Potassium dependent structural changes in the selectivity filter of HERG potassium channels.

The fine tuning of biological electrical signaling is mediated by variations in the rates of opening and closing of gates that control ion flux through different ion channels. Human ether-a-go-go related gene (HERG) potassium channels have uniquely rapid inactivation kinetics which are critical to the role they play in regulating cardiac electrical activity. Here, we exploit the K+ sensitivity of HERG inactivation to determine structures of both a conductive and non-conductive selectivity filter structure of HERG. The conductive state has a canonical cylindrical shaped selectivity filter. The non-conductive state is characterized by flipping of the selectivity filter valine backbone carbonyls to point away from the central axis. The side chain of S620 on the pore helix plays a central role in this process, by coordinating distinct sets of interactions in the conductive, non-conductive, and transition states. Our model represents a distinct mechanism by which ion channels fine tune their activity and could explain the uniquely rapid inactivation kinetics of HERG.

Cyclosporin Structure and Permeability: From A to Z and Beyond.

Cyclosporins are natural or synthetic undecapeptides with a wide range of actual and potential pharmaceutical applications. Several members of the cyclosporin compound family have remarkably high passive membrane permeabilities that are not well-described by simple structural metrics. Here we review experimental studies of cyclosporin structure and permeability, including cyclosporin-metal complexes. We also discuss models for the conformation-dependent permeability of cyclosporins and similar compounds. Finally, we identify current knowledge gaps in the literature and provide recommendations regarding future avenues of exploration.

Molecular Dynamics Simulations and Experimental Results Provide Insight into Clinical Performance Differences between Sandimmune® and Neoral® Lipid-Based Formulations.

OBJECTIVE: Molecular dynamics (MD) simulations provide an in silico method to study the structure of lipid-based formulations (LBFs) and the incorporation of poorly water-soluble drugs within such formulations. In order to validate the ability of MD to effectively model the properties of LBFs, this work investigates the well-known cyclosporine A formulations, Sandimmune® and Neoral®. Sandimmune® exhibits poor dispersibility and its absorption from the gastrointestinal tract is enhanced when administered after food, whereas Neoral® disperses comparatively well and shows no food effect. METHODS: MD simulations were performed of both LBFs to investigate the differences observed in fasted and fed conditions. These conditions were also tested using an in vitro experimental model of dispersion and digestion. RESULTS: These MD simulations were able to show that the food effect observed for Sandimmune® can be explained by large changes in drug solubilization on addition of bile. In contrast, Neoral® is well dispersed in water or in simulated fasted conditions, and this dispersion is relatively unchanged on moving to fed conditions. These differences were confirmed using dispersion and digestion in vitro experimental model. CONCLUSIONS: The current data suggests that MD simulations are a potential method to model the fate of LBFs in the gastrointestinal tract, predict their dispersion and digestion, investigate behaviour of APIs within the formulations, and provide insights into the clinical performance of LBFs.

Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children.

We studied stool specimens from 33 autistic children aged 2-9 years with gastrointestinal (GI) abnormalities and 13 control children without autism and without GI symptoms. We performed quantitative comparison of all Clostridium species and Clostridium perfringens strains from the fecal microbiota by conventional, selective anaerobic culture methods. We isolated C. perfringens strains and performed PCR analysis for the main C. perfringens toxin genes, alpha, beta, beta2, epsilon, iota and C. perfringens enterotoxin gene. Our results indicate that autistic subjects with gastrointestinal disease harbor statistically significantly (p = 0.031) higher counts of C. perfringens in their gut compared to control children. Autistic subjects also harbor statistically significantly (p = 0.015) higher counts of beta2-toxin gene-producing C. perfringens in their gut compared to control children, and the incidence of beta2-toxin gene-producing C. perfringens is significantly higher in autistic subjects compared to control children (p = 0.014). Alpha toxin gene was detected in all C. perfringens strains studied. C. perfringens enterotoxin gene was detected from three autistic and one control subject. Beta, epsilon, and iota toxin genes were not detected from autistic or control subjects.

