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Activated platelets release adenosine trisphosphate (ATP) and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants. However, bioluminescence analysis of ATP possesses poor linearity, the signal is quickly attenuated, and the accuracy of ATP release from platelets is hard to determine accurately enough to be used in a high throughput screening of platelet inhibitors. The present study was designed to optimize bioluminescence analysis of ATP released by platelets and expand its application in high throughput screening of platelet inhibitors. The results showed that accuracy of ATP analysis was significantly improved by adding coenzyme A (CoA) and signal attenuation of ATP analysis was greatly postponed by adding bovine serum albumin (BSA) both in Hank's balanced salt solution (HBSS) and Tyrode's buffer. Furthermore, ATP release of activated platelets and inhibitory effects of Ly294002 and Staurosporine on platelet activation were accurately determined by our optimized bioluminescence analysis of ATP. Thus, we have successfully constructed an optimized bioluminescence analysis of ATP which can be used in high throughput screening of platelet inhibitors. [ABSTRACT FROM AUTHOR]

Plant trichomes originate from epidermal cell, forming protective structure from abiotic and biotic stresses. Different from the unicellular trichome in Arabidopsis, tomato trichomes are multicellular structure and can be classified into seven different types based on cell number, shape and the presence of glandular cells. Despite the importance of tomato trichomes in insect resistance, our understanding of the tomato trichome morphogenesis remains elusive. In this study, we quantitatively analyzed morphological traits of trichomes in tomato and further performed live imaging of cytoskeletons in stably transformed lines with actin and microtubule markers. At different developmental stages, two types of cytoskeletons exhibited distinct patterns in different trichome cells, ranging from transverse, spiral to longitudinal. This gradual transition of actin filament angle from basal to top cells could correlate with the spatial expansion mode in different cells. Further genetic screen for aberrant trichome morphology led to the discovery of a number of independent mutations in SCAR/WAVE and ARP2/3 complex, which resulted in actin bundling and distorted trichomes. Disruption of microtubules caused isotropic expansion while abolished actin filaments entirely inhibited axial extension of trichomes, indicating that microtubules and actin filaments may control distinct aspects of trichome cell expansion. Our results shed light on the roles of cytoskeletons in the formation of multicellular structure of tomato trichomes. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate in order to generate myofibroblasts. When MSCs were seeded in solid collagen scaffolds, they differentiated into myofibroblasts that were observed to strongly bind to the substrate, forming a 3D cell scaffold network that developed tension and shortening after KCl stimulation. Moreover, MSC-laden scaffolds recapitulated the Frank-Starling mechanism so that active tension increased in response to increases in the initial length of the contractile system. This constituted a bioengineering tissue that exhibited the contractile properties observed in both striated and smooth muscles. By using the A. F. Huxley formalism, we determined the myosin crossbridge (CB) kinetics of attachment (f1) and detachment (g1 and g2), maximum myosin ATPase activity, molar myosin concentration, unitary CB force and maximum CB efficiency. CB kinetics were dramatically slow, characterizing the non-muscle myosin type IIA (NMMIIA) present in myofibroblasts. When MSCs were seeded in solid collagen scaffolds functionalized with Arg-Gly-Asp (RGD), contractility increased and CB kinetics were modified, whereas the unitary NMMIIA-CB force and maximum CB efficiency did not change. In conclusion, we provided a non-muscle bioengineering tissue whose molecular mechanical characteristics of NMMIIA were very close to those of a non-muscle contractile tissue such as the human placenta. [ABSTRACT FROM AUTHOR]

Mechanical stimulation appears to play a key role in cartilage homeostasis maintenance, but it can also contribute to osteoarthritis (OA) pathogenesis. Accumulating evidence suggests that cartilage loading in the physiological range contributes to tissue integrity maintenance, whereas excessive or reduced loading have catabolic effects. However, how mechanical stimuli can regulate joint homeostasis is still not completely elucidated and few data are available on human cartilage. We aimed at investigating human OA cartilage response to ex vivo loading at physiological intensity. Cartilage explants from ten OA patients were subjected to ex vivo controlled compression, then recovered and used for gene and protein expression analysis of cartilage homeostasis markers. Compressed samples were compared to uncompressed ones in presence or without interleukin 1β (IL-1β) or interleukin 4 (IL-4). Cartilage explants compressed in combination with IL-4 treatment showed the best histological scores. Mechanical stimulation was able to significantly modify the expression of collagen type II (collagen 2), aggrecan, SOX9 transcription factor, cartilage oligomeric matrix protein (COMP), collagen degradation marker C2C and vascular endothelial growth factor (VEGF). Conversely, ADAMTS4 metallopeptidase, interleukin 4 receptor alpha (IL4Rα), chondroitin sulfate 846 epitope (CS846), procollagen type 2 C-propeptide (CPII) and glycosaminoglycans (GAG) appeared not modulated. Our data suggest that physiological compression of OA human cartilage modulates the inflammatory milieu by differently affecting the expression of components and homeostasis regulators of the cartilage extracellular matrix. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) proteins form Ca2+-permeable, nonselective cation channels, but their role in neuronal Ca2+ homeostasis is elusive. In the present paper, we show that TRPC channels potently regulate synaptic plasticity by changing the presynaptic Ca2+-homeostasis of hippocampal neurons. Specifically, loss of TRPC1/C4/C5 channels decreases basal-evoked secretion, decreases the pool size of readily releasable vesicles, and accelerates synaptic depression during high-frequency stimulation (HFS). In contrast, primary TRPC5 channel-expressing neurons, identified by a novel TRPC5–τ-green fluorescent protein (τGFP) knockin mouse line, show strong short-term enhancement (STE) of synaptic signaling during HFS, indicating a key role of TRPC5 in short-term plasticity. Lentiviral expression of either TRPC1 or TRPC5 turns classic synaptic depression of wild-type neurons into STE, demonstrating that TRPCs are instrumental in regulating synaptic plasticity. Presynaptic Ca2+ imaging shows that TRPC activity strongly boosts synaptic Ca2+ dynamics, showing that TRPC channels provide an additional presynaptic Ca2+ entry pathway, which efficiently regulates synaptic strength and plasticity. [ABSTRACT FROM AUTHOR]

Aims: Glucocorticoid (GC) stimulation has been shown to increase cardiac contractility by elevated intracellular [Ca] but the sources for Ca entry are unclear. This study aims to determine the role of store-operated Ca entry (SOCE) for GC-mediated inotropy. Methods and results: Dexamethasone (Dex) pretreatment significantly increased cardiac contractile force ex vivo in Langendorff-perfused Sprague-Dawley rat hearts (2 mg/kg BW i.p. Dex 24 h prior to experiment). Moreover, Ca transient amplitude as well as fractional shortening were significantly enhanced in Fura-2-loaded isolated rat ventricular myocytes exposed to Dex (1 mg/mL Dex, 24 h). Interestingly, these Dex-dependent effects could be abolished in the presence of SOCE-inhibitors SKF-96356 (SKF, 2 μM) and BTP2 (5 μM). Ca transient kinetics (time to peak, decay time) were not affected by SOCE stimulation. Direct SOCE measurements revealed a negligible magnitude in untreated myocytes but a dramatic increase in SOCE upon Dex-pretreatment. Importantly, the Dex-dependent stimulation of SOCE could be blocked by inhibition of serum and glucocorticoid-regulated kinase 1 (SGK1) using EMD638683 (EMD, 50 μM). Dex preincubation also resulted in increased mRNA expression of proteins involved in SOCE (stromal interaction molecule 2, STIM2, and transient receptor potential cation channels 3/6, TRPC 3/6), which were also prevented in the presence of EMD. Conclusion: Short-term GC-stimulation with Dex improves cardiac contractility by a SOCE-dependent mechanism, which appears to involve increased SGK1-dependent expression of the SOCE-related proteins. Since Ca transient kinetics were unaffected, SOCE appears to influence Ca cycling more by an integrated response across multiple cardiac cycles but not on a beat-to-beat basis. [ABSTRACT FROM AUTHOR]

