Neuroimmunophysiology of the gastrointestinal tract.

Gut physiology is the epicenter of a web of internal communication systems (i.e., neural, immune, hormonal) mediated by cell-cell contacts, soluble factors, and external influences, such as the microbiome, diet, and the physical environment. Together these provide the signals that shape enteric homeostasis and, when they go awry, lead to disease. Faced with the seemingly paradoxical tasks of nutrient uptake (digestion) and retarding pathogen invasion (host defense), the gut integrates interactions between a variety of cells and signaling molecules to keep the host nourished and protected from pathogens. When the system fails, the outcome can be acute or chronic disease, often labeled as "idiopathic" in nature (e.g., irritable bowel syndrome, inflammatory bowel disease). Here we underscore the importance of a holistic approach to gut physiology, placing an emphasis on intercellular connectedness, using enteric neuroimmunophysiology as the paradigm. The goal of this opinion piece is to acknowledge the pace of change brought to our field via single-cell and -omic methodologies and other techniques such as cell lineage tracing, transgenic animal models, methods for culturing patient tissue, and advanced imaging. We identify gaps in the field and hope to inspire and challenge colleagues to take up the mantle and advance awareness of the subtleties, intricacies, and nuances of intestinal physiology in health and disease by defining communication pathways between gut resident cells, those recruited from the circulation, and "external" influences such as the central nervous system and the gut microbiota.

Protease-activated receptor 2 drives migration in a colon cancer cell line but not in noncancerous human epithelial cells.

The inflamed mucosa contains a complex assortment of proteases that may participate in wound healing or the development of inflammation-associated colon cancer. We sought to determine the role of protease-activated receptor 2 (PAR2) in epithelial wound healing in both untransformed and transformed colonic epithelial cells. Monolayers of primary epithelial cells derived from organoids cultivated from patient colonic biopsies and of the T84 colon cancer cell line were grown to confluence, wounded in the presence of a selective PAR2-activating peptide, and healing was visualized by live cell microscopy. Inhibitors of various signaling molecules were used to assess the relevant pathways responsible for wound healing. Activation of PAR2 induced an enhanced wound-healing response in T84 cells but not primary cells. The PAR2-enhanced wound-healing response was associated with the development of lamellipodia in cells at the wound edge, consistent with sheet migration. The response to PAR2 activation in T84 cells was completely dependent on Src kinase activity and partially dependent on Rac1 activity. The Src-associated signaling molecules, focal adhesion kinase, and epidermal growth factor receptor, which typically mediate wound-healing responses, were not involved in the PAR2 response. Experiments repeated in the presence of the inflammatory cytokines TNF and IFNγ revealed a synergistically enhanced PAR2 wound-healing response in T84s but not primary cells. The epithelial response to proteases may be different between primary and cancer cells and is accentuated in the presence of inflammatory cytokines. Our findings have implications for understanding epithelial restitution in the context of inflammatory bowel disease (IBD) and inflammation-associated colon cancer.NEW & NOTEWORTHY Protease-activated receptor 2 enhances wound healing in the T84 colon cancer cell line, but not in primary cells derived from patient biopsies, an effect that is synergistically enhanced in the presence of the inflammatory cytokines TNF and IFNγ.

Exploring avibactam and relebactam inhibition of Klebsiella pneumoniae carbapenemase D179N variant: role of the Ω loop-held deacylation water.

Klebsiella pneumoniae carbapenemase-2 (KPC-2) presents a clinical threat as this ß-lactamase confers resistance to carbapenems. Recent variants of KPC-2 in clinical isolates contribute to concerning resistance phenotypes. Klebsiella pneumoniae expressing KPC-2 D179Y acquired resistance to the ceftazidime/avibactam combination affecting both the ß-lactam and the ß-lactamase inhibitor yet has lowered minimum inhibitory concentrations for all other ß-lactams tested. Furthermore, Klebsiella pneumoniae expressing the KPC-2 D179N variant also manifested resistance to ceftazidime/avibactam yet retained its ability to confer resistance to carbapenems although significantly reduced. This structural study focuses on the inhibition of KPC-2 D179N by avibactam and relebactam and expands our previous analysis that examined ceftazidime resistance conferred by D179N and D179Y variants. Crystal structures of KPC-2 D179N soaked with avibactam and co-crystallized with relebactam were determined. The complex with avibactam reveals avibactam making several hydrogen bonds, including with the deacylation water held in place by Ω loop. These results could explain why the KPC-2 D179Y variant, which has a disordered Ω loop, has a decreased affinity for avibactam. The relebactam KPC-2 D179N complex revealed a new orientation of the diazabicyclooctane (DBO) intermediate with the scaffold piperidine ring rotated ~150° from the standard DBO orientation. The density shows relebactam to be desulfated and present as an imine-hydrolysis intermediate not previously observed. The tetrahedral imine moiety of relebactam interacts with the deacylation water. The rotated relebactam orientation and deacylation water interaction could potentially contribute to KPC-mediated DBO fragmentation. These results elucidate important differences that could aid in the design of novel ß-lactamase inhibitors.

