understanding of the pathophysiology of asthma.

In vivo Post-Cardiac Arrest Myocardial Dysfunction is Supported by CaMKII-Mediated Calcium Long-Term Potentiation and Mitigated by Alda-1, an Agonist of Aldehyde Dehydrogenase Type 2

PubMed Central

Downey, Peter; Zalewski, Adrian; Rubio, Gabriel R.; Liu, Jing; Homburger, Julian R.; Grunwald, Zachary; Qi, Wei; Bollensdorff, Christian; Thanaporn, Porama; Ali, Ayyaz; Riemer, Kirk; Kohl, Peter; Mochly-Rosen, Daria; Gerstenfeld, Edward; Large, Stephen; Ali, Ziad; Ashley, Euan

2016-01-01

Background Survival after sudden cardiac arrest is limited by post-arrest myocardial dysfunction but understanding of this phenomenon is constrained by lack of data from a physiological model of disease. In this study, we established an in vivo model of cardiac arrest and resuscitation, characterized the biology of the associated myocardial dysfunction, and tested novel therapeutic strategies. Methods We developed rodent models of in vivo post-arrest myocardial dysfunction using extra-corporeal membrane oxygenation (ECMO) resuscitation followed by invasive hemodynamics measurement. In post-arrest isolated cardiomyocytes, we assessed mechanical load and Ca2+ induced Ca2+ release (CICR) simultaneously using the micro-carbon-fiber technique and observed reduced function and myofilament calcium sensitivity. We used a novel-designed fiber optic catheter imaging system, and a genetically encoded calcium sensor GCaMP6f, to image CICR in vivo. Results We found potentiation of CICR in isolated cells from this ECMO model and also in cells isolated from an ischemia-reperfusion Langendorff model perfused with oxygenated blood from an arrested animal, but not when reperfused in saline. We established that CICR potentiation begins in vivo. The augmented CICR observed post-arrest was mediated by the activation of Ca2+/calmodulin kinase II (CaMKII). Increased phosphorylation of CaMKII, phospholamban and ryanodine receptor 2 (RyR2) was detected in the post-arrest period. Exogenous adrenergic activation in vivo recapitulated Ca2+ potentiation but was associated with lesser CaMKII activation. Since oxidative stress and aldehydic adduct formation were high post arrest, we tested a small molecule activator of aldehyde dehydrogenase type 2, Alda-1, which reduced oxidative stress, restored calcium and CaMKII homeostasis, and improved cardiac function and post-arrest outcome in vivo. Conclusions Cardiac arrest and reperfusion lead to CaMKII activation and calcium long-term potentiation which support cardiomyocyte contractility in the face of impaired post-ischemic myofilament calcium sensitivity. Alda-1 mitigates these effects, normalizes calcium cycling and improves outcome. PMID:27582424

Hypoxia-induced expression of VE-cadherin and filamin B in glioma cell cultures and pseudopalisade structures.

PubMed

Nissou, Marie-France; El Atifi, Michèle; Guttin, Audrey; Godfraind, Catherine; Salon, Caroline; Garcion, Emmanuel; van der Sanden, Boudewijn; Issartel, Jean-Paul; Berger, François; Wion, Didier

2013-06-01

Most of our knowledge regarding glioma cell biology comes from cell culture experiments. For many years the standards for glioma cell culture were the use of cell lines cultured in the presence of serum and 20 % O2. However, in vivo, normoxia in many brain areas is in close to 3 % O2. Hence, in cell culture, the experimental value referred as the norm is hyperoxic compared to any brain physiological value. Likewise, cells in vivo are not usually exposed to serum, and low-passaged glioma neurosphere cultures maintained in serum-free medium is emerging as a new standard. A consequence of changing the experimental normoxic standard from 20 % O2 to the more brain physiological value of 3 % O2, is that a 3 % O2 normoxic reference point enabled a more rigorous characterization of the level of regulation of genes by hypoxia. Among the glioma hypoxia-regulated genes characterized using this approach we found VE-cadherin that is required for blood vessel formation, and filamin B a gene involved in endothelial cell motility. Both VE-cadherin and filamin B were found expressed in pseudopalisades, a glioblastoma pathognomonic structure made of hypoxic migrating cancer cells. These results provide additional clues on the role played by hypoxia in the acquisition of endothelial traits by glioma cells and on the functional links existing between pseudopalisades, hypoxia, and tumor progression.

Suppression of miR-26a attenuates physiological disturbances arising from exposure of Nile tilapia (Oreochromis niloticus) to ammonia.

PubMed

Zhao, Yan; Zhou, Haotian; Ayisi, Christian Larbi; Wang, Yan; Wang, Jun; Chen, Xiaowu; Zhao, Jinling

2018-04-18

MicroRNAs may affect stress responses because they act as rapid responders at the post-translation level. In this study, we found that miR-26a is abundantly expressed in the brain and gill tissues of tilapia. Expression of miR-26a in the brain decreased significantly with increasing ammonia concentrations using stem-loop qPCR. To analyze the function of miRNA in vivo , miR-26a was stably knocked down with an antagomir in tilapia. Following ammonia challenge, miR-26a antagomir treatment significantly suppressed blood ammonia/[Cl - ]/[K + ] concentration and the reactive oxygen species production, while it markedly enhanced glutamine accumulation and antioxidant enzyme activity in the brain of tilapia, indicating that miR-26a may be involved in the remission of physiological disturbances resulting from ammonia stress. We strongly conclude that there is a direct link between miR-26a and the responses to ammonia in tilapia. Furthermore, bioinformatics analysis and luciferase assays demonstrated that miR-26a regulates HSP70 (heat shock protein 70) and GS (glutamine synthetase) expression by targeting their 3'-UTR and that the suppression of miR-26a could increase the intracellular level of HSP70 and GS in vivo . © 2018. Published by The Company of Biologists Ltd.

Melanopsin mediates light-dependent relaxation in blood vessels.

PubMed

Sikka, Gautam; Hussmann, G Patrick; Pandey, Deepesh; Cao, Suyi; Hori, Daijiro; Park, Jong Taek; Steppan, Jochen; Kim, Jae Hyung; Barodka, Viachaslau; Myers, Allen C; Santhanam, Lakshmi; Nyhan, Daniel; Halushka, Marc K; Koehler, Raymond C; Snyder, Solomon H; Shimoda, Larissa A; Berkowitz, Dan E

2014-12-16

Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.

Physiologically-based Pharmacokinetic(PBPK) Models Application to Screen Environmental Hazards Related to Adverse Outcome Pathways(AOPs)

EPA Science Inventory

PBPK models are useful in estimating exposure levels based on in vitro to in vivo extrapolation (IVIVE) calculations. Linkage of large sets of chemically screened vitro signature effects to in vivo adverse outcomes using IVIVE is central to the concepts of toxicology in the 21st ...

COMPARISON OF IN VIVO DERIVED AND SCALED IN VITRO METABOLIC RATE CONSTANTS FOR SOME VOLATILE ORGANIC COMPOUNDS (VOCS)

EPA Science Inventory

The reliability of physiologically based pharmacokinetic (PBPK) models is directly related to the accuracy of the metabolic rate parameters used as model inputs. When metabolic rate parameters derived from in vivo experiments are unavailable, they can be estimated from in vitro d...

A Surface Groove Essential for Viral Bcl-2 Function During Chronic Infection In Vivo

PubMed Central

Petros, Andrew M; Nettesheim, David; van Dyk, Linda F.; Labrada, Lucia; Speck, Samuel H; Levine, Beth

2005-01-01

Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the γ-herpesvirus 68 (γHV68) Bcl-2 family protein (γHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the γHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type γHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of γHV68 from latency and efficient persistent γHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection. PMID:16201011

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

PubMed

West, Adrian R; Zaman, Nishat; Cole, Darren J; Walker, Matthew J; Legant, Wesley R; Boudou, Thomas; Chen, Christopher S; Favreau, John T; Gaudette, Glenn R; Cowley, Elizabeth A; Maksym, Geoffrey N

2013-01-01

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice.