We compared a published computational model of the action potential of a specific type of human induced pluripotent stem cell -derived cardiomyocytes (hiPSC-CM) with experimental field potential data with regard to their inter-beat interval and the duration of repolarization. In particular, concomitant changes in inter-beat interval and duration of repolarization were calculated after reduction and/or augmentation of specific ion channel conductances as a surrogate for pharmacological manipulation. The observed mismatches between calculations and experimental data indicate that there is information missing about the cellular test system. Based on our results we hypothesize that, among other currents, the actual If (“funny current”) may deviate from the prediction. We show that replacement of the If formulation by alternative equations causes the model predictions to change qualitatively, however, none of the available formulations is actually achieving a satisfactory match with experimental data. We suggest a strategy to clarify whether the mismatch can be completely resolved at all using single cell models and, if yes, how this goal could be reached. [ABSTRACT FROM AUTHOR]

Pituitary endocrine cells fire action potentials (APs) to regulate their cytosolic Ca2+ concentration and hormone secretion rate. Depending on animal species, cell type, and biological conditions, pituitary APs are generated either by TTX-sensitive Na+ currents (INa), high-voltage activated Ca2+ currents (ICa), or by a combination of the two. Previous computational models of pituitary cells have mainly been based on data from rats, where INa is largely inactivated at the resting potential, and spontaneous APs are predominantly mediated by ICa. Unlike in rats, spontaneous INa-mediated APs are consistently seen in pituitary cells of several other animal species, including several species of fish. In the current work we develop a computational model of gonadotropin releasing cells in the teleost fish medaka (Oryzias latipes). The model stands out from previous modeling efforts by being (1) the first model of a pituitary cell in teleosts, (2) the first pituitary cell model that fires sponateous APs that are predominantly mediated by INa, and (3) the first pituitary cell model where the kinetics of the depolarizing currents, INa and ICa, are directly fitted to voltage-clamp data. We explore the firing properties of the model, and compare it to the properties of previous models that fire ICa-based APs. We put a particular focus on how the big conductance K+ current (IBK) modulates the AP shape. Interestingly, we find that IBK can prolong AP duration in models that fire ICa-based APs, while it consistently shortens the duration of the predominantly INa-mediated APs in the medaka gonadotroph model. Although the model is constrained to experimental data from gonadotroph cells in medaka, it may likely provide insights also into other pituitary cell types that fire INa-mediated APs. [ABSTRACT FROM AUTHOR]

Neuronal physiology is particularly sensitive to acute stressors that affect excitability, many of which can trigger seizures and epilepsies. Although intrinsic neuronal homeostasis plays an important role in maintaining overall nervous system robustness and its resistance to stressors, the specific genetic and molecular mechanisms that underlie these processes are not well understood. Here we used a reverse genetic approach in Drosophila to test the hypothesis that specific voltage-gated ion channels contribute to neuronal homeostasis, robustness, and stress resistance. We found that the activity of the voltage-gated potassium channel seizure (sei), an ortholog of the mammalian ERG channel family, is essential for protecting flies from acute heat-induced seizures. Although sei is broadly expressed in the nervous system, our data indicate that its impact on the organismal robustness to acute environmental stress is primarily mediated via its action in excitatory neurons, the octopaminergic system, as well as neuropile ensheathing and perineurial glia. Furthermore, our studies suggest that human mutations in the human ERG channel (hERG), which have been primarily implicated in the cardiac Long QT Syndrome (LQTS), may also contribute to the high incidence of seizures in LQTS patients via a cardiovascular-independent neurogenic pathway. [ABSTRACT FROM AUTHOR]

Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosome-localized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis. [ABSTRACT FROM AUTHOR]

Somatosensory thalamocortical (TC) neurons from the ventrobasal (VB) thalamus are central components in the flow of sensory information between the periphery and the cerebral cortex, and participate in the dynamic regulation of thalamocortical states including wakefulness and sleep. This property is reflected at the cellular level by the ability to generate action potentials in two distinct firing modes, called tonic firing and low-threshold bursting. Although the general properties of TC neurons are known, we still lack a detailed characterization of their morphological and electrical properties in the VB thalamus. The aim of this study was to build biophysically-detailed models of VB TC neurons explicitly constrained with experimental data from rats. We recorded the electrical activity of VB neurons (N = 49) and reconstructed morphologies in 3D (N = 50) by applying standardized protocols. After identifying distinct electrical types, we used a multi-objective optimization to fit single neuron electrical models (e-models), which yielded multiple solutions consistent with the experimental data. The models were tested for generalization using electrical stimuli and neuron morphologies not used during fitting. A local sensitivity analysis revealed that the e-models are robust to small parameter changes and that all the parameters were constrained by one or more features. The e-models, when tested in combination with different morphologies, showed that the electrical behavior is substantially preserved when changing dendritic structure and that the e-models were not overfit to a specific morphology. The models and their analysis show that automatic parameter search can be applied to capture complex firing behavior, such as co-existence of tonic firing and low-threshold bursting over a wide range of parameter sets and in combination with different neuron morphologies. [ABSTRACT FROM AUTHOR]

Cecropia obtusa is popularly used in the Amazonian region and exhibits antioxidant activity. Cosmetic formulations containing C. obtusa extract are commercially available for purchase; however, the chemical composition and the effects of the topical application of the extract are not described in the literature. Therefore, this study aimed to identify the main components of C. obtusa for the first time and to assess the anti aging effect in human fibroblasts and keratinocytes exposed to UVR. The main components in C. obtusa extract were identified by LC-DAD-MS/MS as chlorogenic acid (CGA), luteolin-C-hexoside, luteolin-C-hexose-O-deoxy-hexose, and apigenin-C-hexose-O-deoxy-hexose. C. obtusa extract and CGA decreased the metalloproteinase-1 and protein carbonyl levels and increased the collagen and hyaluronic acid contents. Overall, the extract exhibited better activity than CGA, and we demonstrated the ability of the extract to protect against the UV-induced increase in the pro inflammatory cytokines IL-1β and IL-6, which are potential pathways of the antioxidant and anti aging effect. The chemical characterization added important data to broaden the knowledge related to C. obtusa, and the results suggest that the extract is a promising candidate to be incorporated in topical photochemoprotective formulations. [ABSTRACT FROM AUTHOR]