The best of both worlds: no apparent trade-off between immunity and reproduction in two group-living African mole-rat species.

Co-operatively breeding mammals often exhibit a female reproductive skew and suppression of the subordinate non-breeding group members. According to evolutionary theory and the immunity-fertility axis, an inverse relationship between reproductive investment and survival (through immunocompetence) is expected. As such, this study investigated if a trade-off between immunocompetence and reproduction arises in two co-operatively breeding African mole-rat species, namely the Damaraland mole-rat (Fukomys damarensis) and common mole-rat (Cryptomys hottentotus hottentotus), which possess female reproductive division of labour. This study also attempted to investigate the relationship between the immune and endocrine systems in Damaraland mole-rats. There was no trade-off between reproduction and immunocompetence in co-operatively breeding African mole-rat species, and in the case of the Damaraland mole-rats, breeding females (BFs) possessed increased immunocompetence compared with non-breeding females (NBFs). Furthermore, the increased levels of progesterone possessed by Damaraland mole-rat BFs compared with NBFs appear to be correlated to increased immunocompetence. In comparison, BF and NBF common mole-rats possess similar immunocompetence. The species-specific differences in the immunity-fertility axis may be due to variations in the strengths of reproductive suppression in each species. This article is part of the theme issue 'Evolutionary ecology of inequality'.

Investigation of liver-targeted peripheral focused ultrasound stimulation (pFUS) and its effect on glucose homeostasis and insulin resistance in type 2 diabetes mellitus: a proof of concept, phase 1 trial.

BACKGROUND: Mechanical waves produced by ultrasound pulses have been shown to activate mechanosensitive ion channels and modulate peripheral nerves. However, while peripheral ultrasound neuromodulation has been demonstrated in vitro and in pre-clinical models, there have been few reports of clinical tests. AIM: We modified a diagnostic imaging system for ultrasound neuromodulation in human subjects. We report the first safety and feasibility outcomes in subjects with type 2 diabetes (T2D) mellitus and discuss these outcomes in relation to previous pre-clinical results. DESIGN: The study was performed as an open label feasibility study to assess the effects of hepatic ultrasound (targeted to the porta hepatis) on glucometabolic parameters in subjects with T2D. Stimulation (peripheral focused ultrasound stimulation treatment) was performed for 3 days (i.e. 15 min per day), preceded by a baseline examination and followed by a 2-week observation period. METHODS: Multiple metabolic assays were employed including measures of fasting glucose and insulin, insulin resistance and glucose metabolism. The safety and tolerability were also assessed by monitoring adverse events, changes in vital signs, electrocardiogram parameters and clinical laboratory measures. RESULTS AND CONCLUSION: We report post-pFUS trends in several outcomes that were consistent with previous pre-clinical findings. Fasting insulin was lowered, resulting in a reduction of HOMA-IR scores (P-value 0.01; corrected Wilcoxon signed-rank test). Additional safety and exploratory markers demonstrated no device-related adverse impact of pFUS. Our findings demonstrate that pFUS represents a promising new treatment modality that could be used as a non-pharmaceutical adjunct or even alternative to current drug treatments in diabetes.

Application of Mixed-Solvent Molecular Dynamics Simulations for Prediction of Allosteric Sites on G Protein-Coupled Receptors.