PubMed

Gioscia-Ryan, Rachel A; LaRocca, Thomas J; Sindler, Amy L; Zigler, Melanie C; Murphy, Michael P; Seals, Douglas R

2014-06-15

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth.

PubMed

Motes, Christy M; Pechter, Priit; Yoo, Cheol Min; Wang, Yuh-Shuh; Chapman, Kent D; Blancaflor, Elison B

2005-12-01

Plant development is regulated by numerous chemicals derived from a multitude of metabolic pathways. However, we know very little about the biological effects and functions of many of these metabolites in the cell. N-Acylethanolamines (NAEs) are a group of lipid mediators that play important roles in mammalian physiology. Despite the intriguing similarities between animals and plants in NAE metabolism and perception, not much is known about the precise function of these metabolites in plant physiology. In plants, NAEs have been shown to inhibit phospholipase Dalpha (PLDalpha) activity, interfere with abscisic acid-induced stomatal closure, and retard Arabidopsis seedling development. 1-Butanol, an antagonist of PLD-dependent phosphatidic acid production, was reported to induce defects in Arabidopsis seedling development that were somewhat similar to effects induced by elevated levels of NAE. This raised the possibility that the impact of NAE on seedling growth could be mediated in part via its influence on PLD activity. To begin to address this possibility, we conducted a detailed, comparative analysis of the effects of 1-butanol and N-lauroylethanolamine (NAE 12:0) on Arabidopsis root cell division, in vivo cytoskeletal organization, seed germination, and seedling growth. Although both NAE 12:0 and 1-butanol induced profound cytoskeletal and morphological alterations in seedlings, there were distinct differences in their overall effects. 1-Butanol induced more pronounced modifications in cytoskeletal organization, seedling growth, and cell division at concentrations severalfold higher than NAE 12:0. We propose that these compounds mediate their differential effects on cellular organization and seedling growth, in part through the differential modulation of specific PLD isoforms.

Integration of Life-Stage Physiologically Based Pharmacokinetic Models with Adverse Outcome Pathways and Environmental Exposure Models to Screen for Environmental Hazards

EPA Science Inventory

A Life-stage Physiologically-Based Pharmacokinetic (PBPK) model was developed to include descriptions of several life-stage events such as pregnancy, fetal development, the neonate and child growth. The overall modeling strategy was used for in vitro to in vivo (IVIVE) extrapolat...

Physiological Parameters for Oral Delivery and In vitro Testing

PubMed Central

Mudie, Deanna M.; Amidon, Gordon L.; Amidon, Gregory E.

2010-01-01

Pharmaceutical solid oral dosage forms must undergo dissolution in the intestinal fluids of the gastrointestinal tract before they can be absorbed and reach the systemic circulation. Therefore, dissolution is a critical part of the drug-delivery process. The rate and extent of drug dissolution and absorption depend on the characteristics of the active ingredient as well as properties of the dosage form. Just as importantly, characteristics of the physiological environment such as buffer species, pH, bile salts, gastric emptying rate, intestinal motility, and hydrodynamics can significantly impact dissolution and absorption. While significant progress has been made since 1970 when the first compendial dissolution test was introduced (USP Apparatus 1), current dissolution testing does not take full advantage of the extensive physiologic information that is available. For quality control purposes, where the question is one of lot-to-lot consistency in performance, using nonphysiologic test conditions that match drug and dosage form properties with practical dissolution media and apparatus may be appropriate. However, where in vitro – in vivo correlations are desired, it is logical to consider and utilize knowledge of the in vivo condition. This publication critically reviews the literature that is relevant to oral human drug delivery. Physiologically relevant information must serve as a basis for the design of dissolution test methods and systems that are more representative of the human condition. As in vitro methods advance in their physiological relevance, better in vitro - in vivo correlations will be possible. This will, in turn, lead to in vitro systems that can be utilized to more effectively design dosage forms that have improved and more consistent oral bioperformance. PMID:20822152

The role of Drosophila Piezo in mechanical nociception.

PubMed

Kim, Sung Eun; Coste, Bertrand; Chadha, Abhishek; Cook, Boaz; Patapoutian, Ardem

2012-02-19

Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain. Ion channels have a role in neuronal mechanotransduction in invertebrates; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, no mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in Drosophila melanogaster and Caenorhabditis elegans; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members are mechanotransducers in C. elegans and potentially in D. melanogaster; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. Here we study the physiological role of the single Piezo member in D. melanogaster (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, we found that Dmpiezo and ppk function in parallel pathways in ppk-positive cells, and that mechanical nociception is abolished in the absence of both channels. These data demonstrate the physiological relevance of the Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception.

[Biological function of trophology and the pathogenesis of metabolic syndrome--syndrome of overeating. Phylogenetically theory of general pathology, role of leptin and adiponectin].

PubMed

Titov, V N

2014-01-01

Metabolic syndrome (overeating) is a phylogenetically-determined succession of symptoms with the same pathogenesis. There is only one etiological factor, namely, increased consumption of physiologically optimal food. Enterocytes and omental fat cells are a phylogenetically early paracrine-regulated cell community that realizes the biological reactions of exo- and endotrophy. Visceral obesity, high levels of unesterified fatty acids (FA), formation of a pool of micellar FA in the blood, integration of these FA into endothelial cell plasma membrane and enlargement of adipocytes are the causes of hydrodynamic pressure elevation. Toll-like receptors recognize the associates between albumin and greater than physiological number of FA as "foreing" and initiate inflammatory response. "Endoplasm stress" develops in lipid-overloaded cells, protein synthesis (folding) in them is impaired and apoptosis-like cell death is activated. Visceral fat is a phylogenetically early depot of FA to fulfill the biological function of homeostasis, trophology, endoecology and adaptation; it is regulated at the level of paracrine communities and is anatomically limited. The subcutaneous fat depot fulfills the phylogenetically late function of locomotion; the depot size is not anatomically limited. Visceral fat cells have no receptors for phylogenetically late insulin (INS); specialized adipocyes bearing INS and GLUT4 receptors are cells that form the subcutaneous depot. These cells are regulated by phylogenetically late humoral factors at the entire body level. Leptin is an initiator of humoral hypothalamic regulation of in vivo number of ontogenetically programmed number of visceral INS-insensitive fat cells. It prevents "endoplasm stress" and apoptosis, being designed to regulate the amount of consumed food. Leptin initiates storage of FA from visceral pool into subcutaneous pool. Adiponectin is a phylogenetically late humoral hypothalamic regulatory factor that controls optimal number of fat cells in vivo. Its biological role consists in regulation of the number (proliferation) of insulin-dependent adipocytes in subcutaneous fatty tissue.

Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables

PubMed Central

Ishida, Masahiko; Hara, Masakazu; Fukino, Nobuko; Kakizaki, Tomohiro; Morimitsu, Yasujiro

2014-01-01

Unique secondary metabolites, glucosinolates (S-glucopyranosyl thiohydroximates), are naturally occurring S-linked glucosides found mainly in Brassicaceae plants. They are enzymatically hydrolyzed to produce sulfate ions, D-glucose, and characteristic degradation products such as isothiocyanates. The functions of glucosinolates in the plants remain unclear, but isothiocyanates possessing a pungent or irritating taste and odor might be associated with plant defense from microbes. Isothiocyanates have been studied extensively in experimental in vitro and in vivo carcinogenesis models for their cancer chemopreventive properties. The beneficial isothiocyanates, glucosinolates that are functional for supporting human health, have received attention from many scientists studying plant breeding, plant physiology, plant genetics, and food functionality. This review presents a summary of recent topics related with glucosinolates in the Brassica family, along with a summary of the chemicals, metabolism, and genes of glucosinolates in Brassicaceae. The bioavailabilities of isothiocyanates from certain functional glucosinolates and the importance of breeding will be described with emphasis on glucosinolates. PMID:24987290

Widespread immunological functions of mast cells: fact or fiction?