Optogenetics enables the selective activation of genetically-targeted neuronal populations using light-sensitive ion channels. Genetic strategies using Cre-dependent mouse strains, especially the Ai32 line expressing Channelrhodopsin (ChR2)-EYFP fusion protein, have been a popular means to drive opsin expression in a cell-type specific manner. Here we report a low level of leaky ‘off-target’ (Cre-independent) ChR2-EYFP expression in Ai32/Ai32 homozygous mice throughout the nervous system. This leaky off-target expression was characterized in multiple prevalent nervous system regions using anti-EYFP immunostaining. Expression of full-length ChR2-EYFP protein was confirmed using immunoprecipitation followed by Western blotting. Notably, light stimulation of these ChR2-EYFP expressing neurons in the spinal cord dorsal horn did not induce detectable photocurrents in juvenile 4-week old mice. Given the wide use of the Ai32 line by many labs, our results suggest researchers should be vigilant of possible off-target ChR2-EYFP expression in their region of interest, especially when generating Ai32/Ai32 homozygotes to drive high levels of ChR2-EYFP expression in adult mice. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis occurs in many forms of heart disease and is considered to be one of the main arrhythmogenic factors. Regions with a high density of fibroblasts are likely to cause blocks of wave propagation that give rise to dangerous cardiac arrhythmias. Therefore, studies of the wave propagation through these regions are very important, yet the precise mechanisms leading to arrhythmia formation in fibrotic cardiac tissue remain poorly understood. Particularly, it is not clear how wave propagation is organized at the cellular level, as experiments show that the regions with a high percentage of fibroblasts (65-75%) are still conducting electrical signals, whereas geometric analysis of randomly distributed conducting and non-conducting cells predicts connectivity loss at 40% at the most (percolation threshold). To address this question, we used a joint in vitro-in silico approach, which combined experiments in neonatal rat cardiac monolayers with morphological and electrophysiological computer simulations. We have shown that the main reason for sustainable wave propagation in highly fibrotic samples is the formation of a branching network of cardiomyocytes. We have successfully reproduced the morphology of conductive pathways in computer modelling, assuming that cardiomyocytes align their cytoskeletons to fuse into cardiac syncytium. The electrophysiological properties of the monolayers, such as conduction velocity, conduction blocks and wave fractionation, were reproduced as well. In a virtual cardiac tissue, we have also examined the wave propagation at the subcellular level, detected wavebreaks formation and its relation to the structure of fibrosis and, thus, analysed the processes leading to the onset of arrhythmias. [ABSTRACT FROM AUTHOR]

Primary cilia of renal epithelial cells express several members of the transient receptor potential (TRP) class of cation-conducting channel, including TRPC1, TRPM3, TRPM4, TRPP2, and TRPV4. Some cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by defects in TRPP2 (also called polycystin-2, PC2, or PKD2). A large-conductance, TRPP2-dependent channel in renal cilia has been well described, but it is not known whether this channel includes any other protein subunits. To study this question, we investigated the pharmacology of the TRPP2-dependent channel through electrical recordings from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. The pharmacology was found to match that of TRPM3 channels. The ciliary TRPP2-dependent channel is known to be activated by depolarization and by increasing cytoplasmic Ca2+. This activation was greatly enhanced by external pregnenolone sulfate, an agonist of TRPM3 channels. Pregnenolone sulfate did not change the single-channel current-voltage relation. The channels were effectively blocked by isosakuranetin, a specific inhibitor of TRPM3 channels. Both pregnenolone sulfate and isosakuranetin were effective at concentrations as low as 1 μM. Knocking out TRPM3 by CRISPR/Cas9 genome editing eliminated the ciliary channel. Thus the channel is both TRPM3-dependent and TRPP2-dependent, suggesting that it may include both types of subunit. Knocking out TRPM3 did not change the level of TRPP2 protein in the cilia, so it is unlikely that the absence of functional ciliary channels results from a failure of trafficking. [ABSTRACT FROM AUTHOR]

Vocal fold fibroblasts (VFF) constitute the main cell type of the vocal fold’s lamina propria, produce the extracellular matrix and thereby determine the tissue characteristics. To study VFF behavior under in vitro conditions it is important to mimic the dynamic environment of the in vivo state. The aim of our study was to develop and validate a novel phonomimetic bioreactor system mainly based on commercially available components. The use of cell culture dishes with flexible silicone bottoms in combination with a suitable loudspeaker made it possible to expose the cells to various kinds of phonatory stimuli. The fundamental vibration characteristics of silicone membranes were investigated with and without cell culture medium by laser Doppler vibrometry. Human VFF were seeded in flexible-bottomed plates and placed in a custom-made housing containing a loudspeaker. After the cells were exposed to a predefined audio stimulation protocol, cell viability was assessed and gene as well as protein expression levels were compared to static controls. Laser Doppler vibrometry revealed that addition of cell culture medium changed the resonance frequencies of vibrating membranes. Gene expression of hyaluronan synthase 2, collagen III, fibronectin and TGFβ-1 was significantly upregulated in VFF exposed to vibration, compared to static control. Vibration also significantly upregulated collagen I gene and protein expression. We present a new type of phonomimetic bioreactor. Compared to previous models, our device is easy to assemble and cost-effective, yet can provide a wide spectrum of phonatory stimuli based on the entire dynamic range of the human voice. Gene expression data of VFF cultured in our phonomimetic bioreactor show a significant effect of vibration on ECM metabolism, which illustrates the efficacy of our device. [ABSTRACT FROM AUTHOR]

Loss of function of the tumor suppressor p53 is known to increase the rate of migration of cells transiting the narrow pores of the traditional Boyden chamber assay. Here by contrast we investigate how p53 impacts the rate of cellular migration within a 2D confluent cell layer and a 3D collagen-embedded multicellular spheroid. We use two human carcinoma cell lines, the bladder carcinoma EJ and the colorectal carcinoma HCT116. In the confluent 2-D cell layer, for both EJ and HCT cells the migratory speeds and effective diffusion coefficients for the p53 null cells were significantly smaller than in p53-expressing cells. Compared to p53 expressers, p53-null cells exhibited more organized cortical actin rings together with reduced front-rear cell polarity. Furthermore, loss of p53 caused cells to exert smaller traction forces upon their substrates, and reduced formation of cryptic lamellipodia. In the 3D multicellular spheroid, loss of p53 consistently reduced collective cellular migration into surrounding collagen matrix. As regards the role of p53 in cellular migration, extrapolation from the Boyden chamber assay to other cellular microenvironments is seen to be fraught even in terms of the sign of the effect. Together, these paradoxical results show that the effects of p53 on cellular migration are context-dependent. [ABSTRACT FROM AUTHOR]

Brain coding strategies are enabled by the balance of synaptic inputs that individual neurons receive as determined by the networks in which they reside. Inhibitory cell types contribute to brain function in distinct ways but recording from specific, inhibitory cell types during behaviour to determine their contributions is highly challenging. In particular, the in vivo activities of vasoactive intestinal peptide-expressing interneuron specific 3 (IS3) cells in the hippocampus that only target other inhibitory cells are unknown at present. We perform a massive, computational exploration of possible synaptic inputs to IS3 cells using multi-compartment models and optimized synaptic parameters. We find that asynchronous, in vivo-like states that are sensitive to additional theta-timed inputs (8 Hz) exist when excitatory and inhibitory synaptic conductances are approximately equally balanced and with low numbers of activated synapses receiving correlated inputs. Specifically, under these balanced conditions, the input resistance is larger with higher mean spike firing rates relative to other activated synaptic conditions investigated. Incoming theta-timed inputs result in strongly increased spectral power relative to baseline. Thus, using a generally applicable computational approach we predict the existence and features of background, balanced states in hippocampal circuits. [ABSTRACT FROM AUTHOR]

