A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks

Background: A variety of mechanisms that govern connexin channel gating and permeability regulate coupling in gap junction networks. Mutations in connexin genes have been linked to several pathologies, including cardiovascular anomalies, peripheral neuropathy, skin disorders, cataracts and deafness. Gap junction coupling and its patho–physiological alterations are commonly assayed by microinjection experiments with fluorescent tracers, which typically require several minutes to allow dye transfer to a limited number of cells. Comparable or longer time intervals are required by fluorescence recovery after photobleaching experiments. Paired electrophysiological recordings have excellent time resolution but provide extremely limited spatial information regarding network connectivity. Results: Here, we developed a rapid and sensitive method to assay gap junction communication using a combination of single cell electrophysiology, large–scale optical recordings and a digital phase–sensitive detector to extract signals with a known frequency from Vf2.1.Cl, a novel fluorescent sensor of plasma membrane potential. Tests performed in HeLa cell cultures confirmed that suitably encoded Vf2.1.Cl signals remained confined within the network of cells visibly interconnected by fluorescently tagged gap junction channels. We used this method to visualize instantly intercellular connectivity over the whole field of view (hundreds of cells) in cochlear organotypic cultures from postnatal mice. A simple resistive network model reproduced accurately the spatial dependence of the electrical signals throughout the cellular network. Our data suggest that each pair of cochlear non−sensory cells of the lesser epithelial ridge is coupled by ~1500 gap junction channels, on average. Junctional conductance was reduced by 14% in cochlear cultures harboring the T5M mutation of connexin30, which induces a moderate hearing loss in connexin30T5M/T5M knock–in mice, and by 91% in cultures from connexin30−/− mice, which are profoundly deaf. Conclusions: Our methodology allows greater sensitivity (defined as the minimum magnitude of input signal required to produce a specified output signal having a specified signal−to−noise ratio) and better time resolution compared to classical tracer–based techniques. It permitted us to dynamically visualize intercellular connectivity down to the 10th order in non−sensory cell networks of the developing cochlea. We believe that our approach is of general interest and can be seamlessly extended to a variety of biological systems, as well as to other connexin−related disease condition

Calcium signaling in the cochlea - Molecular mechanisms and physiopathological implications

Calcium ions (Ca2+) regulate numerous and diverse aspects of cochlear and vestibular physiology. This review focuses on the Ca2+ control of mechanotransduction and synaptic transmission in sensory hair cells, as well as on Ca2+ signalling in non-sensory cells of the developing cochlea

Cachaça distillation investigated on the basis of model systems

This work reports experimental tests using commercial spirits that were diluted and had their initial composition modified in order to better measure the distillation behavior of selected minor compounds of importance for the quality of alcoholic beverages. Such compounds were added to the commercial cachaça and the corresponding model wine, obtained after the spirits' dilution, was distilled. In this way a more precise distillation profile could be determined for those minor compounds. The alembic heating was performed by electrical resistance and the corresponding heat transfer coefficient was determined by analyzing the thermal behavior of the still during the distillations. A simulation algorithm was developed, including the mass and enthalpy balances as well as the heat transfer flux to the boiling wine. Good agreement between experimental and simulated results was obtained for the distillate rate, alcoholic graduation, temperature and most minor component profiles.429440Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

An unusual cause of gross hematuria, improved by recumbency

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Plant Polyphenols and Exendin-4 Prevent Hyperactivity and TNF-alpha Release in LPS-Treated In vitro Neuron/Astrocyte/Microglial Networks

Increasing evidence supports a decisive role for neuroinflammation in the neurodegenerative process of several central nervous system (CNS) disorders. Microglia are essential mediators of neuroinflammation and can regulate a broad spectrum of cellular responses by releasing reactive oxygen intermediates, nitric oxide, proteases, excitatory amino acids, and cytokines. We have recently shown that also in ex-vivo cortical networks of neurons, astrocytes and microglia, an increased level of tumor necrosis factor-alpha (TNF-α) was detected a few hours after exposure to the bacterial endotoxin lipopolysaccharide (LPS). Simultaneously, an atypical “seizure-like” neuronal network activity was recorded by multi-electrode array (MEA) electrophysiology. These effects were prevented by minocycline, an established anti-inflammatory antibiotic. We show here that the same inhibitory effect against LPS-induced neuroinflammation is exerted also by natural plant compounds, polyphenols, such as curcumin (CU, curcuma longa), crocin (CR, saffron), and resveratrol (RE, grape), as well as by the glucagon like peptide-1 receptor (GLP-1R) agonist exendin-4 (EX-4). The drugs tested also caused per-se early transient (variable) changes of network activity. Since it has been reported that LPS-induced neuroinflammation causes rearrangements of glutamate transporters in astrocytes and microglia, we suggest that neural activity could be putatively increased by an imbalance of glial glutamate transporter activity, leading to prolonged synaptic glutamatergic dysregulation