PubMed

Rodewald, Hans-Reimer; Feyerabend, Thorsten B

2012-07-27

Immunological functions of mast cells are currently considered to be much broader than the original role of mast cells in IgE-driven allergic disease. The spectrum of proposed mast cell functions includes areas as diverse as the regulation of innate and adaptive immune responses, protective immunity against viral, microbial, and parasitic pathogens, autoimmunity, tolerance to graft rejection, promotion of or protection from cancer, wound healing, angiogenesis, cardiovascular diseases, diabetes, obesity, and others. The vast majority of in vivo mast cell data have been based on mast cell-deficient Kit mutant mice. However, work in new mouse mutants with unperturbed Kit function, which have a surprisingly normal immune system, has failed to corroborate some key immunological aspects, formerly attributed to mast cells. Here, we consider the implications of these recent developments for the state of the field as well as for future work, aiming at deciphering the physiological functions of mast cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Engineering Silicone Rubbers for In vitro Studies: Creating AAA Models and ILT Analogues with Physiological Properties

PubMed Central

Corbett, T.J.; Doyle, B.J.; Callanan, A.; Walsh, M.T.; McGloughlin, T.M

2010-01-01

Background In vitro studies of abdominal aortic aneurysm (AAA) have been widely reported. Frequently mock artery models with intraluminal thrombus (ILT) analogues are used to mimic the AAA in vivo. While the models used may be physiological, their properties are frequently either not reported or investigated. Method of Approach This study is concerned with the testing and characterisation of previously used vessel analogue materials and the development of new materials for the manufacture of AAA models. These materials were used in conjunction with a previously validated injection moulding technique to manufacture AAA models of ideal geometry. To determine the model properties (stiffness (β) and compliance) the diameter change of each AAA model was investigated under incrementally increasing internal pressures and compared to published in vivo studies to determine if the models behaved physiologically. A FEA study was implemented to determine if the pressure – diameter change behaviour of the models could be predicted numerically. ILT analogues were also manufactured and characterised. Ideal models were manufactured with ILT analogue internal to the aneurysm region and the effect of the ILT analogue on the model compliance and stiffness was investigated. Results The wall materials had similar properties to aortic tissue at physiological pressures (Einit 2.22MPa and 1.57MPa (aortic tissue: 1.8MPa)). ILT analogues had similar Young’s modulus to the medial layer of ILT (0.24 and 0.33MPa (ILT: 0.28MPa)). All models had aneurysm sac compliance in the physiological range (2.62 – 8.01×10-4/mmHg (AAA in vivo: 1.8 – 9.4×10-4/mmHg)). The necks of our AAA models had similar stiffness to healthy aortas (20.44 – 29.83 (healthy aortas in vivo: 17.5±5.5)). Good agreement was seen between the diameter changes due to pressurisation in the experimental and FEA wall models with a maximum error of 7.3% at 120mmHg. It was also determined that the inclusion of ILT analogue in the sac of our models could have an effect on the compliance of the model neck. Conclusions Ideal AAA models with physiological properties were manufactured. The behaviour of these models due to pressurisation was predicted using FEA, validating this technique for the future design of realistic, physiological AAA models. Addition of ILT analogues in the aneurysm sac was shown to affect neck behaviour. This could have implications for endovascular AAA repair due to the importance of the neck for stent-graft fixation. PMID:20524746

Modeling the Blood-Brain Barrier in a 3D triple co-culture microfluidic system.

PubMed

Adriani, G; Ma, D; Pavesi, A; Goh, E L K; Kamm, R D

2015-01-01

The need for a blood-brain barrier (BBB) model that accurately mimics the physiological characteristics of the in-vivo situation is well-recognized by researchers in academia and industry. However, there is currently no in-vitro model allowing studies of neuronal growth and/or function influenced by factors from the blood that cross through the BBB. Therefore, we established a 3D triple co-culture microfluidic system using human umbilical vein endothelial cells (HUVEC) together with primary rat astrocytes and neurons. Immunostaining confirmed the successful triple co-culture system consisting of an intact BBB with tight intercellular junctions in the endothelial monolayer. The BBB selective permeability was determined by a fluorescent-based assay using dextrans of different molecular weights. Finally, neuron functionality was demonstrated by calcium imaging.

Nano-graphene oxide composite for in vivo imaging

PubMed Central

Oh, Seo Yeong; Vilian, AT Ezhil; Lee, Ilsong; Han, Young-Kyu; Park, Jeong Hoon; Roh, Changhyun; Huh, Yun Suk

2018-01-01

Introduction Positron emission tomography (PET) tracers has the potential to revolutionize cancer imaging and diagnosis. PET tracers offer non-invasive quantitative imaging in biotechnology and biomedical applications, but it requires radioisotopes as radioactive imaging tracers or radiopharmaceuticals. Method This paper reports the synthesis of 18F-nGO-PEG by covalently functionalizing PEG with nano-graphene oxide, and its excellent stability in physiological solutions. Using a green synthesis route, nGO is then functionalized with a biocompatible PEG polymer to acquire high stability in PBS and DMEM. Results and discussion The radiochemical safety of 18F-nGO-PEG was measured by a reactive oxygen species and cell viability test. The biodistribution of 18F-nGO-PEG could be observed easily by PET, which suggested the significantly high sensitivity tumor uptake of 18F-nGO-PEG and in a tumor bearing CT-26 mouse compared to the control. 18F-nGO-PEG was applied successfully as an efficient radiotracer or drug agent in vivo using PET imaging. This article is expected to assist many researchers in the fabrication of 18F-labeled graphene-based bio-conjugates with high reproducibility for applications in the biomedicine field. PMID:29379283

ALDH1B1 links alcohol consumption and diabetes.

PubMed

Singh, Surendra; Chen, Ying; Matsumoto, Akiko; Orlicky, David J; Dong, Hongbin; Thompson, David C; Vasiliou, Vasilis

2015-08-07

Aldehyde dehydrogenase 1B1 (ALDH1B1) is a mitochondrial enzyme sharing 65% and 72% sequence identity with ALDH1A1 and ALDH2 proteins, respectively. Compared to the latter two ALDH isozymes, little is known about the physiological functions of ALDH1B1. Studies in humans indicate that ALDH1B1 may be associated with alcohol sensitivity and stem cells. Our recent in vitro studies using human ALDH1B1 showed that it metabolizes acetaldehyde and retinaldehyde. To investigate the in vivo role of ALDH1B1, we generated and characterized a global Aldh1b1 knockout mouse line. These knockout (KO) mice are fertile and show overtly good health. However, ethanol pharmacokinetic analysis revealed ∼40% increase in blood acetaldehyde levels in KO mice. Interestingly, the KO mice exhibited higher fasting blood glucose levels. Collectively, we show for the first time the functional in vivo role of ALDH1B1 in acetaldehyde metabolism and in maintaining glucose homeostasis. This mouse model is a valuable tool to investigate the mechanism by which alcohol may promote the development of diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Apoptosis-mediated endothelial toxicity but not direct calcification or functional changes in anti-calcification proteins defines pathogenic effects of calcium phosphate bions

NASA Astrophysics Data System (ADS)

Kutikhin, Anton G.; Velikanova, Elena A.; Mukhamadiyarov, Rinat A.; Glushkova, Tatiana V.; Borisov, Vadim V.; Matveeva, Vera G.; Antonova, Larisa V.; Filip'Ev, Dmitriy E.; Golovkin, Alexey S.; Shishkova, Daria K.; Burago, Andrey Yu.; Frolov, Alexey V.; Dolgov, Viktor Yu.; Efimova, Olga S.; Popova, Anna N.; Malysheva, Valentina Yu.; Vladimirov, Alexandr A.; Sozinov, Sergey A.; Ismagilov, Zinfer R.; Russakov, Dmitriy M.; Lomzov, Alexander A.; Pyshnyi, Dmitriy V.; Gutakovsky, Anton K.; Zhivodkov, Yuriy A.; Demidov, Evgeniy A.; Peltek, Sergey E.; Dolganyuk, Viatcheslav F.; Babich, Olga O.; Grigoriev, Evgeniy V.; Brusina, Elena B.; Barbarash, Olga L.; Yuzhalin, Arseniy E.