Aims: Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) predisposes to ventricular tachyarrhythmias (VTs) during high heart rates due to physical or psychological stress. The essential role of catecholaminergic effects on ventricular cardiomyocytes in this situation is well documented, but the importance of heart rate per se for arrhythmia initiation in CPVT1 is largely unexplored. Methods and results: Sixteen CPVT1 patients performed a bicycle stress-test. Occurrence of VT triggers, i.e. premature ventricular complexes (PVC), depended on high heart rate, with individual thresholds. Atrial pacing above the individual PVC threshold in three patients did not induce PVCs. The underlying mechanism for the clinical observation was explored using cardiomyocytes from mice with the RyR2-R2474S (RyR2-RS) mutation, which exhibit exercise-induced VTs. While rapid pacing increased the number of Ca2+ waves in both RyR2-RS and wild-type (p<0.05), β-adrenoceptor (βAR) stimulation induced more Ca2+ waves in RyR2-RS (p<0.05). Notably, Ca2+ waves occurred despite decreased sarcoplasmic reticulum (SR) Ca2+ content in RyR2-RS (p<0.05), suggesting increased cytosolic RyR2 Ca2+ sensitivity. A computational model of mouse ventricular cardiomyocyte electrophysiology reproduced the cellular CPVT1 phenotype when RyR2 Ca2+ sensitivity was increased. Importantly, diastolic fluctuations in phosphorylation of RyR2 and SR Ca2+ content determined Ca2+ wave initiation. These factors were modulated towards increased propensity for arrhythmia initiation by increased pacing rates, but even more by βAR stimulation. Conclusion: In CPVT1, VT propensity depends on individual heart rate thresholds for PVCs. Through converging data from clinical exercise stress-testing, cellular studies and computational modelling, we confirm the heart rate-independent pro-arrhythmic effects of βAR stimulation in CPVT1, but also identify an independent and synergistic contribution from effects of high heart rate. [ABSTRACT FROM AUTHOR]

Microdomains that emerge from spatially constricted spread of biochemical signaling components play a central role in several neuronal computations. Although dendrites, endowed with several voltage-gated ion channels, form a prominent structural substrate for microdomain physiology, it is not known if these channels regulate the spatiotemporal spread of signaling microdomains. Here, we employed a multiscale, morphologically realistic, conductance-based model of the hippocampal pyramidal neuron that accounted for experimental details of electrical and calcium-dependent biochemical signaling. We activated synaptic N-Methyl--Aspartate receptors through theta-burst stimulation (TBS) or pairing (TBP) and assessed microdomain propagation along a signaling pathway that included calmodulin, calcium/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1. We found that the spatiotemporal spread of the TBS-evoked microdomain in phosphorylated CaMKII (pCaMKII) was amplified in comparison to that of the corresponding calcium microdomain. Next, we assessed the role of two dendritically expressed inactivating channels, one restorative (A-type potassium) and another regenerative (T-type calcium), by systematically varying their conductances. Whereas A-type potassium channels suppressed the spread of pCaMKII microdomains by altering the voltage response to TBS, T-type calcium channels enhanced this spread by modulating TBS-induced calcium influx without changing the voltage. Finally, we explored cross-dependencies of these channels with other model components, and demonstrated the heavy mutual interdependence of several biophysical and biochemical properties in regulating microdomains and their spread. Our conclusions unveil a pivotal role for dendritic voltage-gated ion channels in actively amplifying or suppressing biochemical signals and their spatiotemporal spread, with critical implications for clustered synaptic plasticity, robust information transfer and efficient neural coding. [ABSTRACT FROM AUTHOR]

Cellular regulatory networks are not static, but continuously reconfigure in response to stimuli via alterations in protein abundance and confirmation. However, typical computational approaches treat them as static interaction networks derived from a single time point. Here, we provide methods for learning the dynamic modulation of relationships between proteins from static single-cell data. We demonstrate our approach using TGFß induced epithelial-to-mesenchymal transition (EMT) in murine breast cancer cell line, profiled with mass cytometry. We take advantage of the asynchronous rate of transition to EMT in the data and derive a pseudotime EMT trajectory. We propose methods for visualizing and quantifying time-varying edge behavior over the trajectory, and a metric of edge dynamism to predict the effect of drug perturbations on EMT. [ABSTRACT FROM AUTHOR]

Densin is a scaffold protein known to associate with key elements of neuronal signaling. The present study examines the distribution of densin at the ultrastructural level in order to reveal potential sites that can support specific interactions of densin. Immunogold electron microscopy on hippocampal cultures shows intense labeling for densin at postsynaptic densities (PSDs), but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was ~27 nm, with the majority of label (90%) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (90 mM K+ for 2 min) promoted a slight shift of densin label within the PSD complex resulting in 77% of label remaining within 40 nm from the postsynaptic membrane. Densin molecules firmly embedded within the PSD may target a minor pool of CaMKII to substrates at the PSD core. [ABSTRACT FROM AUTHOR]

Hemodynamics may play an essential role in the initiation and progression of abdominal aortic aneurysm (AAA). We aimed to study the mechanism of self-healing process by the changes of hemodynamics and pathology in an enlarging AAA in rabbits. Seventy-two rabbits were randomly divided into three groups. Rabbits underwent extrinsic coarctation and received a 10-minute elastase incubation in Group A and Group B. Absorbable suture used in Group A was terminated by balloon dilation at week 4. Diameter was measured after 1, 3, 5, and 15 weeks, computational fluid dynamics analysis was performed at week 3 and week 15. Rabbits were sacrificed after 1, 5, and 15 weeks for pathological and quantitative studies. The higher velocity magnitude, intensified bulk flow and obvious vortex formation were observed in Group A at week 3 instead of week 15. Both low wall shear stress and high relative residence time increased in Group B, however, high oscillatory shear index had relatively less increase compared with Group A. Aortic diameter reached a plateau at 5 weeks in Group A, which was significantly lower than in week 15 in Group B. Intimal hyperplasia, intima-media thickness increased significantly in Group A at week 5, significantly higher than in week 15 in Group B. Marked destruction of elastin fibers and smooth muscle cells occurred at week 1, and increased significantly at week 15 in Group A. Aneurysm exhibited strong expression of matrix metalloproteinase 9 and mouse anti-rabbit macrophage 11 at week 1, and showed a tendency to decrease. Matrix metalloproteinase 2 expression decreased significantly in Group B at week 15 compared with week 5 and Group A. In conclusion, the self-healing of rabbit AAA may attributed to the regeneration of smooth muscle cells. The turbulence flow caused by coarctation is associated with continuous growth of rabbit AAA and prevents the self-healing phenomenon. [ABSTRACT FROM AUTHOR]

Research in photobiology is currently limited by a lack of devices capable of delivering precise and tunable irradiation to cells in a high-throughput format. This limits researchers to using expensive commercially available or custom-built light sources which make it difficult to replicate, standardize, optimize, and scale experiments. Here we present an open-source Microplate Photoirradiation System (MPS) developed to enable high-throughput light experiments in standard 96 and 24-well microplates for a variety of applications in photobiology research. This open-source system features 96 independently controlled LEDs (4 LEDs per well in 24-well), Wi-Fi connected control and programmable graphical user interface (GUI) for control and programming, automated calibration GUI, and modular control and LED boards for maximum flexibility. A web-based GUI generates light program files containing irradiation parameters for groups of LEDs. These parameters are then uploaded wirelessly, stored and used on the MPS to run photoirradiation experiments inside any incubator. A rapid and semi-quantitative porphyrin metabolism assay was also developed to validate the system in wild-type fibroblasts. Protoporphyrin IX (PpIX) fluorescence accumulation was induced by incubation with 5-aminolevulinic acid (ALA), a photosensitization method leveraged clinically to destroy malignant cell types in a process termed photodynamic therapy (PDT), and cells were irradiated with 405nm light with varying irradiance, duration and pulsation parameters. Immediately after light treatment with the MPS, subsequent photobleaching was measured in live, adherent cells in both 96-well and a 24-well microplates using a microplate reader. Results demonstrate the utility and reliability of the Microplate Photoirradiation System to irradiate cells with precise irradiance and timing parameters in order to measure PpIx photobleaching kinetics in live adherent cells and perform comparable experiments with both 24 and 96 well microplate formats. The high-throughput capability of the MPS enabled measurement of enough irradiance conditions in a single microplate to fit PpIX fluorescence to a bioexponential decay model of photobleaching, as well as reveal a dependency of photobleaching on duty-cycle—but not frequency—in a pulsed irradiance regimen. [ABSTRACT FROM AUTHOR]