Hofmeister Ion-Induced Changes in Water Structure Correlate with Changes in Solvation of an Aggregated Protein Complex.

RecA is a naturally aggregating Escherichia coli protein that catalyzes the strand exchange reaction utilized in DNA repair. Previous studies have shown that the presence of salts influence RecA activity, aggregation, and stability and that salts stabilize RecA in an inverse-anionic Hofmeister series. Here we utilized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and circular dichroism (CD) to investigate how various Hofmeister salts alter the water structure and RecA solvation and aggregation. Spectroscopic studies performed in water and deuterium oxide suggest that salts alter water O-(1)H and O-(2)H stretch and bend vibrations as well as protein amide I (or I') and amide II (or II') vibrations. Anions have a much larger influence on water vibrations than cations. Water studies also show increased water-water and/or water-ion interactions in the presence of strongly hydrated SO4(2-) salts and evidence for decreased interactions with weakly hydrated Cl(-) and ClO4(-) salts. Salt-water difference infrared spectra show that kosmotropic salts are more hydrated than chaotropic salts. Interestingly, this is the opposite trend to the changes in protein solvation. Infrared spectra of RecA show that vibrations associated with protein desolvation were observed in the presence of SO4(2-) salts. Conversely, vibrations associated with protein solvation were observed in the presence of Cl(-) and ClO4(-) salts. Difference infrared studies on the dehydration of model proteins aided in identifying changes in RecA-solvent interactions. This study provides evidence that salt-induced changes in water vibrations correlate to changes in protein solvent interactions and thermal stability.

Free energy landscape of a minimalist salt bridge model.

Salt bridges are essential to protein stability and dynamics. Despite the importance, there has been scarce of detailed discussion on how salt bridge partners interact with each other in distinct solvent exposed environments. In this study, employing a recent generalized orthogonal space tempering (gOST) method, we enabled efficient molecular dynamics simulation of repetitive breaking and reforming of salt bridge structures within a minimalist salt-bridge model, the Asp-Arg dipeptide and thereby were able to map its detailed free energy landscape in aqueous solution. Free energy surface analysis shows that although individually-solvated states are more favorable, salt-bridge states still occupy a noticeable portion of the overall population. Notably, the competing forces, e.g. intercharge attractions that drive the formation of salt bridges and solvation forces that pull the charged groups away from each other, are energetically comparable. As the result, the salt bridge stability is highly tunable by local environments; for instance when local water molecules are perturbed to interact more strongly with each other, the population of the salt-bridge states is likely to increase. Our results reveal the critical role of local solvent structures in modulating salt-bridge partner interactions and imply the importance of water fluctuations on conformational dynamics that involves solvent accessible salt bridge formations.

Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers.

The health benefits of pomegranate (POM) consumption are attributed to ellagitannins and their metabolites, formed and absorbed in the intestine by the microbiota. In this study twenty healthy participants consumed 1000 mg of POM extract daily for four weeks. Based on urinary and fecal content of the POM metabolite urolithin A (UA), we observed three distinct groups: (1) individuals with no baseline UA presence but induction of UA formation by POM extract consumption (n = 9); (2) baseline UA formation which was enhanced by POM extract consumption (N = 5) and (3) no baseline UA production, which was not inducible (N = 6). Compared to baseline the phylum Actinobacteria was increased and Firmicutes decreased significantly in individuals forming UA (producers). Verrucomicrobia (Akkermansia muciniphila) was 33 and 47-fold higher in stool samples of UA producers compared to non-producers at baseline and after 4 weeks, respectively. In UA producers, the genera Butyrivibrio, Enterobacter, Escherichia, Lactobacillus, Prevotella, Serratia and Veillonella were increased and Collinsella decreased significantly at week 4 compared to baseline. The consumption of pomegranate resulted in the formation of its metabolites in some but not all participants. POM extract consumption may induce health benefits secondary to changes in the microbiota.