2016-06-01

Calcium phosphate bions (CPB) are biomimetic mineralo-organic nanoparticles which represent a physiological mechanism regulating the function, transport and disposal of calcium and phosphorus in the human body. We hypothesised that CPB may be pathogenic entities and even a cause of cardiovascular calcification. Here we revealed that CPB isolated from calcified atherosclerotic plaques and artificially synthesised CPB are morphologically and chemically indistinguishable entities. Their formation is accelerated along with the increase in calcium salts-phosphates/serum concentration ratio. Experiments in vitro and in vivo showed that pathogenic effects of CPB are defined by apoptosis-mediated endothelial toxicity but not by direct tissue calcification or functional changes in anti-calcification proteins. Since the factors underlying the formation of CPB and their pathogenic mechanism closely resemble those responsible for atherosclerosis development, further research in this direction may help us to uncover triggers of this disease.

A new paradigm in respiratory hygiene: modulating respiratory secretions to contain cough bioaerosol without affecting mucus clearance.

PubMed

Zayas, Gustavo; Valle, Juan C; Alonso, Mauricio; Alfaro, Henry; Vega, Daniel; Bonilla, Gloria; Reyes, Miguel; King, Malcolm

2007-08-13

Several strategies and devices have been designed to protect health care providers from acquiring transmissible respiratory diseases while providing care. In modulating the physical characteristics of the respiratory secretions to minimize the aerosolization that facilitates transmission of airborne diseases, a fundamental premise is that the prototype drugs have no adverse effect on the first line of respiratory defense, clearance of mucus by ciliary action. To assess and demonstrate the primary mechanism of our mucomodulators (XLs), we have built our evidence moving from basic laboratory studies to an ex-vivo model and then to an in-vivo large animal model. We exposed anesthetized dogs without hypersecretion to different dose concentrations of aerosolized XL "B", XL "D" and XL "S". We assessed: cardio-respiratory pattern, tracheal mucus clearance, airway patency, and mucus viscoelastic changes. Exposure of frog palate mucus to XLs did not affect the clearance of mucus by ciliary action. Dogs maintained normal cardio-respiratory pattern with XL administration. Tracheal mucociliary clearance in anesthetized dogs indicated a sustained 40% mean increase. Tracheal mucus showed increased filance, and there was no mucus retention in the airways. The ex-vivo frog palate and the in-vivo mammalian models used in this study, appear to be appropriate and complement each other to better assess the effects that our mucomodulators exert on the mucociliary clearance defence mechanism. The physiological function of the mucociliary apparatus was not negatively affected in any of the two epithelial models. Airway mucus crosslinked by mucomodulators is better cleared from an intact airway and normally functioning respiratory system, either due to enhanced interaction with cilia or airflow-dependent mechanisms. Data obtained in this study allow us to assure that we have complied with the fundamental requirement criteria established in the initial phase of developing the concept of mucomodulation: Can we modulate the physical characteristics of the respiratory secretions to reduce aerosolization without affecting normal mucociliary clearance function, or even better improving it?

Major amyloid-β-degrading enzymes, endothelin-converting enzyme-2 and neprilysin, are expressed by distinct populations of GABAergic interneurons in hippocampus and neocortex.

PubMed

Pacheco-Quinto, Javier; Eckman, Christopher B; Eckman, Elizabeth A

2016-12-01

Impaired clearance of amyloid-β peptide (Aβ) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aβ in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aβ that may be functionally significant. To better understand the differential regulation of Aβ concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aβ degradation is consistent with the possibility that Aβ may have a physiological function related to the regulation of inhibitory signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Pharmacology of P2X channels.

PubMed

Gever, Joel R; Cockayne, Debra A; Dillon, Michael P; Burnstock, Geoffrey; Ford, Anthony P D W

2006-08-01

Significant progress in understanding the pharmacological characteristics and physiological importance of homomeric and heteromeric P2X channels has been achieved in recent years. P2X channels, gated by ATP and most likely trimerically assembled from seven known P2X subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacological classification holds true in native cells and in vivo is less well-established. Nevertheless, several potent and selective P2X antagonists have been discovered in recent years and shown to be efficacious in various animal models including those for visceral organ function, chronic inflammatory and neuropathic pain, and inflammation. The recent advancement of drug candidates targeting P2X channels into human trials, confirms the medicinal exploitability of this novel target family and provides hope that safe and effective medicines for the treatment of disorders involving P2X channels may be identified in the near future.

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

PubMed Central

Kang, Hong-Jun; Vassilopoulos, Athanassios

2016-01-01

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context. PMID:26966987

Function and dysfunction of CNG channels: insights from channelopathies and mouse models.

PubMed

Biel, Martin; Michalakis, Stylianos

2007-06-01

Channels directly gated by cyclic nucleotides (CNG channels) are important cellular switches that mediate influx of Na+ and Ca2+ in response to increases in the intracellular concentration of cAMP and cGMP. In photoreceptors and olfactory receptor neurons, these channels serve as final targets for cGMP and cAMP signaling pathways that are initiated by the absorption of photons and the binding of odorants, respectively. CNG channels have been also found in other types of neurons and in non-excitable cells. However, in most of these cells, the physiological role of CNG channels has yet to be determined. CNG channels have a complex heteromeric structure. The properties of individual subunits that assemble in specific stoichiometries to the native channels have been extensively investigated in heterologous expression systems. Recently, mutations in human CNG channel genes leading to inherited diseases (so-called channelopathies) have been functionally characterized. Moreover, mouse knockout models were generated to define the role of CNG channel proteins in vivo. In this review, we will summarize recent insights into the physiological and pathophysiological role of CNG channel proteins that have emerged from genetic studies in mice and humans.

A quantitative analysis of statistical power identifies obesity end points for improved in vivo preclinical study design.

PubMed

Selimkhanov, J; Thompson, W C; Guo, J; Hall, K D; Musante, C J

2017-08-01

The design of well-powered in vivo preclinical studies is a key element in building the knowledge of disease physiology for the purpose of identifying and effectively testing potential antiobesity drug targets. However, as a result of the complexity of the obese phenotype, there is limited understanding of the variability within and between study animals of macroscopic end points such as food intake and body composition. This, combined with limitations inherent in the measurement of certain end points, presents challenges to study design that can have significant consequences for an antiobesity program. Here, we analyze a large, longitudinal study of mouse food intake and body composition during diet perturbation to quantify the variability and interaction of the key metabolic end points. To demonstrate how conclusions can change as a function of study size, we show that a simulated preclinical study properly powered for one end point may lead to false conclusions based on secondary end points. We then propose the guidelines for end point selection and study size estimation under different conditions to facilitate proper power calculation for a more successful in vivo study design.

Visualization of Nicotine Adenine Dinucleotide Redox Homeostasis with Genetically Encoded Fluorescent Sensors.