Experiments have shown that the relaxation phase of cardiac sarcomeres during an isometric twitch is prolonged in muscles that reached a higher peak tension. However, the mechanism is not completely understood. We hypothesize that the binding of calcium to troponin is enhanced by the tension in the thin filament, thus contributing to the prolongation of contraction upon higher peak tension generation. To test this hypothesis, we developed a computational model of sarcomere mechanics that incorporates tension-dependence of calcium binding. The model was used to simulate isometric twitch experiments with time dependency in the form of a two-state cross-bridge cycle model and a transient intracellular calcium concentration. In the simulations, peak isometric twitch tension appeared to increase linearly by 51.1 KPa with sarcomere length from 1.9 μm to 2.2 μm. Experiments showed an increase of 47.3 KPa over the same range of sarcomere lengths. The duration of the twitch also increased with both sarcomere length and peak intracellular calcium concentration, likely to be induced by the inherently coupled increase of the peak tension in the thin filament. In the model simulations, the time to 50% relaxation (tR50) increased over the range of sarcomere lengths from 1.9 μm to 2.2 μm by 0.11s, comparable to the increased duration of 0.12s shown in experiments. Model simulated tR50 increased by 0.12s over the range of peak intracellular calcium concentrations from 0.87 μM to 1.45 μM. Our simulation results suggest that the prolongation of contraction at higher tension is a result of the tighter binding of Ca2+ to troponin in areas under higher tension, thus delaying the deactivation of the troponin. [ABSTRACT FROM AUTHOR]

The vascular endothelium is considered as a key cell compartment for the response to ionizing radiation of normal tissues and tumors, and as a promising target to improve the differential effect of radiotherapy in the future. Following radiation exposure, the global endothelial cell response covers a wide range of gene, miRNA, protein and metabolite expression modifications. Changes occur at the transcriptional, translational and post-translational levels and impact cell phenotype as well as the microenvironment by the production and secretion of soluble factors such as reactive oxygen species, chemokines, cytokines and growth factors. These radiation-induced dynamic modifications of molecular networks may control the endothelial cell phenotype and govern recruitment of immune cells, stressing the importance of clearly understanding the mechanisms which underlie these temporal processes. A wide variety of time series data is commonly used in bioinformatics studies, including gene expression, protein concentrations and metabolomics data. The use of clustering of these data is still an unclear problem. Here, we introduce kernels between Gaussian processes modeling time series, and subsequently introduce a spectral clustering algorithm. We apply the methods to the study of human primary endothelial cells (HUVECs) exposed to a radiotherapy dose fraction (2 Gy). Time windows of differential expressions of 301 genes involved in key cellular processes such as angiogenesis, inflammation, apoptosis, immune response and protein kinase were determined from 12 hours to 3 weeks post-irradiation. Then, 43 temporal clusters corresponding to profiles of similar expressions, including 49 genes out of 301 initially measured, were generated according to the proposed method. Forty-seven transcription factors (TFs) responsible for the expression of clusters of genes were predicted from sequence regulatory elements using the MotifMap system. Their temporal profiles of occurrences were established and clustered. Dynamic network interactions and molecular pathways of TFs and differential genes were finally explored, revealing key node genes and putative important cellular processes involved in tissue infiltration by immune cells following exposure to a radiotherapy dose fraction. [ABSTRACT FROM AUTHOR]

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30–40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation. [ABSTRACT FROM AUTHOR]

Excess glutamate during intense neuronal activity is not instantly cleared and may accumulate in the extracellular space. This has various long-term consequences such as ectopic signaling, modulation of synaptic efficacy and excitotoxicity; the latter implicated in various neurodevelopmental and neurodegenerative diseases. In this study, the quantitative imaging of glutamate homeostasis of hippocampal slices from methyl-CpG binding protein 2 knock-out (Mecp2-/y) mice, a model of Rett syndrome (RTT), revealed unusual repetitive glutamate transients. They appeared in phase with bursts of action potentials in the CA1 neurons. Both glutamate transients and bursting activity were suppressed by the blockade of sodium, AMPA and voltage-gated calcium channels (T- and R-type), and enhanced after the inhibition of HCN channels. HCN and calcium channels in RTT and wild-type (WT) CA1 neurons displayed different voltage-dependencies and kinetics. Both channels modulated postsynaptic integration and modified the pattern of glutamate spikes in the RTT hippocampus. Spontaneous glutamate transients were much less abundant in the WT preparations, and, when observed, had smaller amplitude and frequency. The basal ambient glutamate levels in RTT were higher and transient glutamate increases (spontaneous and evoked by stimulation of Schaffer collaterals) decayed slower. Both features indicate less efficient glutamate uptake in RTT. To explain the generation of repetitive glutamate spikes, we designed a novel model of glutamate-induced glutamate release. The simulations correctly predicted the patterns of spontaneous glutamate spikes observed under different experimental conditions. We propose that pervasive spontaneous glutamate release is a hallmark of Mecp2-/y hippocampus, stemming from and modulating the hyperexcitability of neurons. [ABSTRACT FROM AUTHOR]

Megakaryocytes (Mks) derive from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and develop into large, polyploid cells that eventually give rise to platelets. As Mks mature, they migrate from the bone marrow niche into the vasculature, where they are exposed to shear forces from blood flow, releasing Mk particles (platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs)) into circulation. We have previously shown that transcription factor p53 is important in Mk maturation, and that physiological levels of shear promote Mk particle generation and platelet biogenesis. Here we examine the role of p53 in the Mk shear-stress response. We show that p53 is acetylated in response to shear in both immature and mature Mks, and that decreased expression of deacetylase HDAC1, and increased expression of the acetyltransferases p300 and PCAF might be responsible for these changes. We also examined the hypothesis that p53 might be involved in the shear-induced Caspase 3 activation, phosphatidylserine (PS) externalization, and increased biogenesis of PLPs, PPTs, and MkMPs. We show that p53 is involved in all these shear-induced processes. We show that in response to shear, acetyl-p53 binds Bax, cytochrome c is released from mitochondria, and Caspase 9 is activated. We also show that shear-stimulated Caspase 9 activation and Mk particle biogenesis depend on transcription-independent p53-induced apoptosis (TIPA), but PS externalization is not. This is the first report to show that shear flow stimulates TIPA and that Caspase 9 activation and Mk-particle biogenesis are directly modulated by TIPA. [ABSTRACT FROM AUTHOR]