The development of small molecule allosteric modulators acting at G protein-coupled receptors (GPCRs) is becoming increasingly attractive. Such compounds have advantages over traditional drugs acting at orthosteric sites on these receptors, in particular target specificity. However, the number and locations of druggable allosteric sites within most clinically relevant GPCRs are unknown. In the present study, we describe the development and application of a mixed-solvent molecular dynamics (MixMD)-based method for the identification of allosteric sites on GPCRs. The method employs small organic probes with druglike qualities to identify druggable hotspots in multiple replicate short-timescale simulations. As proof of principle, we first applied the method retrospectively to a test set of five GPCRs (cannabinoid receptor type 1, C-C chemokine receptor type 2, M2 muscarinic receptor, P2Y purinoceptor 1, and protease-activated receptor 2) with known allosteric sites in diverse locations. This resulted in the identification of the known allosteric sites on these receptors. We then applied the method to the µ-opioid receptor. Several allosteric modulators for this receptor are known, although the binding sites for these modulators are not known. The MixMD-based method revealed several potential allosteric sites on the mu-opioid receptor. Implementation of the MixMD-based method should aid future efforts in the structure-based drug design of drugs targeting allosteric sites on GPCRs. SIGNIFICANCE STATEMENT: Allosteric modulation of G protein-coupled receptors (GPCRs) has the potential to provide more selective drugs. However, there are limited structures of GPCRs bound to allosteric modulators, and obtaining such structures is problematic. Current computational methods utilize static structures and therefore may not identify hidden or cryptic sites. Here we describe the use of small organic probes and molecular dynamics to identify druggable allosteric hotspots on GPCRs. The results reinforce the importance of protein dynamics in allosteric site identification.

Intestinal distension orchestrates neuronal activity in the enteric nervous system of adult mice.

The enteric nervous system (ENS) regulates the motor, secretory and defensive functions of the gastrointestinal tract. Enteric neurons integrate mechanical and chemical inputs from the gut lumen to generate complex motor outputs. How intact enteric neural circuits respond to changes in the gut lumen is not well understood. We recorded intracellular calcium in live-cell confocal recordings in neurons from intact segments of mouse intestine in order to investigate neuronal response to luminal mechanical and chemical stimuli. Wnt1-, ChAT- and Calb1-GCaMP6 mice were used to record neurons from the jejunum and colon. We measured neuronal calcium response to KCl (75 mM), veratridine (10 µM), 1,1-dimethyl-4-phenylpiperazinium (DMPP; 100 µM) or luminal nutrients (Ensure®), in the presence or absence of intraluminal distension. In the jejunum and colon, distension generated by the presence of luminal content (chyme and faecal pellets, respectively) renders the underlying enteric circuit unresponsive to depolarizing stimuli. In the distal colon, high levels of distension inhibit neuronal response to KCl, while intermediate levels of distension reorganize Ca2+ response in circumferentially propagating slow waves. Mechanosensitive channel inhibition suppresses distension-induced Ca2+ elevations, and calcium-activated potassium channel inhibition restores neuronal response to KCl, but not DMPP in the distended colon. In the jejunum, distension prevents a previously unknown tetrodotoxin-resistant neuronal response to luminal nutrient stimulation. Our results demonstrate that intestinal distension regulates the excitability of ENS circuits via mechanosensitive channels. Physiological levels of distension locally silence or synchronize neurons, dynamically regulating the excitability of enteric neural circuits based on the content of the intestinal lumen. KEY POINTS: How the enteric nervous system of the gastrointestinal tract responds to luminal distension remains to be fully elucidated. Here it is shown that intestinal distension modifies intracellular calcium levels in the underlying enteric neuronal network, locally and reversibly silencing neurons in the distended regions. In the distal colon, luminal distension is integrated by specific mechanosensitive channels and coordinates the dynamics of neuronal activation within the enteric network. In the jejunum, distension suppresses the neuronal calcium responses induced by luminal nutrients. Physiological levels of distension dynamically regulate the excitability of enteric neuronal circuits.

Role of CB1 receptors in the acute regulation of small intestinal permeability: effects of high-fat diet.