PubMed

Zhao, Yuzheng; Zhang, Zhuo; Zou, Yejun; Yang, Yi

2018-01-20

Beyond their roles as redox currency in living organisms, pyridine dinucleotides (NAD + /NADH and NADP + /NADPH) are also precursors or cosubstrates of great significance in various physiologic and pathologic processes. Recent Advances: For many years, it was challenging to develop methodologies for monitoring pyridine dinucleotides in situ or in vivo. Recent advances in fluorescent protein-based sensors provide a rapid, sensitive, specific, and real-time readout of pyridine dinucleotide dynamics in single cells or in vivo, thereby opening a new era of pyridine dinucleotide bioimaging. In this article, we summarize the developments in genetically encoded fluorescent sensors for NAD + /NADH and NADP + /NADPH redox states, as well as their applications in life sciences and drug discovery. The strengths and weaknesses of individual sensors are also discussed. These sensors have the advantages of being specific and organelle targetable, enabling real-time monitoring and subcellular-level quantification of targeted molecules in living cells and in vivo. NAD + /NADH and NADP + /NADPH have distinct functions in metabolic and redox regulation, and thus, a comprehensive evaluation of metabolic and redox states must be multiplexed with a combination of various metabolite sensors in a single cell. Antioxid. Redox Signal. 28, 213-229.

The effects of exercise on human articular cartilage

PubMed Central

Eckstein, F; Hudelmaier, M; Putz, R

2006-01-01

The effects of exercise on articular hyaline articular cartilage have traditionally been examined in animal models, but until recently little information has been available on human cartilage. Magnetic resonance imaging now permits cartilage morphology and composition to be analysed quantitatively in vivo. This review briefly describes the methodological background of quantitative cartilage imaging and summarizes work on short-term (deformational behaviour) and long-term (functional adaptation) effects of exercise on human articular cartilage. Current findings suggest that human cartilage deforms very little in vivo during physiological activities and recovers from deformation within 90 min after loading. Whereas cartilage deformation appears to become less with increasing age, sex and physical training status do not seem to affect in vivo deformational behaviour. There is now good evidence that cartilage undergoes some type of atrophy (thinning) under reduced loading conditions, such as with postoperative immobilization and paraplegia. However, increased loading (as encountered by elite athletes) does not appear to be associated with increased average cartilage thickness. Findings in twins, however, suggest a strong genetic contribution to cartilage morphology. Potential reasons for the inability of cartilage to adapt to mechanical stimuli include a lack of evolutionary pressure and a decoupling of mechanical competence and tissue mass. PMID:16637874

Development of a flow feedback pulse duplicator system with rhesus monkey arterial input impedance characteristics

NASA Technical Reports Server (NTRS)

Schaub, J. D.; Koenig, S. C.; Schroeder, M. J.; Ewert, D. L.; Drew, G. A.; Swope, R. D.; Convertino, V. A. (Principal Investigator)

1999-01-01

An in vitro pulsatile pump flow system that is capable of producing physiologic pressures and flows in a mock circulatory system tuned to reproduce the first nine harmonics of the input impedance of a rhesus monkey was developed and tested. The system was created as a research tool for evaluating cardiovascular function and for the design, testing, and evaluation of electrical-mechanical cardiovascular models and chronically implanted sensors. The system possesses a computerized user interface for controlling a linear displacement pulsatile pump in a controlled flow loop format to emulate in vivo cardiovascular characteristics. Evaluation of the pump system consisted of comparing its aortic pressure and flow profiles with in vivo rhesus hemodynamic waveforms in the time and frequency domains. Comparison of aortic pressure and flow data between the pump system and in vivo data showed good agreement in the time and frequency domains, however, the pump system produced a larger pulse pressure. The pump system can be used for comparing cardiovascular parameters with predicted cardiovascular model values and for evaluating such items as vascular grafts, heart valves, biomaterials, and sensors. This article describes the development and evaluation of this feedback controlled cardiovascular dynamics simulation modeling system.

New techniques for motion-artifact-free in vivo cardiac microscopy

PubMed Central

Vinegoni, Claudio; Lee, Sungon; Aguirre, Aaron D.; Weissleder, Ralph

2015-01-01

Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases. PMID:26029116

The effects of exercise on human articular cartilage.

PubMed

Eckstein, F; Hudelmaier, M; Putz, R

2006-04-01

The effects of exercise on articular hyaline articular cartilage have traditionally been examined in animal models, but until recently little information has been available on human cartilage. Magnetic resonance imaging now permits cartilage morphology and composition to be analysed quantitatively in vivo. This review briefly describes the methodological background of quantitative cartilage imaging and summarizes work on short-term (deformational behaviour) and long-term (functional adaptation) effects of exercise on human articular cartilage. Current findings suggest that human cartilage deforms very little in vivo during physiological activities and recovers from deformation within 90 min after loading. Whereas cartilage deformation appears to become less with increasing age, sex and physical training status do not seem to affect in vivo deformational behaviour. There is now good evidence that cartilage undergoes some type of atrophy (thinning) under reduced loading conditions, such as with postoperative immobilization and paraplegia. However, increased loading (as encountered by elite athletes) does not appear to be associated with increased average cartilage thickness. Findings in twins, however, suggest a strong genetic contribution to cartilage morphology. Potential reasons for the inability of cartilage to adapt to mechanical stimuli include a lack of evolutionary pressure and a decoupling of mechanical competence and tissue mass.

In vivo imaging of small animals with optical tomography and near-infrared fluorescent probes

NASA Astrophysics Data System (ADS)

Palmer, Matthew R.; Shibata, Yasushi; Kruskal, Jonathan B.; Lenkinski, Robert E.

2002-06-01

A developmental optical tomography has been designed for imaging small animals in vivo using near IR fluorophores. The system employs epi-illumination via a 450 W Xe arc lamp, filtered and collimated to illuminate a 10 cm square movable stage. Emission light is filtered then collected by a high- resolution, high quantum efficiency, cooled CCD camera. Stage movement and image acquisition are under the control of a personal computer running system integration and automation software. During an experiment, the anesthetized animal is secured to the stage and up to 200 projections can be acquired over 180 degrees rotation. Angular sampling of the light distribution at a point on the surface is used to determine relative contributions form ballistic and diffuse photons. We have employed the system to investigate a number of applications of in-vivo fluorescent imaging. In dynamic studies, hepatic function has been visualized in nude mice following intravenous injection of indocyanine green (ICG) and cerebrospinal fluid flow as been measured by injection of ICG-lipoprotein conjugate in the subarachnoid space of the lumbar spine followed by dynamic imaging of the brain. Further applications in physiological imaging, cancer detection, and molecular imaging are under investigation in our laboratory.

Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins

PubMed Central

Tiede, L M; Cook, E A; Morsey, B; Fox, H S

2011-01-01

Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1–11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons. PMID:22190005

Mitochondrial NAD(P)H In vivo: Identifying Natural Indicators of Oxidative Phosphorylation in the (31)P Magnetic Resonance Spectrum.

PubMed

Conley, Kevin E; Ali, Amir S; Flores, Brandon; Jubrias, Sharon A; Shankland, Eric G

2016-01-01

Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P)) are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis, and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P)(+) and NAD(P)H), which are compartmentalized between cytosol and mitochondria. Here we provide evidence for detection of NAD(P)(+) and NAD(P)H in separate mitochondrial and cytosol pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy ((31)P MRS). These NAD(P) pools are identified by chemical standards (NAD(+), NADP(+), and NADH) and by physiological tests. A unique resonance reflecting mitochondrial NAD(P)H is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(P)H with oxidation is matched by a stoichiometric rise in the NAD(P)(+) peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(P)H peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus, non-invasive detection of NAD(P)(+) and NAD(P)H in cytosol vs. mitochondria yields natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

Osteocyte calcium signals encode strain magnitude and loading frequency in vivo.