The protein von Willebrand factor (VWF) is key for the adhesion of blood platelets to sites of vascular injury. Recent studies have shown that the release of oxidative agents during inflammation increases the platelet-tethering activity of VWF contributing to a pro-thrombotic state. This has been linked to the oxidation of methionine residues in the A1, A2 and A3 domains of VWF. The A1 domain binds to platelet surface receptors glycoprotein Ib α (GpIbα). This interaction has been shown to be inhibited under static conditions by the neighboring A2 domain. Tensile force exerted by blood flow unfolds the A2 domain normally leading to its cleavage by the metalloprotease ADAMTS13 preventing pathological thrombus formation. However, oxidizing conditions inhibit proteolysis through ADAMTS13. Here, molecular dynamics simulations tested the hypothesis whether methionine oxidation induced by inflammatory conditions favors unfolding of the A2 domain contributing to the experimentally observed activation of VWF. The results indicate that oxidation of methionine residues located near the C-terminal helix of the A2 domain reduce the force necessary to initiate unfolding. Furthermore, oxidation of methionine residues shifts the thermodynamic equilibrium of the A2 domain fold towards the denatured state. This work suggests a mechanism whereby oxidation reduces the kinetic and thermodynamic stability of the A2 domain removing its inhibitory function on the binding of the A1 domain to GpIbα. [ABSTRACT FROM AUTHOR]

Large conductance voltage- and calcium-gated channels (BK) control fundamental processes, including smooth muscle contractility and artery diameter. We used a baboon (Papio spp) model of pregnancy that is similar to that of humans to characterize BK channels in the middle cerebral artery and its branches in near-term (165 dGa) primate fetuses and corresponding pregnant mothers. In cell-attached patches (K+pipette = 135 mM) on freshly isolated fetal cerebral artery myocytes, BK currents were identified by large conductance, and voltage- and paxilline-sensitive effects. Their calcium sensitivity was confirmed by a lower Vhalf (transmembrane voltage needed to reach half-maximal current) in inside-out patches at 30 versus 3 μM [Ca2+]free. Immunostaining against the BK channel-forming alpha subunit revealed qualitatively similar levels of BK alpha protein-corresponding fluorescence in fetal and maternal myocytes. Fetal and maternal BK currents recorded at 3 μM [Ca2+]free from excised membrane patches had similar unitary current amplitude, and Vhalf. However, subtle differences between fetal and maternal BK channel phenotypes were detected in macroscopic current activation kinetics. To assess BK function at the organ level, fetal and maternal artery branches were pressurized in vitro at 30 mmHg and probed with the selective BK channel blocker paxilline (1 μM). The degree of paxilline-induced constriction was similar in fetal and maternal arteries, yet the constriction of maternal arteries was achieved sooner. In conclusion, we present a first identification and characterization of fetal cerebral artery BK channels in myocytes from primates. Although differences in BK channels between fetal and maternal arteries exist, the similarities reported herein advance the idea that vascular myocyte BK channels are functional near-term, and thus may serve as pharmacological targets during the perinatal-neonatal period. [ABSTRACT FROM AUTHOR]

Cartilage tissue engineering typically involves the combination of a biodegradable polymeric support material with chondrocytes. The culture environment in which cell–material constructs are created and stored is an important factor. The aim of the present study was to investigate the effects of combined stimuli on cartilage zonal organization which is important to maintain cartilage functions such as lubrication and cushion. For that purpose, we developed a joint mimicking loading system which was composed of compression and shear stress. To mimic the joint loading condition, we manufactured a stimuli system that has a device similar to the shape of a femoral condyle in human knee. The fibrin/hyaluronic acid mixture with chondrocytes were dropped into support made of silicon, and placed under the device. The cartilage explants were stimulated with the joint mimicking loading system for 1 hour per day over the course of 4 weeks. The amounts of GAG and collagen in the stimulated tissue were more than that of the static cultured tissue. Cells and collagen were arranged horizontally paralleled to the surface by stimuli, while it did not happen in the control group. The results of this study suggests that mechanical load exerting in the joint play a crucial role in stimulation of extracellular matrix (ECM) production as well as its functional rearrangement. [ABSTRACT FROM AUTHOR]

Centrifugation is the primary preparation step for isolating red blood cells (RBCs) from whole blood, including for use in studies focused on transduction of adenosine triphosphate (ATP), an important vasodilatory signaling molecule. Despite the wide use of centrifugation, little work has focused on how the centrifugation itself affects release of ATP from RBCs prior to subsequent experimentation. Here we report that both the centrifugation force and duration have a pronounced impact on the concentration of ATP present in the packed RBCs following centrifugation. Multiple subsequent centrifugations yield extracellular ATP concentrations comparable to the amount released during the initial centrifugation, suggesting this effect is cumulative. Pairwise measurements of hemoglobin and ATP suggest the presence of ATP is primarily due to an increase in centrifugation-induced hemolysis. These results indicate that common centrifugation parameters, within the ranges explored here, can release ATP in quantities comparable to the low end of the range of values measured in typical ATP transduction experiments, potentially complicating experimental interpretation of those results. [ABSTRACT FROM AUTHOR]

The article focuses on the role of noncanonical placental Fc receptors in modulating transplacental transfer of maternal Immunoglobulin G (IgG). Topics discussed include role of IgG in protecting fetus against infectious diseases, identification of Fc region amino acid after mutational analyses, and production of maternal IgG-based therapeutics and vaccines.

We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 μM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs. [ABSTRACT FROM AUTHOR]

Ligamentum flavum (LF) hypertrophy in lumbar spinal canal stenosis (LSCS) is characterized by a loss of elastic fibers and fibrosis. Chronic inflammation is thought to be responsible for the histological change but the mechanism underlying elastic fiber degradation remains unclear. Given that matrix metalloproteinase (MMP)-2 and -9 have elastolytic activity and are partly regulated by inflammatory cytokines such as interleukin (IL)-6, in this study, we investigated whether MMPs mediate LF degeneration using 52 LF samples obtained during lumbar surgery, including 31 LSCS and 21 control specimens. We confirmed by histological analysis that the LSCS samples exhibited severe degenerative changes compared with the controls. We found that MMP-2 was upregulated in LF tissue from patients with LSCS at the mRNA and protein levels, whereas MMP-9 expression did not differ between the two groups. The MMP-2 level was positively correlated with LF thickness and negatively correlated with the area occupied by elastic fibers. IL-6 mRNA expression was also increased in LF tissue from patients with LSCS and positively correlated with that of MMP-2. Signal transducer and activator of transcription (STAT)3, a component of the IL-6 signaling pathway, was activated in hypertrophied LF tissues. Our in vitro experiments using fibroblasts from LF tissue revealed that IL-6 increased MMP-2 expression, secretion, and activation via induction of STAT3 signaling, and this effect was reversed by STAT3 inhibitor treatment. Moreover, elastin degradation was promoted by IL-6 stimulation in LF fibroblast culture medium. These results indicate that MMP-2 induction by IL-6/STAT3 signaling in LF fibroblasts can degrade elastic fibers, leading to LF degeneration in LSCS. [ABSTRACT FROM AUTHOR]