The endocannabinoid system of the gastrointestinal tract is involved in the control of intestinal barrier function. Whether the cannabinoid 1 (CB1) receptor is expressed on the intestinal epithelium and acutely regulates barrier function has not been determined. Here, we tested the hypothesis that ligands of the CB1 receptor acutely modulate small intestinal permeability and that this is associated with altered distribution of tight junction proteins. We examined the acute effects of CB1 receptor ligands on small intestinal permeability both in chow-fed and 2-wk high-fat diet (HFD)-fed mice using Ussing chambers. We assessed the distribution of CB1 receptor and tight junction proteins using immunofluorescence and the expression of CB1 receptor using PCR. A low level of CB1 expression was found on the intestinal epithelium. CB1 receptor was highly expressed on enteric nerves in the lamina propria. Neither the CB1/CB2 agonist CP55,940 nor the CB1 neutral antagonist AM6545 altered the flux of 4kDa FITC dextran (FD4) across the jejunum or ileum of chow-fed mice. Remarkably, both CP55,940 and AM6545 reduced FD4 flux across the jejunum and ileum in HFD-fed mice that have elevated baseline intestinal permeability. These effects were absent in CB1 knockout mice. CP55,940 reduced the expression of claudin-2, whereas AM6545 had little effect on claudin-2 expression. Neither ligand altered the expression of ZO-1. Our data suggest that CB1 receptor on the intestinal epithelium regulates tight junction protein expression and restores barrier function when it is increased following exposure to a HFD for 2 wk.NEW & NOTEWORTHY The endocannabinoid system of the gastrointestinal tract regulates homeostasis by acting as brake on motility and secretion. Here we show that when exposed to a high fat diet, intestinal permeability is increased and activation of the CB1 receptor on the intestinal epithelium restores barrier function. This work further highlights the role of the endocannabinoid system in regulating intestinal homeostasis when it is perturbed.

The genetic heterogeneity and drug resistance mechanisms of relapsed refractory multiple myeloma.

Multiple myeloma is the second most common hematological malignancy. Despite significant advances in treatment, relapse is common and carries a poor prognosis. Thus, it is critical to elucidate the genetic factors contributing to disease progression and drug resistance. Here, we carry out integrative clinical sequencing of 511 relapsed, refractory multiple myeloma (RRMM) patients to define the disease's molecular alterations landscape. The NF-κB and RAS/MAPK pathways are more commonly altered than previously reported, with a prevalence of 45-65% each. In the RAS/MAPK pathway, there is a long tail of variants associated with the RASopathies. By comparing our RRMM cases with untreated patients, we identify a diverse set of alterations conferring resistance to three main classes of targeted therapy in 22% of our cohort. Activating mutations in IL6ST are also enriched in RRMM. Taken together, our study serves as a resource for future investigations of RRMM biology and potentially informs clinical management.

Role of the Endocannabinoid System in the Regulation of Intestinal Homeostasis.

The maintenance of intestinal homeostasis is fundamentally important to health. Intestinal barrier function and immune regulation are key determinants of intestinal homeostasis and are therefore tightly regulated by a variety of signaling mechanisms. The endocannabinoid system is a lipid mediator signaling system widely expressed in the gastrointestinal tract. Accumulating evidence suggests the endocannabinoid system is a critical nexus involved in the physiological processes that underlie the control of intestinal homeostasis. In this review we will illustrate how the endocannabinoid system is involved in regulation of intestinal permeability, fluid secretion, and immune regulation. We will also demonstrate a reciprocal regulation between the endocannabinoid system and the gut microbiome. The role of the endocannabinoid system is complex and multifaceted, responding to both internal and external factors while also serving as an effector system for the maintenance of intestinal homeostasis.

In silico analysis of SARS-CoV-2 proteins as targets for clinically available drugs.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires treatments with rapid clinical translatability. Here we develop a multi-target and multi-ligand virtual screening method to identify FDA-approved drugs with potential activity against SARS-CoV-2 at traditional and understudied viral targets. 1,268 FDA-approved small molecule drugs were docked to 47 putative binding sites across 23 SARS-CoV-2 proteins. We compared drugs between binding sites and filtered out compounds that had no reported activity in an in vitro screen against SARS-CoV-2 infection of human liver (Huh-7) cells. This identified 17 "high-confidence", and 97 "medium-confidence" drug-site pairs. The "high-confidence" group was subjected to molecular dynamics simulations to yield six compounds with stable binding poses at their optimal target proteins. Three drugs-amprenavir, levomefolic acid, and calcipotriol-were predicted to bind to 3 different sites on the spike protein, domperidone to the Mac1 domain of the non-structural protein (Nsp) 3, avanafil to Nsp15, and nintedanib to the nucleocapsid protein involved in packaging the viral RNA. Our "two-way" virtual docking screen also provides a framework to prioritize drugs for testing in future emergencies requiring rapidly available clinical drugs and/or treating diseases where a moderate number of targets are known.