PubMed

Lewis, Karl J; Frikha-Benayed, Dorra; Louie, Joyce; Stephen, Samuel; Spray, David C; Thi, Mia M; Seref-Ferlengez, Zeynep; Majeska, Robert J; Weinbaum, Sheldon; Schaffler, Mitchell B

2017-10-31

Osteocytes are considered to be the major mechanosensory cells of bone, but how osteocytes in vivo process, perceive, and respond to mechanical loading remains poorly understood. Intracellular calcium (Ca 2+ ) signaling resulting from mechanical stimulation has been widely studied in osteocytes in vitro and in bone explants, but has yet to be examined in vivo. This is achieved herein by using a three-point bending device which is capable of delivering well-defined mechanical loads to metatarsal bones of living mice while simultaneously monitoring the intracellular Ca 2+ responses of individual osteocytes by using a genetically encoded fluorescent Ca 2+ indicator. Osteocyte responses are imaged by using multiphoton fluorescence microscopy. We investigated the in vivo responses of osteocytes to strains ranging from 250 to 3,000 [Formula: see text] and frequencies from 0.5 to 2 Hz, which are characteristic of physiological conditions reported for bone. At all loading frequencies examined, the number of responding osteocytes increased strongly with applied strain magnitude. However, Ca 2+ intensity within responding osteocytes did not change significantly with physiological loading magnitudes. Our studies offer a glimpse into how these critical bone cells respond to mechanical load in vivo, as well as provide a technique to determine how the cells encode magnitude and frequency of loading. Published under the PNAS license.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke.

PubMed

Talley Watts, Lora; Zheng, Wei; Garling, R Justin; Frohlich, Victoria C; Lechleiter, James Donald

2015-06-23

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Unperturbed vs. post-transplantation hematopoiesis: both in vivo but different

PubMed Central

Busch, Katrin; Rodewald, Hans-Reimer

2016-01-01

Purpose of review Hematopoietic stem cell (HSC) transplantation has yielded tremendous information on experimental properties of HSCs. Yet, it remains unclear whether transplantation reflects the physiology of hematopoiesis. A limitation is the difficulty in accessing HSC functions without isolation, in-vitro manipulation and readout for potential. New genetic fate mapping and clonal marking techniques now shed light on hematopoiesis under physiological conditions. Recent findings Transposon-based genetic marks were introduced across the entire hematopoietic system to follow the clonal dynamics of these tags over time. A polyclonal source downstream from stem cells was found responsible for the production of at least granulocytes. In independent experiments, HSCs were genetically marked in adult mice, and the kinetics of label emergence throughout the system was followed over time. These experiments uncovered that during physiological steady-state hematopoiesis large numbers of HSCs yield differentiated progeny. Individual HSCs were active only rarely, indicating their very slow periodicity of differentiation rather than quiescence. Summary Noninvasive genetic experiments in mice have identified a major role of stem and progenitor cells downstream from HSCs as drivers of adult hematopoiesis, and revealed that post-transplantation hematopoiesis differs quantitatively from normal steady-state hematopoiesis. PMID:27213498

Species Extrapolation of Life-Stage Physiologically-Based Pharmacokinetic (PBPK) Models to Investigate the Developmental Toxicology of Ethanol Using In vitro to In vivo (IVIVE) Methods

EPA Science Inventory

To provide useful alternatives to in vivo animal studies, in vitro assays for dose-response assessments of xenobiotic chemicals must use concentrations in media and target tissues that are within biologically-plausible limits. Determining these concentrations is a complex matter,...

The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory

PubMed Central

Cicvaric, Ana; Yang, Jiaye; Krieger, Sigurd; Khan, Deeba; Kim, Eun-Jung; Dominguez-Rodriguez, Manuel; Cabatic, Maureen; Molz, Barbara; Acevedo Aguilar, Juan Pablo; Milicevic, Radoslav; Smani, Tarik; Breuss, Johannes M.; Kerjaschki, Dontscho; Pollak, Daniela D.; Uhrin, Pavel; Monje, Francisco J.

2016-01-01

Abstract Introduction: Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized. Materials and methods: Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin. Results: Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF. Discussion: This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology.Key messagesPodoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions.Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation.Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well as a role for podoplanin in plasticity-related brain neuronal functions is here proposed. PMID:27558977

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

PubMed Central

Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

2014-01-01

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

Transepithelial nasal potential difference (NPD) measurements in cystic fibrosis (CF).

PubMed

Sands, Dorota

2013-01-01

The main underlying physiologic abnormality in cystic fibrosis (CF) is dysfunction of the CF transmembrane conductance regulator (CFTR), which results in abnormal transport of sodium and chloride across epithelial surfaces. CFTR function could be tested in vivo using measurements of nasal transepithelial potential difference (PD). Nasal measurements show characteristic features of CF epithelia, including hyperpolarized baseline readings (basal PD), excessive depolarization in response to sodium channel inhibitors, such as amiloride (ΔAmiloride), and little or no chloride (Cl-) secretion in response to isoproterenol in a chloride-free solution (ΔCl- free-isoproterenol). PD test is applied for CF diagnosis and monitoring of new therapeutic modulations and corrections.

Dynamic modulation of innate immunity programming and memory.

PubMed

Yuan, Ruoxi; Li, Liwu

2016-01-01

Recent progress harkens back to the old theme of immune memory, except this time in the area of innate immunity, to which traditional paradigm only prescribes a rudimentary first-line defense function with no memory. However, both in vitro and in vivo studies reveal that innate leukocytes may adopt distinct activation states such as priming, tolerance, and exhaustion, depending upon the history of prior challenges. The dynamic programming and potential memory of innate leukocytes may have far-reaching consequences in health and disease. This review aims to provide some salient features of innate programing and memory, patho-physiological consequences, underlying mechanisms, and current pressing issues.

Design Principles of Bioresorbable Polymeric Scaffolds.

PubMed

Kossuth, Mary Beth; Perkins, Laura E L; Rapoza, Richard J

2016-07-01

The concept for a bioresorbable vascular scaffold combines the best features of the first 3 generations of percutaneous coronary intervention (namely), balloon angioplasty, bare metallic stents, and drug-eluting stents, into a single device. The principles of operation of a BRS follow 3 phases of functionality that reflect the different physiologic requirements over time; revascularization, restoration, and resorption. Most BRS designs make use of the continuum of hydrolytic degradation in aliphatic polyesters, such as poly(l-lactide), in which molecular weight, strength, and mass decrease progressively in 3 distinct stages, consistent with the in vivo requirements of each performance phase. Copyright © 2016 Elsevier Inc. All rights reserved.

Excitatory amino acid neurotoxicity and neurodegenerative disease.

PubMed

Meldrum, B; Garthwaite, J

1990-09-01

The progress over the last 30 years in defining the role of excitatory amino acids in normal physiological function and in the abnormal neuronal activity of epilepsy has been reviewed in earlier articles in this series. In the last five years it has become clear that excitatory amino acids also play a role in a wide range of neurodegenerative processes. The evidence is clearest where the degenerative process is acute, but is more controversial for slow degenerative processes. In this article Brian Meldrum and John Garthwaite review in vivo and in vitro studies of the cytotoxicity of amino acids and summarize the contribution of such toxicity to acute and chronic neurodegenerative disorders.

Functional Characterization of Resting and Adenovirus-Induced Reactive Astrocytes in Three-Dimensional Culture

PubMed Central

Woo, Junsung; Im, Sun-Kyoung; Chun, Heejung; Jung, Soon-Young; Oh, Soo-Jin; Choi, Nakwon

2017-01-01

Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native in vivo counterparts. Moreover, we also induced reactive astrocytosis in culture by infecting with high-titer adenovirus to mimic pathophysiological conditions in vivo. Adenoviral infection induced morphological changes in astrocytes, increased passive conductance, and increased GABA content as well as tonic GABA release, which are characteristics of reactive gliosis. Together, our study presents a powerful in vitro model resembling both physiological and pathophysiological conditions in vivo, and thereby provides a versatile experimental tool for studying various neurological diseases that accompany reactive astrocytes. PMID:28680301

A proposed food breakdown classification system to predict food behavior during gastric digestion.