Bladder small DRG neurons, which are putative nociceptors pivotal to urinary bladder function, express more than a dozen different ionic membrane mechanisms: ion channels, pumps and exchangers. Small-conductance Ca2+-activated K+ (SKCa) channels which were earlier thought to be gated solely by intracellular Ca2+ concentration ([Ca]i) have recently been shown to exhibit inward rectification with respect to membrane potential. The effect of SKCa inward rectification on the excitability of these neurons is unknown. Furthermore, studies on the role of KCa channels in repetitive firing and their contributions to different types of afterhyperpolarization (AHP) in these neurons are lacking. In order to study these phenomena, we first constructed and validated a biophysically detailed single compartment model of bladder small DRG soma constrained by physiological data. The model includes twenty-two major known membrane mechanisms along with intracellular Ca2+ dynamics comprising Ca2+ diffusion, cytoplasmic buffering, and endoplasmic reticulum (ER) and mitochondrial mechanisms. Using modelling studies, we show that inward rectification of SKCa is an important parameter regulating neuronal repetitive firing and that its absence reduces action potential (AP) firing frequency. We also show that SKCa is more potent in reducing AP spiking than the large-conductance KCa channel (BKCa) in these neurons. Moreover, BKCa was found to contribute to the fast AHP (fAHP) and SKCa to the medium-duration (mAHP) and slow AHP (sAHP). We also report that the slow inactivating A-type K+ channel (slow KA) current in these neurons is composed of 2 components: an initial fast inactivating (time constant ∼ 25-100 ms) and a slow inactivating (time constant ∼ 200-800 ms) current. We discuss the implications of our findings, and how our detailed model can help further our understanding of the role of C-fibre afferents in the physiology of urinary bladder as well as in certain disorders. [ABSTRACT FROM AUTHOR]

Two-pore domain potassium channels (K2P) constitute major candidates for the regulation of background potassium currents in mammalian cells. Channels of the TREK subfamily are also well positioned to play an important role in sensory transduction due to their sensitivity to a large number of physiological and physical stimuli (pH, mechanical, temperature). Following our previous report describing the molecular expression of different K2P channels in the vagal sensory system, here we confirm that TREK channels are functionally expressed in neurons from the mouse nodose ganglion (mNG). Neurons were subdivided into three groups (A, Ah and C) based on their response to tetrodotoxin and capsaicin. Application of the TREK subfamily activator riluzole to isolated mNG neurons evoked a concentration-dependent outward current in the majority of cells from all the three subtypes studied. Riluzole increased membrane conductance and hyperpolarized the membrane potential by approximately 10 mV when applied to resting neurons. The resting potential was similar in all three groups, but C cells were clearly less excitable and showed smaller hyperpolarization-activated currents at -100 mV and smaller sustained currents at -30 mV. Our results indicate that the TREK subfamily of K2P channels might play an important role in the maintenance of the resting membrane potential in sensory neurons of the autonomic nervous system, suggesting its participation in the modulation of vagal reflexes. [ABSTRACT FROM AUTHOR]

Many types of large cells have multiple nuclei. In skeletal muscle fibers, the nuclei are distributed along the cell to maximize their internuclear distances. This myonuclear positioning is crucial for cell function. Although microtubules, microtubule associated proteins, and motors have been implicated, mechanisms responsible for myonuclear positioning remain unclear. We used a combination of rough interacting particle and detailed agent-based modeling to examine computationally the hypothesis that a force balance generated by microtubules positions the muscle nuclei. Rather than assuming the nature and identity of the forces, we simulated various types of forces between the pairs of nuclei and between the nuclei and cell boundary to position the myonuclei according to the laws of mechanics. We started with a large number of potential interacting particle models and computationally screened these models for their ability to fit biological data on nuclear positions in hundreds of Drosophila larval muscle cells. This reverse engineering approach resulted in a small number of feasible models, the one with the best fit suggests that the nuclei repel each other and the cell boundary with forces that decrease with distance. The model makes nontrivial predictions about the increased nuclear density near the cell poles, the zigzag patterns of the nuclear positions in wider cells, and about correlations between the cell width and elongated nuclear shapes, all of which we confirm by image analysis of the biological data. We support the predictions of the interacting particle model with simulations of an agent-based mechanical model. Taken together, our data suggest that microtubules growing from nuclear envelopes push on the neighboring nuclei and the cell boundaries, which is sufficient to establish the nearly-uniform nuclear spreading observed in muscle fibers. [ABSTRACT FROM AUTHOR]

Animal locomotion is mediated by a sensory system referred to as proprioception. Defects in the proprioceptive coordination of locomotion result in uncontrolled and inefficient movements. However, the molecular mechanisms underlying proprioception are not fully understood. Here, we identify two transient receptor potential cation (TRPC) channels, trp-1 and trp-2, as necessary and sufficient for proprioceptive responses in C. elegans head steering locomotion. Both channels are expressed in the SMDD neurons, which are required and sufficient for head bending, and mediate coordinated head steering by sensing mechanical stretches due to the contraction of head muscle and orchestrating dorsal head muscle contractions. Moreover, the SMDD neurons play dual roles to sense muscle stretch as well as to control muscle contractions. These results demonstrate that distinct locomotion patterns require dynamic and homeostatic modulation of feedback signals between neurons and muscles. [ABSTRACT FROM AUTHOR]

When core body temperature increases, appetite and food consumption decline. A higher core body temperature can occur during exercise, during exposure to warm environmental temperatures, or during a fever, yet the mechanisms that link relatively warm temperatures to appetite suppression are unknown. A recent study in PLOS Biology demonstrates that neurons in the mouse hypothalamus that express pro-opiomelanocortin (POMC), a neural population well known to suppress food intake, also express a temperature-sensitive ion channel, transient receptor potential vanilloid 1 (TRPV1). Slight increases in body temperature cause a TRPV1-dependent increase in activity in POMC neurons, which suppresses feeding in mice. Taken together, this study suggests a novel mechanism linking body temperature and food-seeking behavior. [ABSTRACT FROM AUTHOR]

The circuit organization within the mammalian brainstem respiratory network, specifically within and between the pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes, and the roles of these circuits in respiratory pattern generation are continuously debated. We address these issues with a combination of optogenetic experiments and modeling studies. We used transgenic mice expressing channelrhodopsin-2 under the VGAT-promoter to investigate perturbations of respiratory circuit activity by site-specific photostimulation of inhibitory neurons within the pre-BötC or BötC. The stimulation effects were dependent on the intensity and phase of the photostimulation. Specifically: (1) Low intensity (≤ 1.0 mW) pulses delivered to the pre-BötC during inspiration did not terminate activity, whereas stronger stimulations (≥ 2.0 mW) terminated inspiration. (2) When the pre-BötC stimulation ended in or was applied during expiration, rebound activation of inspiration occurred after a fixed latency. (3) Relatively weak sustained stimulation (20 Hz, 0.5–2.0 mW) of pre-BötC inhibitory neurons increased respiratory frequency, while a further increase of stimulus intensity (> 3.0 mW) reduced frequency and finally (≥ 5.0 mW) terminated respiratory oscillations. (4) Single pulses (0.2–5.0 s) applied to the BötC inhibited rhythmic activity for the duration of the stimulation. (5) Sustained stimulation (20 Hz, 0.5–3.0 mW) of the BötC reduced respiratory frequency and finally led to apnea. We have revised our computational model of pre-BötC and BötC microcircuits by incorporating an additional population of post-inspiratory inhibitory neurons in the pre-BötC that interacts with other neurons in the network. This model was able to reproduce the above experimental findings as well as previously published results of optogenetic activation of pre-BötC or BötC neurons obtained by other laboratories. The proposed organization of pre-BötC and BötC circuits leads to testable predictions about their specific roles in respiratory pattern generation and provides important insights into key circuit interactions operating within brainstem respiratory networks. [ABSTRACT FROM AUTHOR]