Molnupiravir; molecular and functional descriptors of mitochondrial safety.

Molnupiravir is an orally active nucleoside analog antiviral drug that recently was approved by the U.S. FDA for emergency treatment of adult patients infected with the SARS-CoV-2 (COVID-19) virus and at risk for severe progression. The active form of the drug, N-hydroxycytidine (NHC) triphosphate competes for incorporation by RNA-dependent RNA-polymerase (RdRp) into the replicating viral genome resulting in mutations and arrest of the replicating virus. Historically, some nucleoside analog antiviral drugs have been found to lack specificity for the virus and also inhibit replication and/or expression of the mitochondrial genome. The objective of the present study was to test whether molnupiravir and/or NHC also target mitochondrial DNA polymerase gamma (PolG) or RNA polymerase (POLRMT) activity to inhibit the replication and/or expression of the mitochondrial genome leading to impaired mitochondrial function. Human-derived HepG2 cells were exposed for 48 h in culture to increasing concentrations of either molnupiravir or NHC after which cytotoxicity, mtDNA copy number and mitochondrial gene expression were determined. The phenotypic endpoint, mitochondrial respiration, was measured with the Seahorse® XF96 Extracellular Flux Analyzer. Both molnupiravir and NHC were cytotoxic at concentrations of ≥10 µM. However, at non-cytotoxic concentrations, neither significantly altered mitochondrial gene dose or transcription, or mitochondrial respiration. From this we conclude that mitochondrial toxicity is not a primary off target in the mechanism of cytotoxicity for either molnupiravir or its active metabolite NHC in the HepG2 cell line.

Structural Characterization of the D179N and D179Y Variants of KPC-2 ß-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) present a global clinical threat, as these ß-lactamases confer resistance to carbapenems and oxyimino-cephalosporins. Recent clinically identified KPC variants with substitutions at Ambler position D179, located in the Ω loop, are resistant to the ß-lactam/ß-lactamase inhibitor combination ceftazidime-avibactam, but susceptible to meropenem-vaborbactam. To gain insights into ceftazidime-avibactam resistance conferred by D179N/Y variants of KPC-2, crystal structures of these variants were determined. The D179N KPC-2 structure revealed that the change of the carboxyl to an amide moiety at position 179 disrupted the salt bridge with R164 present in wild-type KPC-2. Additional interactions were disrupted in the Ω loop, causing a decrease in the melting temperature. Shifts originating from N179 were also transmitted toward the active site, including ∼1-Å shifts of the deacylation water and interacting residue N170. The structure of the D179Y KPC-2 ß-lactamase revealed more drastic changes, as this variant exhibited disorder of the Ω loop, with other flanking regions also being disordered. We postulate that the KPC-2 variants can accommodate ceftazidime because the Ω loop is displaced in D179Y or can be more readily displaced in D179N KPC-2. To understand why the ß-lactamase inhibitor vaborbactam is less affected by the D179 variants than avibactam, we determined the crystal structure of D179N KPC-2 in complex with vaborbactam, which revealed wild-type KPC-2-like vaborbactam-active site interactions. Overall, the structural results regarding KPC-2 D179 variants revealed various degrees of destabilization of the Ω loop that contribute to ceftazidime-avibactam resistance, possible substrate-assisted catalysis of ceftazidime, and meropenem and meropenem-vaborbactam susceptibility.

Computational Identification of Possible Allosteric Sites and Modulators of the SARS-CoV-2 Main Protease.