PubMed

Bornhorst, Gail M; Ferrua, Maria J; Singh, R Paul

2015-05-01

The pharmaceutical industry has implemented the Biopharmaceutics Classification System (BCS), which is used to classify drug products based on their solubility and intestinal permeability. The BCS can help predict drug behavior in vivo, the rate-limiting mechanism of absorption, and the likelihood of an in vitro-in vivo correlation. Based on this analysis, we have proposed a Food Breakdown Classification System (FBCS) framework that can be used to classify solid foods according to their initial hardness and their rate of softening during physiological gastric conditions. The proposed FBCS will allow for prediction of food behavior during gastric digestion. The applicability of the FBCS framework in differentiating between dissimilar solid foods was demonstrated using four example foods: raw carrot, boiled potato, white rice, and brown rice. The initial hardness and rate of softening parameter (softening half time) were determined for these foods as well as their hypothesized FBCS class. In addition, we have provided future suggestions as to the methodological and analytical challenges that need to be overcome prior to widespread use and adoption of this classification system. The FBCS gives a framework that may be used to classify food products based on their material properties and their behavior during in vitro gastric digestion, and may also be used to predict in vivo food behavior. As consumer demand increases for functional and "pharma" food products, the food industry will need widespread testing of food products for their structural and functional performance during digestion. © 2015 Institute of Food Technologists®

S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins.

PubMed

Dorwart, Michael R; Wray, Robin; Brautigam, Chad A; Jiang, Youxing; Blount, Paul

2010-12-07

While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C(8)E(5) detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.

Evidence of isometric function of the flexor hallucis longus muscle in normal gait.

PubMed

Kirane, Y M; Michelson, J D; Sharkey, N A

2008-01-01

Studying mechanics of the muscles spanning multiple joints provides insights into intersegmental dynamics and movement coordination. Multiarticular muscles are thought to function at "near-isometric" lengths to transfer mechanical energy between the adjacent body segments. Flexor hallucis longus (FHL) is a multiarticular flexor of the great toe; however, its potential isometric function has received little attention. We used a robotic loading apparatus to investigate FHL mechanics during simulated walking in cadaver feet, and hypothesized that physiological force transmission across the foot can occur with isometric FHL function. The extrinsic foot tendons, stripped of the muscle fibers, were connected to computer-controlled linear actuators. The FHL activity was controlled using force-feedback (FC) based upon electromyographic data from healthy subjects, and subsequently, isometric positional feedback (PC), maintaining the FHL myotendinous junction stationary during simulated walking. Tendon forces and excursions were recorded, as were the strains within the first metatarsal. Forces in the metatarsal and metatarsophalangeal joint were derived from these strains. The FHL tendon excursion under FC was 6.57+/-3.13mm. The forces generated in the FHL tendon, metatarsal and metatarsophalangeal joint with the FHL under isometric PC were not significantly different in pattern from FC. These observations provide evidence that physiological forces could be generated along the great toe with isometric FHL function. A length servo mechanism such as the stretch reflex could likely control the isometric FHL function during in vivo locomotion; this could have interesting implications regarding the conditions of impaired stretch reflex such as spastic paresis and peripheral neuropathies.

Detection of low-amplitude in vivo intrinsic signals from an optical imager of retinal function

NASA Astrophysics Data System (ADS)

Barriga, Eduardo S.; T'so, Dan; Pattichis, Marios; Kwon, Young; Kardon, Randy; Abramoff, Michael; Soliz, Peter

2006-02-01

In the early stages of some retinal diseases, such as glaucoma, loss of retinal activity may be difficult to detect with today's clinical instruments. Many of today's instruments focus on detecting changes in anatomical structures, such as the nerve fiber layer. Our device, which is based on a modified fundus camera, seeks to detect changes in optical signals that reflect functional changes in the retina. The functional imager uses a patterned stimulus at wavelength of 535nm. An intrinsic functional signal is collected at a near infrared wavelength. Measured changes in reflectance in response to the visual stimulus are on the order of 0.1% to 1% of the total reflected intensity level, which makes the functional signal difficult to detect by standard methods because it is masked by other physiological signals and by imaging system noise. In this paper, we analyze the video sequences from a set of 60 experiments with different patterned stimuli from cats. Using a set of statistical techniques known as Independent Component Analysis (ICA), we estimate the signals present in the videos. Through controlled simulation experiments, we quantify the limits of signal strength in order to detect the physiological signal of interest. The results of the analysis show that, in principle, signal levels of 0.1% (-30dB) can be detected. The study found that in 86% of the animal experiments the patterned stimuli effects on the retina can be detected and extracted. The analysis of the different responses extracted from the videos can give an insight of the functional processes present during the stimulation of the retina.

Progress in Integrative Biomaterial Systems to Approach Three-Dimensional Cell Mechanotransduction

PubMed Central

Zhang, Ying; Liao, Kin; Li, Chuan; Lai, Alvin C.K.; Foo, Ji-Jinn

2017-01-01

Mechanotransduction between cells and the extracellular matrix regulates major cellular functions in physiological and pathological situations. The effect of mechanical cues on biochemical signaling triggered by cell–matrix and cell–cell interactions on model biomimetic surfaces has been extensively investigated by a combination of fabrication, biophysical, and biological methods. To simulate the in vivo physiological microenvironment in vitro, three dimensional (3D) microstructures with tailored bio-functionality have been fabricated on substrates of various materials. However, less attention has been paid to the design of 3D biomaterial systems with geometric variances, such as the possession of precise micro-features and/or bio-sensing elements for probing the mechanical responses of cells to the external microenvironment. Such precisely engineered 3D model experimental platforms pave the way for studying the mechanotransduction of multicellular aggregates under controlled geometric and mechanical parameters. Concurrently with the progress in 3D biomaterial fabrication, cell traction force microscopy (CTFM) developed in the field of cell biophysics has emerged as a highly sensitive technique for probing the mechanical stresses exerted by cells onto the opposing deformable surface. In the current work, we first review the recent advances in the fabrication of 3D micropatterned biomaterials which enable the seamless integration with experimental cell mechanics in a controlled 3D microenvironment. Then, we discuss the role of collective cell–cell interactions in the mechanotransduction of engineered tissue equivalents determined by such integrative biomaterial systems under simulated physiological conditions. PMID:28952551

Simultaneous in vivo positron emission tomography and magnetic resonance imaging.

PubMed

Catana, Ciprian; Procissi, Daniel; Wu, Yibao; Judenhofer, Martin S; Qi, Jinyi; Pichler, Bernd J; Jacobs, Russell E; Cherry, Simon R

2008-03-11

Positron emission tomography (PET) and magnetic resonance imaging (MRI) are widely used in vivo imaging technologies with both clinical and biomedical research applications. The strengths of MRI include high-resolution, high-contrast morphologic imaging of soft tissues; the ability to image physiologic parameters such as diffusion and changes in oxygenation level resulting from neuronal stimulation; and the measurement of metabolites using chemical shift imaging. PET images the distribution of biologically targeted radiotracers with high sensitivity, but images generally lack anatomic context and are of lower spatial resolution. Integration of these technologies permits the acquisition of temporally correlated data showing the distribution of PET radiotracers and MRI contrast agents or MR-detectable metabolites, with registration to the underlying anatomy. An MRI-compatible PET scanner has been built for biomedical research applications that allows data from both modalities to be acquired simultaneously. Experiments demonstrate no effect of the MRI system on the spatial resolution of the PET system and <10% reduction in the fraction of radioactive decay events detected by the PET scanner inside the MRI. The signal-to-noise ratio and uniformity of the MR images, with the exception of one particular pulse sequence, were little affected by the presence of the PET scanner. In vivo simultaneous PET and MRI studies were performed in mice. Proof-of-principle in vivo MR spectroscopy and functional MRI experiments were also demonstrated with the combined scanner.

MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3β expression.