Neuronal information processing is regulated by fast and localized fluctuations of brain states. Brain states reliably switch between distinct spatiotemporal signatures at a network scale even though they are composed of heterogeneous and variable rhythms at a cellular scale. We investigated the mechanisms of this network control in a conductance-based population model that reliably switches between active and oscillatory mean-fields. Robust control of the mean-field properties relies critically on a switchable negative intrinsic conductance at the cellular level. This conductance endows circuits with a shared cellular positive feedback that can switch population rhythms on and off at a cellular resolution. The switch is largely independent from other intrinsic neuronal properties, network size and synaptic connectivity. It is therefore compatible with the temporal variability and spatial heterogeneity induced by slower regulatory functions such as neuromodulation, synaptic plasticity and homeostasis. Strikingly, the required cellular mechanism is available in all cell types that possess T-type calcium channels but unavailable in computational models that neglect the slow kinetics of their activation. [ABSTRACT FROM AUTHOR]

Mutations in the dentin sialophosphoprotein (DSPP) gene cause dentinogenesis imperfecta. After synthesis, DSPP is proteolytically processed into NH2- and COOH-terminal fragments. The NH2-terminal fragment of DSPP is highly glycosylated but not phosphorylated, whereas the COOH-terminal fragment (named “dentin phosphoprotein” or “DPP”) is highly phosphorylated but not glycosylated. These two fragments are believed to perform distinct roles in dentin formation. To analyze the functions of DPP in dentinogenesis, we created “Dspp-/-;DPP Tg mice”, which expressed transgenic DPP driven by a Type I collagen promoter but lacked the endogenous Dspp gene. We characterized the dentin of the Dspp-/-;DPP Tg mice using X-ray radiography, histology, scanning electron microscopy, double fluorochrome labeling, immunohistochemistry and in situ hybridization. Micro-computed tomography analyses revealed that at postnatal 6 months, the transgenic expression of DPP increased the dentin thickness of the Dspp-null mice by 97.1% and restored the dentin material density by 29.5%. Histological analyses showed that the Dspp-null mice manifested an abnormal widening of the predentin while the predentin in Dspp-/-;DPP Tg mice was narrower than in the Dspp-null mice. Scanning electron microscopy analyses showed that the dentinal tubules in the Dspp-/-;DPP Tg mice were better organized than in the Dspp-null mice. The double fluorochrome labeling analyses demonstrated that the dentin mineral deposition rate in the Dspp-/-;DPP Tg mice was significantly improved compared to that in the Dspp-null mice. These findings indicate that the transgenic expression of DPP partially rescued the dentin defects of the DSPP-null mice, suggesting that DPP may promote dentin formation and that the coordinated actions between DPP and the NH2-terminal fragment of DSPP may be necessary for dentinogenesis. [ABSTRACT FROM AUTHOR]

Vandetanib, a multi-kinase inhibitor used for the treatment of various cancers, has been reported to induce several adverse cardiac effects. However, the underlying mechanisms of vandetanib-induced cardiotoxicity are unclear. This study aimed to investigate the mechanism of vandetanib-induced cardiotoxicity using intracellular electrophysiological recordings on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), rabbit Purkinje fibers, and HEK293 cells transiently expressing human ether-a-go-go-related gene (hERG; the rapidly activating delayed rectifier K+ channel, IKr), KCNQ1/KCNE1 (the slowly activating delayed rectifier K+ current, IKs), KCNJ2 (the inwardly rectifying K+ current, IK1) or SCN5A (the inward Na+ current, INa). Purkinje fiber assays and ion channel studies showed that vandetanib at concentrations of 1 and 3 μM inhibited the hERG currents and prolonged the action potential duration. Alanine scanning and in silico hERG docking studies demonstrated that Y652 and F656 in the hERG S6 domain play critical roles in vandetanib binding. In hiPSC-CMs, vandetanib markedly reduced the maximum rate of depolarization during the AP upstroke. Ion channel studies revealed that hiPSC-CMs were more sensitive to inhibition of the INa by vandetanib than in a heterogeneously expressed HEK293 cell model, consistent with the changes in the AP parameters of hiPSC-CMs. The subclasses of Class I antiarrhythmic drugs inhibited INa currents in a dose-dependent manner in hiPSC-CMs and SCN5A-encoded HEK293 cells. The inhibitory potency of vandetanib for INa was much higher in hiPSC-CMs (IC50: 2.72 μM) than in HEK293 cells (IC50: 36.63 μM). These data suggest that AP and INa assays using hiPSC-CMs are useful electrophysiological models for prediction of drug-induced cardiotoxicity. [ABSTRACT FROM AUTHOR]

Most weakly electric fish navigate and communicate by sensing electric signals generated by their muscle-derived electric organs. Adults of one lineage (Apteronotidae), which discharge their electric organs in excess of 1 kHz, instead have an electric organ derived from the axons of specialized spinal neurons (electromotorneurons [EMNs]). EMNs fire spontaneously and are the fastest-firing neurons known. This biophysically extreme phenotype depends upon a persistent sodium current, the molecular underpinnings of which remain unknown. We show that a skeletal muscle–specific sodium channel gene duplicated in this lineage and, within approximately 2 million years, began expressing in the spinal cord, a novel site of expression for this isoform. Concurrently, amino acid replacements that cause a persistent sodium current accumulated in the regions of the channel underlying inactivation. Therefore, a novel adaptation allowing extreme neuronal firing arose from the duplication, change in expression, and rapid sequence evolution of a muscle-expressing sodium channel gene. [ABSTRACT FROM AUTHOR]

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV. [ABSTRACT FROM AUTHOR]

The membrane potential helps determine pulmonary artery smooth muscle cell (PASMC) contraction. The Kv7 channel activators, retigabine and flupirtine, are thought to dilate pulmonary arteries by hyperpolarising PASMC. Zinc pyrithione activates Kv7 channels by a mechanism distinct from retigabine and with different Kv7 subunit selectivity. This study aimed to determine if zinc pyrithione selectively activates Kv7 channels in rat PASMC to evoke pulmonary artery dilation. Zinc pyrithione relaxed pulmonary arteries with half-maximal effect at 4.3μM. At 10μM it activated pronounced voltage-dependent K+ current and hyperpolarized PASMCs by around 10mV. Tetraethylammonium ions (TEA, 10mM) and paxilline (1μM) abolished both the current and hyperpolarisation. XE991 (10μM) blocked the hyperpolarization and reduced the current by 30%. Iberiotoxin (50nM) had no effect on the hyperpolarisation, but reduced the current by 40%. The XE991-sensitive current activated with an exponential time course (time constant 17ms), whereas the iberiotoxin-sensitive current followed a bi-exponential time course (time constants 6 and 57ms), suggesting that the drugs blocked different components of the zinc pyrithione-induced current. Zinc pyrithione therefore appears to activate at least two types of K+ channel in PASMC; an XE991, TEA and paxilline-sensitive Kv7 channel and a TEA, paxilline and iberiotoxin-sensitive BKCa channel. Both could contribute to the relaxing effect of zinc pyrithione on pulmonary artery. [ABSTRACT FROM AUTHOR]

LRRC55 (leucine-rich repeat-containing protein 55) protein is an auxiliary γ subunit of BK (Big conductance potassium channel) channels, which leftward shifts GVs of BK channels around 50 mV in the absence of cytosolic Ca2+. LRRC55 protein is also the only γ subunit of BK channels that is expressed in mammalian nervous system. However, the expression pattern of LRRC55 gene in adult mammalian brain remains elusive. In this study, we investigated the distribution of LRRC55 mRNA in the adult mouse brain by using in situ hybridization. We found that LRRC55 mRNA is richly expressed in the adult mouse medial habenula nucleus (MHb), cerebellum and pons. However, the potential role of LRRC55 in MHb and cerebellum could be different based on the function of BK channels in these brain regions. [ABSTRACT FROM AUTHOR]