Replicating Intermittent Fasting in Human Skeletal Muscle Cells: A Pilot Study

Type 2 diabetes (T2D), the most common form of diabetes (90-95% of diagnoses), is marked by decreased insulin sensitivity (insulin resistance) or a defect in insulin secretion. T2D disrupts nutrient signaling where the body cannot maintain adequate blood glucose levels. Inability to receive glucose in skeletal muscle due to insulin resistance in T2D results in oxidative stress and increased muscle atrophy. Properly regulated glucose uptake is pivotal for healthy aging and maintenance of the skeletal muscle system. PURPOSE: The purpose of this study is to examine the effects of nutrient deprivation on human skeletal muscle metabolism, with an emphasis on oxidative stress and atrophy markers in healthy and T2D cell models. METHODS: Healthy human skeletal muscle myoblast cells (HSMM) and diabetic human skeletal muscle myoblast cells (DHSMM) (Lonza Inc, Walkersville MD) were cultured in a 37°C with 5% CO2 incubator in a T-75 flask. At confluency, cells were transferred into four 24-well plates and were incubated for 48h with standard culture media (Lonza Inc, Walkersville MD). The cells were then incubated for 12 or 24 h in media containing varying serum concentrations: 5%, 10%, and 15%. The media contained either fetal bovine serum (FBS) (Lonza Inc, Walkersville MD) or pooled human serum (HS) from either healthy or diabetic patients (Doctors Regional, Corpus Christi TX). Following the 24 hours, cell viability and density were determined, and sandwich enzyme-linked immunosorbent assay kits (RayBiotech, Norcross GA) were performed to measure the amount of superoxide dismutase (SOD1) present in each sample. RESULTS: A treatment effect was found using T2D HS which had a significant influence on mean SOD1 levels (range of SOD1 pg/mL; p=0.0423). There was no significant effect of time between 12h and 24h (p=0.1100). In the FBS models, a significant effect of concentration HSMM is seen (p=0.0263). Incubation time had little effect on FBS DHSMM (p=0.2671) and HSMM (p=0.2780) models. CONCLUSION: As serum concentration increases, the level of SOD1 present in the samples also increases. This suggests that treatment concentration may influence the activity of SOD1. This may be due to exogenous SOD1 already present in the serum. However, we did not asses the rate of appearance and decay of SOD1 already present in the serum. Incubation time shows little difference in all models. These results suggest that compositional environment can influence SOD1 levels and that a higher concentration may promote oxidative stress more so than a lower concentration environment

Connexin-Mediated Signaling in Nonsensory Cells Is Crucial for the Development of Sensory Inner Hair Cells in the Mouse Cochlea

Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca(2+) signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26(Sox10-Cre)), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients. SIGNIFICANCE STATEMENT: The correct development and function of the mammalian cochlea relies not only on the sensory hair cells, but also on the surrounding nonsensory cells. Although the nonsensory cells have been largely implicated in the general homeostasis in the mature cochlea, their involvement in the initial functional differentiation of the sensory inner hair cells is less clear. Using mutant mouse models for the most common form of congenital deafness in humans, which are knock-outs for the gap-junction channels connexin 26 and connexin 30 genes, we show that defects in nonsensory cells prevented the functional maturation of inner hair cells. In connexin knock-outs, inner hair cells remained stuck at a prehearing stage of development and, as such, are unable to process sound information

Inferring Foraging Areas of Nesting Loggerhead Turtles Using Satellite Telemetry and Stable Isotopes