PubMed

Jiang, J-J; Liu, C-M; Zhang, B-Y; Wang, X-W; Zhang, M; Saijilafu; Zhang, S-R; Hall, P; Hu, Y-W; Zhou, F-Q

2015-08-27

MicroRNAs are emerging to be important epigenetic factors that control axon regeneration. Here, we report that microRNA-26a (miR-26a) is a physiological regulator of mammalian axon regeneration in vivo. We demonstrated that endogenous miR-26a acted to target specifically glycogen synthase kinase 3β (GSK3β) in adult mouse sensory neurons in vitro and in vivo. Inhibition of endogenous miR-26a in sensory neurons impaired axon regeneration in vitro and in vivo. Moreover, the regulatory effect of miR-26a was mediated by increased expression of GSK3β because downregulation or pharmacological inhibition of GSK3β fully rescued axon regeneration. Our results also suggested that the miR-26a-GSK3β pathway regulated axon regeneration at the neuronal soma by controlling gene expression. We provided biochemical and functional evidences that the regeneration-associated transcription factor Smad1 acted downstream of miR-26a and GSK3β to control sensory axon regeneration. Our study reveals a novel miR-26a-GSK3β-Smad1 signaling pathway in the regulation of mammalian axon regeneration. Moreover, we provide the first evidence that, in addition to inhibition of GSK3β kinase activity, maintaining a lower protein level of GSK3β in neurons by the microRNA is necessary for efficient axon regeneration.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans.

PubMed

Lagido, Cristina; McLaggan, Debbie; Glover, L Anne

2015-10-16

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode's ATP levels.(1-3) Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.(4,5) In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

Identification of an ovarian voltage-activated Na+-channel type: hints to involvement in luteolysis.

PubMed

Bulling, A; Berg, F D; Berg, U; Duffy, D M; Stouffer, R L; Ojeda, S R; Gratzl, M; Mayerhofer, A

2000-07-01

An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24-48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24-48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.

Functional Brace in ACL Surgery: Force Quantification in an In Vivo Study

PubMed Central

LaPrade, Robert F.; Venderley, Melanie B.; Dahl, Kimi D.; Dornan, Grant J.; Turnbull, Travis Lee

2017-01-01

Background: A need exists for a functional anterior cruciate ligament (ACL) brace that dynamically supports the knee joint to match the angle-dependent forces of a native ACL, especially in the early postoperative period. Purpose/Hypothesis: The purpose of this study was to quantify the posteriorly directed external forces applied to the anterior proximal tibia by both a static and a dynamic force ACL brace. The proximal strap forces applied by the static force brace were hypothesized to remain relatively constant regardless of knee flexion angle compared with those of the dynamic force brace. Study Design: Controlled laboratory study. Methods: Seven healthy adult males (mean age, 27.4 ± 3.4 years; mean height, 1.8 ± 0.1 m; mean body mass, 84.1 ± 11.3 kg) were fitted with both a static and a dynamic force ACL brace. Participants completed 3 functional activities: unloaded extension, sit-to-stand, and stair ascent. Kinematic data were collected using traditional motion-capture techniques while posteriorly directed forces applied to the anterior aspect of both the proximal and distal tibia were simultaneously collected using a customized pressure-mapping technique. Results: The mean posteriorly directed forces applied to the proximal tibia at 30° of flexion by the dynamic force brace during unloaded extension (80.2 N), sit-to-stand (57.5 N), and stair ascent (56.3 N) activities were significantly larger, regardless of force setting, than those applied by the static force brace (10.1 N, 9.5 N, and 11.9 N, respectively; P < .001). Conclusion: The dynamic force ACL brace, compared with the static force brace, applied significantly larger posteriorly directed forces to the anterior proximal tibia in extension, where the ACL is known to experience larger in vivo forces. Further studies are required to determine whether the physiological behavior of the brace will reduce anterior knee laxity and improve long-term patient outcomes. Clinical Relevance: ACL braces that dynamically restrain the proximal tibia in a manner similar to physiological ACL function may improve pre- and postoperative treatment. PMID:28748195

Imaging Reactive Oxygen Species-Induced Modifications in Living Systems.

PubMed

Maulucci, Giuseppe; Bačić, Goran; Bridal, Lori; Schmidt, Harald Hhw; Tavitian, Bertrand; Viel, Thomas; Utsumi, Hideo; Yalçın, A Süha; De Spirito, Marco

2016-06-01

Reactive Oxygen Species (ROS) may regulate signaling, ion channels, transcription factors, and biosynthetic processes. ROS-related diseases can be due to either a shortage or an excess of ROS. Since the biological activity of ROS depends on not only concentration but also spatiotemporal distribution, real-time imaging of ROS, possibly in vivo, has become a need for scientists, with potential for clinical translation. New imaging techniques as well as new contrast agents in clinically established modalities were developed in the previous decade. An ideal imaging technique should determine ROS changes with high spatio-temporal resolution, detect physiologically relevant variations in ROS concentration, and provide specificity toward different redox couples. Furthermore, for in vivo applications, bioavailability of sensors, tissue penetration, and a high signal-to-noise ratio are additional requirements to be satisfied. None of the presented techniques fulfill all requirements for clinical translation. The obvious way forward is to incorporate anatomical and functional imaging into a common hybrid-imaging platform. Antioxid. Redox Signal. 24, 939-958.

Assessment of the probiotic potential of a dairy product fermented by Propionibacterium freudenreichii in piglets.

PubMed

Cousin, Fabien J; Foligné, Benoît; Deutsch, Stéphanie-Marie; Massart, Sébastien; Parayre, Sandrine; Le Loir, Yves; Boudry, Gaëlle; Jan, Gwénaël

2012-08-15

Dairy propionibacteria, including Propionibacterium freudenreichii , display promising probiotic properties, including immunomodulation. These properties are highly strain-dependent and rarely studied in a fermented dairy product. We screened 10 strains, grown in a newly developed fermented milk ultrafiltrate, for immunomodulatory properties in vitro. The most anti-inflammatory strain, P. freudenreichii BIA129, was further tested on piglets. P. freudenreichii -fermented product improved food intake and growth of piglets. Colonic mucosa explants of treated pigs secreted less interleukin 8 (-25%, P < 0.05) and tumor necrosis factor α (-20%, P < 0.05), either in basal conditions or after a lipopolysaccharide challenge. By contrast, the gut structure, barrier function (measured ex vivo in Ussing chambers), microbial diversity (assessed by 16S rRNA pyrosequencing), and colonic short-chain fatty acid content were unchanged, assuming maintenance of normal intestinal physiology. In conclusion, this work confirms in vivo probiotic properties of dairy propionibacteria-fermented products, which are promising for the prevention or healing of inflammatory bowel diseases.

Detection of protease activity in cells and animals.

PubMed

Verdoes, Martijn; Verhelst, Steven H L

2016-01-01

Proteases are involved in a wide variety of biologically and medically important events. They are entangled in a complex network of processes that regulate their activity, which makes their study intriguing, but challenging. For comprehensive understanding of protease biology and effective drug discovery, it is therefore essential to study proteases in models that are close to their complex native environments such as live cells or whole organisms. Protease activity can be detected by reporter substrates and activity-based probes, but not all of these reagents are suitable for intracellular or in vivo use. This review focuses on the detection of proteases in cells and in vivo. We summarize the use of probes and substrates as molecular tools, discuss strategies to deliver these tools inside cells, and describe sophisticated read-out techniques such as mass spectrometry and various imaging applications. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Actin Cytoskeletal Disruption following Cryopreservation Alters the Biodistribution of Human Mesenchymal Stromal Cells In Vivo

PubMed Central

Chinnadurai, Raghavan; Garcia, Marco A.; Sakurai, Yumiko; Lam, Wilbur A.; Kirk, Allan D.; Galipeau, Jacques; Copland, Ian B.

2014-01-01

Summary Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic. PMID:25068122