In this study, we target the main protease (Mpro) of the SARS-CoV-2 virus as it is a crucial enzyme for viral replication. Herein, we report three plausible allosteric sites on Mpro that can expand structure-based drug discovery efforts for new Mpro inhibitors. To find these sites, we used mixed-solvent molecular dynamics (MixMD) simulations, an efficient computational protocol that finds binding hotspots through mapping the surface of unbound proteins with 5% cosolvents in water. We have used normal mode analysis to support our claim of allosteric control for these sites. Further, we have performed virtual screening against the sites with 361 hits from Mpro screenings available through the National Center for Advancing Translational Sciences (NCATS). We have identified the NCATS inhibitors that bind to the remote sites better than the active site of Mpro, and we propose these molecules may be allosteric regulators of the system. After identifying our sites, new X-ray crystal structures were released that show fragment molecules in the sites we found, supporting the notion that these sites are accurate and druggable.

Event-Based Tracking Consensus for Multiagent Systems With Volatile Control Gain.

This work investigates the tracking consensus problem of multiagent systems over directed networks, where the control gains follow certain volatile patterns. Some event-based consensus protocols are formulated so as to reduce the redundant execution of control. By using an extended differential inequality with a time-dependent coefficient, criteria for tracking consensus under time- and state-dependent triggering conditions are constructed, respectively. It is proved that the time average of the control gain, together with the agent dynamics, network topology, and triggering conditions, governs the consensus despite the fluctuation of control gain. The derived theorem can be utilized to ensure consensus with intermittent strategies aiming to lessen the burden in communications, including aperiodic on-off control with periodic perturbation and pulse-modulated on-off control. Unlike existing works, the requirement of a positive lower bound of control ratios is removed and, thus, a wide range of control gain patterns is possible, signifying higher flexibility in intermittent policy design. Finally, numerical examples are provided to further illustrate the theoretical results.

Protease-activated receptor-2 activation enhances epithelial wound healing via epidermal growth factor receptor.

The intestinal barrier function relies on the presence of a single layer of epithelial cells. Barrier dysfunction is associated with the inflammatory bowel diseases (IBD). Understanding the mechanisms involved in intestinal wound healing in order to sustain the barrier function has a great therapeutic potential. Activation of protease-activated receptor-2 (PAR2) induces COX-2 expression in intestinal epithelial cells via EGFR transactivation. COX-2 is well known for its protective effects in the gastrointestinal tract. Therefore, we hypothesized that PAR-2 activation induces a wound healing response in intestinal epithelial cells through COX-2-derived lipid mediators and EGFR transactivation. Immunofluorescence and calcium assay were used to characterize CMT-93 mouse colonic epithelial cell line for PAR2 expression and its activity, respectively. Treatment with PAR2 activating peptide 2-furoyl-LIGRLO-NH2 (2fLI), but not by its inactive reverse-sequence peptide (2fO) enhanced wound closure in scratch wounded monolayers. The EGFR tyrosine kinase inhibitor (PD153035), broad-spectrum matrix metalloproteinase inhibitor (GM6001) and Src tyrosine kinase inhibitor (PP2) inhibited PAR2-induced wound healing. However, PAR2 activation did not induce COX-2 expression in CMT-93 cells and inhibition of COX-2 by COX-2 selective inhibitor (NS-398) did not alter PAR2-induced wound healing. In conclusion, PAR2 activation drives wound healing in CMT-93 cells via EGFR transactivation. Matrix metalloproteinases and Src tyrosine kinase activity may involve in EGFR transactivation and PAR2-induced wound healing is independent of COX-2 activity. These findings provide a mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases.

Cells and mediators of inflammation as effectors of epithelial repair in the inflamed intestine.

Mucosal and histological healing have become the gold standards for assessing the efficacy of therapy in patients living with inflammatory bowel diseases (IBD). Despite these being the accepted goals in therapy, the mechanisms that underlie the healing of the mucosa after an inflammatory insult are not well understood, and many patients fail to meet this therapeutic endpoint. Here we review the emerging evidence that mediators (e.g., prostaglandins, cytokines, proteases, reactive oxygen, and nitrogen species) and innate immune cells (e.g., neutrophils and monocytes/macrophages), that are involved in the initiation of the inflammatory response, are also key players in the mechanisms underlying mucosal healing to resolve chronic inflammation in the colon. The dual function mediators comprise an inflammation/repair program that returns damaged tissue to homeostasis. Understanding details of the dual mechanisms of these mediators and cells may provide the basis for the development of drugs that can help to stimulate epithelial repair in patients affected by IBD.