In recent years, the use of intrinsic markers such as stable isotopes to link breeding and foraging grounds of migratory species has increased. Nevertheless, several assumptions still must be tested to interpret isotopic patterns found in the marine realm. We used a combination of satellite telemetry and stable isotope analysis to (i) identify key foraging grounds used by female loggerheads nesting in Florida and (ii) examine the relationship between stable isotope ratios and post-nesting migration destinations. We collected tissue samples for stable isotope analysis from 14 females equipped with satellite tags and an additional 57 untracked nesting females. Telemetry identified three post-nesting migratory pathways and associated non-breeding foraging grounds: (1) a seasonal continental shelf-constrained migratory pattern along the northeast U. S. coastline, (2) a non-breeding residency in southern foraging areas and (3) a residency in the waters adjacent to the breeding area. Isotopic variability in both delta C-13 and delta N-15 among individuals allowed identification of three distinct foraging aggregations. We used discriminant function analysis to examine how well delta C-13 and delta N-15 predict female post-nesting migration destination. The discriminant analysis classified correctly the foraging ground used for all but one individual and was used to predict putative feeding areas of untracked turtles. We provide the first documentation that the continental shelf of the Mid-and South Atlantic Bights are prime foraging areas for a large number (61%) of adult female loggerheads from the largest loggerhead nesting population in the western hemisphere and the second largest in the world. Our findings offer insights for future management efforts and suggest that this technique can be used to infer foraging strategies and residence areas in lieu of more expensive satellite telemetry, enabling sample sizes that are more representative at the population level

Pathophysiological changes in inner hair cell ribbon synapses in the ageing mammalian cochlea

Mammalian cochlear inner hair cells (IHCs) are specialized sensory receptors able to provide dynamic coding of sound signals. This ability is largely conferred by their ribbon synapses, which tether a large number of vesicles at the IHC's presynaptic active zones, allowing high rates of sustained synaptic transmission onto the afferent fibres. How the physiological and morphological properties of ribbon synapses change with age remains largely unknown. Here, we have investigated the biophysical and morphological properties of IHC ribbon synapses in the ageing cochlea (9–12 kHz region) of four mouse strains commonly used in hearing research: early‐onset progressive hearing loss (C57BL/6J and C57BL/6NTac) and ‘good hearing’ strains (C57BL/6NTacCdh23+ and C3H/HeJ). We found that with age, both modiolar and pillar sides of the IHC exhibited a loss of ribbons, but there was an increased volume of those that remained. These morphological changes, which only occurred after 6 months of age, were correlated with the level of hearing loss in the different mouse strains, being most severe for C57BL/6NTac and C57BL/6J, less so for C57BL/6NTacCdh23+ and absent for C3H/HeJ strains. Despite the age‐related reduction in ribbon number in three of the four strains, the size and kinetics of Ca2+‐dependent exocytosis, as well as the replenishment of synaptic vesicles, in IHCs was not affected. The degree of vesicle release at the fewer, but larger, individual remaining ribbon synapses colocalized with the post‐synaptic afferent terminals is likely to increase, indicating the presence of a previously unknown degree of functional compensation in the ageing mouse cochlea

Preliminary studies on enniatin B1 and zearalenone in aquaculture fish from markets in Spain

Mycotoxins are secondary metabolites produced by fungi and mainly by Fusarium spp., Aspergillus spp. and Penicillium spp (1). Fusarium spp, is known producer of emerging mycotoxins: beauvericin and enniatins (A, A1, B, B1). These mycotoxins can be found in feed for farmed fish (2) and when ingested, could arrive in the animal muscle, becoming a risk to human consumer. The European Commission defined maximum Levels (MLs) for mycotoxins in feed (3), but few data are available in literature regarding their presence in farmed fishes. The aim of this study is to investigate the presence of mycotoxins, in particular enniatin B and zearalenone, in two common species of aquaculture fish, sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) with a multiresidue LC-tandem MS methods. A total of 25 sea basses and 25 sea breams were randomly collected from different markets in Valencia, to simulate mean fish consumption of a Spanish consumer. The chemical analysis was performed on head, bowel and muscle samples purified and extracted using a QuEChERS technique. Our preliminary data show the occurrence of the studied compounds in samples analysed and the scattered presence of enniatin B in head, bowel and muscle with a prevalence of 46% in sea bream and 36% in sea bass (concentration range 0.13-26.81 \ub5g g-1). Zearalenone was found in sea bream head and bowel with a prevalence of 7.0 % (concentration range 2.26-6.83 \ub5g g-1) and 13.8% (concentration range 1.04-8.75 \ub5g g-1) respectively and in sea bass head with a prevalence of 4.0% (concentration range 2.38-5.15 \ub5g g-1) and bowel (prevalence 97%; concentration range 1.22-8.33 \ub5g g-1). These preliminary results show the presence of the studied contaminants, with a preferential localization in sea bream