Antimicrobial Activity of Pomegranate and Green Tea Extract on Propionibacterium Acnes, Propionibacterium Granulosum, Staphylococcus Aureus and Staphylococcus Epidermidis.

We used pomegranate extract (POMx), pomegranate juice (POM juice) and green tea extract (GT) to establish in vitro activities against bacteria implicated in the pathogenesis of acne. Minimum inhibitory concentrations (MIC) of 94 Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus aureus, and Staphylococcus epidermidis strains were determined by Clinical and Laboratory Standards Institute-approved agar dilution technique. Total phenolics content of the phytochemicals was determined using the Folin-Ciocalteu method and the polyphenol composition by HPLC. Bacteria were identified by 16S rRNA sequence analysis. GT MIC of 400 µg/ml or less was obtained for 98% of the strains tested. 64% of P. acnes strains had POMx MICs at 50 µg/ml whereas 36% had MIC >400 µg/ml. POMx, POM juice, and GT showed inhibitory activity against all the P. granulosum strains at ≤100 µg/ml. POMx and GT inhibited all the S. aureus strains at 400 µg/ml or below, and POM juice had an MIC of 200 µg/ml against 17 S. aureus strains. POMx inhibited S. epidermidis strains at 25 µg/ml, whereas POM juice MICs were ≥200 µg/ml. The antibacterial properties of POMx and GT on the most common bacteria associated with the development and progression of acne suggest that these extracts may offer a better preventative/therapeutic regimen with fewer side effects than those currently available.

Pomegranate extract exhibits in vitro activity against Clostridium difficile.

OBJECTIVE: To determine the possible utility of pomegranate extract in the management or prevention of Clostridium difficile infections or colonization. METHOD: The activity of pomegranate was tested against 29 clinical C. difficile isolates using the Clinical and Laboratory Standards Institute-approved agar dilution technique. Total phenolics content of the pomegranate extract was determined by Folin-Ciocalteau colorimetric method and final concentrations of 6.25 to 400 µg/mL gallic acid equivalent were achieved in the agar. RESULTS: All strains had MICs at 12.5 to 25 mg/mL gallic acid equivalent range. Our results suggest antimicrobial in vitro activity for pomegranate extract against toxigenic C. difficile. CONCLUSION: Pomegranate extract may be a useful contributor to the management and prevention of C. difficile disease or colonization.

Xylooligosaccharide increases bifidobacteria but not lactobacilli in human gut microbiota.

This study was conducted to determine the tolerance and effects of the prebiotic xylooligosaccharide (XOS) on the composition of human colonic microbiota, pH and short chain fatty acids (SCFA) in order to determine whether significant changes in the microbiota would be achievable without side effects. Healthy adult subjects (n = 32) were recruited in a double-blind, randomized, placebo-controlled study. Subjects received 1.4 g XOS, 2.8 g XOS or placebo in daily doses. The study consisted of a 2 week run-in, an 8 week intervention, and a 2 week washout phase. Stool samples were collected at baseline, after 4 and 8 weeks of intervention and 2 weeks after cessation of intervention. Samples were subjected to culture, pyrosequencing of community DNA, pH and SCFA analyses. Tolerance was evaluated by daily symptom charts. XOS was tolerated without significant gastrointestinal side effects. Bifidobacterium counts increased in both XOS groups compared to the placebo subjects, the 2.8 g per day group showed significantly greater increases than the 1.4 g per day group. Total anaerobic counts and Bacteroides fragilis group counts were significantly higher in the 2.8 g per day XOS group. There were no significant differences in the counts of Lactobacillus, Enterobacteriaceae and Clostridium between the three groups. XOS intervention had no significant effect on stool pH, SCFA or lactic acid. Pyrosequencing showed no notable shifts in bacterial diversity. XOS supplementation may be beneficial to gastrointestinal microbiota and 2.8 g per day may be more effective than 1.4 g per day. The low dose required and lack of gastrointestinal side effects makes the use of XOS as a food supplement feasible.