The dietary fibre rhamnogalacturonan improves intestinal epithelial barrier function in a microbiota-independent manner.

BACKGROUND AND PURPOSE: Dietary fibre comprises a complex group of polysaccharides that are indigestible but are fermented by gut microbiota, promoting beneficial effects to the intestinal mucosa indirectly through the production of short chain fatty acids. We found that a polysaccharide, rhamnogalacturonan (RGal), from the plant Acmella oleracea, has direct effects on intestinal epithelial barrier function. Our objective was to determine the mechanism whereby RGal enhances epithelial barrier function. EXPERIMENTAL APPROACH: Monolayers of colonic epithelial cell lines (Caco-2, T84) and of human primary cells from organoids were mounted in Ussing chambers to assess barrier function. The cellular mechanism of RGal effects on barrier function was determined using inhibitors of TLR-4 and PKC isoforms. KEY RESULTS: Apically applied RGal (1000 µg ml-1 ) significantly enhanced barrier function as shown by increased transepithelial electrical resistance (TER) and reduced fluorescein isothiocyanate (FITC)-dextran flux in Caco-2, T84 and human primary cell monolayers, and accelerated tight junction reassembly in Caco-2 cells in a calcium switch assay. RGal also reversed the barrier-damaging effects of inflammatory cytokines on FITC-dextran flux and preserved the tight junction distribution of occludin. RGal activated TLR4 in TLR4-expressing HEK reporter cells, an effect that was inhibited by the TLR4 inhibitor, C34. The effect of RGal was also dependent on PKC, specifically the isoforms PKCδ and PKCζ. CONCLUSION AND IMPLICATIONS: RGal enhances intestinal epithelial barrier function through activation of TLR4 and PKC signalling pathways. Elucidation of RGal mechanisms of action could lead to new, dietary approaches to enhance mucosal healing in inflammatory bowel diseases.

Remdesivir; molecular and functional measures of mitochondrial safety.

Remdesivir is one of a few antiviral drugs approved for treating severe cases of coronavirus 2 (SARS-CoV-2) infection in hospitalized patients. The prodrug is a nucleoside analog that interferes with viral replication by inhibiting viral RNA-dependent RNA polymerase. The drug has also been shown to be a weak inhibitor of human mitochondrial RNA polymerase, leaving open the possibility of mitochondrial off-targets and toxicity. The investigation was designed to explore whether remdesivir causes mitochondrial toxicity, using both genomic and functional parameters in the assessment. Human-derived HepG2 liver cells were exposed for up to 48 h in culture to increasing concentrations of remdesivir. At sub-cytotoxic concentrations (<1 µM), the drug failed to alter either the number of copies or the expression of the mitochondrial genome. mtDNA copy number was unaffected as was the relative rates of expression of mtDNA-encoded and nuclear encoded subunits of complexes I and IV of the mitochondrial respiratory chain. Consistent with this is the observation that remdesivir was without effect on mitochondrial respiration, including basal respiration, proton leak, maximum uncoupled respiration, spare respiratory capacity or coupling efficiency. We conclude that although remdesivir has weak inhibitory activity towards mitochondrial RNA polymerase, mitochondria are not primary off-targets for the mechanism of cytotoxicity of the drug.

Mixed-solvent molecular dynamics simulation-based discovery of a putative allosteric site on regulator of G protein signaling 4.

Regulator of G protein signaling 4 (RGS4) is an intracellular protein that binds to the Gα subunit ofheterotrimeric G proteins and aids in terminating G protein coupled receptor signaling. RGS4 has been implicated in pain, schizophrenia, and the control of cardiac contractility. Inhibitors of RGS4 have been developed but bind covalently to cysteine residues on the protein. Therefore, we sought to identify alternative druggable sites on RGS4 using mixed-solvent molecular dynamics simulations, which employ low concentrations of organic probes to identify druggable hotspots on the protein. Pseudo-ligands were placed in consensus hotspots, and perturbation with normal mode analysis led to the identification and characterization of a putative allosteric site, which would be invaluable for structure-based drug design of non-covalent, small molecule inhibitors. Future studies on the mechanism of this allostery will aid in the development of novel therapeutics targeting RGS4.