Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but ...whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown.
Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation.
HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (r2 = 0.35, p = 0.043).
Ex induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.
Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor ...subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.
Understanding sources of microbial contamination in outpatient rehabilitation (REHAB) clinics is important to patients and healthcare providers.
The purpose of this study was to characterize the ...microbiome of an outpatient REHAB clinic and examine relationships between clinic factors and contamination.
Forty commonly contacted surfaces in an outpatient REHAB clinic were observed for frequency of contact and swiped using environmental sample collection kits. Surfaces were categorized based on frequency of contact and cleaning and surface type. Total bacterial and fungal load was assessed using primer sets specific for the 16S rRNA and ITS genes, respectively. Bacterial samples were sequenced using the Illumina system and analyzed using Illumina-utils, Minimum Entropy Decomposition, QIIME2 (for alpha and beta diversity), LEfSe and ANCOM-BC for taxonomic differential abundance and ADONIS to test for differences in beta diversity (p<0.05).
Porous surfaces had more bacterial DNA compared to non-porous surfaces (median non-porous = 0.0016ng/μL, 95%CI = 0.0077-0.00024ng/μL, N = 15; porous = 0.0084 ng/μL, 95%CI = 0.0046-0.019 ng/μL, N = 18. p = 0.0066,DNA. Samples clustered by type of surface with non-porous surfaces further differentiated by those contacted by hand versus foot. ADONIS two-way ANOVA showed that the interaction of porosity and contact frequency (but neither alone) had a significant effect on 16S communities (F = 1.7234, R2 = 0.0609, p = 0.032).
Porosity of surfaces and the way they are contacted may play an underestimated, but important role in microbial contamination. Additional research involving a broader range of clinics is required to confirm results. Results suggest that surface and contact-specific cleaning and hygiene measures may be needed for optimal sanitization in outpatient REHAB clinics.
The cholinergic signalling system has been an attractive pathway to seek targets for modulation of arousal, cognition, and attention which are compromised in neurodegenerative and neuropsychiatric ...diseases. The acetylcholine muscarinic receptor M1 and M4 subtypes which are highly expressed in the central nervous system, in cortex, hippocampus and striatum, key areas of cognitive and neuropsychiatric control, have received particular attention. Historical muscarinic drug development yielded first generation agonists with modest selectivity for these two receptor targets over M2 and M3 receptors, the major peripheral sub-types hypothesised to underlie the dose-limiting clinical side effects. More recent compound screening and medicinal chemistry optimization of orthosteric and allosteric agonists, and positive allosteric modulators binding to sites distinct from the highly homologous acetylcholine binding pocket have yielded a collection of highly selective tool compounds for preclinical validation studies. Several M1 selective ligands have progressed to early clinical development and in time will hopefully lead to useful therapeutics for treating symptoms of Alzheimer's disease and related disorders.
This article is part of the Special Issue entitled ‘Neuropharmacology on Muscarinic Receptors’.
•M1 and M4 receptors remain attractive targets for neurodegenerative and neuropsychiatric diseases.•Targeting M1 receptors has focused on newer approaches such as the development of selective positive allosteric modulators.•M4 receptors have received less attention, but agonists and positive allosteric modulators are being pursued.
Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell–cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the ...exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell–cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT–CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.
Background Stroke is the leading cause of severe disability and many survivors report long-term physical or cognitive impairments that may impact their ability to achieve community mobility (CM).
To ...determine the extent to which people with chronic stroke achieve CM compared to age-matched norms or non-neurologically impaired controls. Methods The StrokEDGE outcome measures were searched to identify validated tools that included >25% of items addressing CM. MEDLINE, CINAHL, Google Scholar, PubMed, PEDro and the Cochrane databases were searched from 2001 to 2015 with the identified outcome measures cross-referenced against search terms related to stroke and CM.
utilized a validated CM outcome measure, chronic (>3 months post) stroke survivors, and randomized controlled trial, observational or cohort study design. One reviewer screened the studies and performed data extraction and three performed quality appraisal. Fourteen studies met all inclusion criteria. Results Stroke survivors have impaired CM as demonstrated by 30-83% of normative or non-stroke subject CM scores. As time post-stroke increased, CM improved only slightly. Factors found to correlate with the CM were age, education, general well-being, emotional state, motor function and coordination, independence in activities of daily living, balance, endurance and driving status. Limitations of this review include a relatively high functioning cohort, no meta-analysis and reliance on outcome measures not specifically designed to measure CM. Conclusion Survivors of stroke may experience a significant decrease in CM compared to people without neurological injury. Rehabilitation addressing motor function, coordination, independence in activities of daily living, balance and endurance may be important for achieving higher levels of CM. Outcome measures directly addressing CM are needed.
Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with ...their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.
We present a study of the dust-to-gas ratios in five nearby galaxies: NGC 628 (M74), NGC 6503, NGC 7793, UGC 5139 (Holmberg I), and UGC 4305 (Holmberg II). Using Hubble Space Telescope broadband ...WFC3/UVIS UV and optical images from the Treasury program Legacy ExtraGalactic UV Survey (LEGUS) combined with archival HST/Advanced Camera for Surveys data, we correct thousands of individual stars for extinction across these five galaxies using an isochrone-matching (reddening-free Q) method. We generate extinction maps for each galaxy from the individual stellar extinctions using both adaptive and fixed resolution techniques and correlate these maps with neutral H i and CO gas maps from the literature, including the H i Nearby Galaxy Survey and the HERA CO-Line Extragalactic Survey. We calculate dust-to-gas ratios and investigate variations in the dust-to-gas ratio with galaxy metallicity. We find a power-law relationship between dust-to-gas ratio and metallicity, consistent with other studies of dust-to-gas ratio compared to metallicity. We find a change in the relation when H2 is not included. This implies that underestimation of N H 2 in low-metallicity dwarfs from a too-low CO-to-H2 conversion factor XCO could have produced too low a slope in the derived relationship between dust-to-gas ratio and metallicity. We also compare our extinctions to those derived from fitting the spectral energy distribution (SED) using the Bayesian Extinction and Stellar Tool for NGC 7793 and find systematically lower extinctions from SED fitting as compared to isochrone matching.
Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction ...blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration (Ca2+i) triggers hemichannel opening, making use of peptides that are identical to a short amino‐acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)‐expressing cells showed that an increase in Ca2+i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on Ca2+i elevation within a window situated around 500 nM. Our results indicate that Ca2+i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.
Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated ...hemichannel-mediated ATP responses triggered by changes of intracellular Ca(2+) (Ca(2+)(i)) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when Ca(2+)(i) was in the 500nM range but the responses disappeared with larger Ca(2+)(i) transients. Ca(2+)-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca(2+)-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of Ca(2+)(i) triggers hemichannel opening, not by a direct action of Ca(2+) on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high Ca(2+)(i) and acting to restrain cellular ATP loss.
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