Role of N-Cadherin cis and trans Interfaces in?the Dynamics of Adherens Junctions in Living Cells

Cadherins, Ca²⁺-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca²⁺-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca²⁺-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca²⁺-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.     

Microbiota Dynamics in Patients Treated with Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembraneous colitis and is responsible for a large and increasing fraction of hospital-acquired infections. Fecal microbiota transplantation (FMT) is an alternate treatment option for recurrent C. difficile infection (RCDI) refractory to antibiotic therapy. It has recently been discussed favorably in the clinical and scientific communities and is receiving increasing public attention. However, short- and long-term health consequences of FMT remain a concern, as the effects of the transplanted microbiota on the patient remain unknown. To shed light on microbial events associated with RCDI and treatment by FMT, we performed fecal microbiota analysis by 16S rRNA gene amplicon pyrosequencing of 14 pairs of healthy donors and RCDI patients treated successfully by FMT. Post-FMT patient and healthy donor samples collected up to one year after FMT were studied longitudinally, including one post-FMT patient with antibiotic-associated relapse three months after FMT. This analysis allowed us not only to confirm prior reports that RCDI is associated with reduced diversity and compositional changes in the fecal microbiota, but also to characterize previously undocumented post-FMT microbiota dynamics. Members of the Streptococcaceae, Enterococcaceae, or Enterobacteriaceae were significantly increased and putative butyrate producers, such as Lachnospiraceae and Ruminococcaceae were significantly reduced in samples from RCDI patients before FMT as compared to post-FMT patient and healthy donor samples. RCDI patient samples showed more case-specific variations than post-FMT patient and healthy donor samples. However, none of the bacterial groups were invariably associated with RCDI or successful treatment by FMT. Overall microbiota compositions in post-FMT patients, specifically abundances of the above-mentioned Firmicutes, continued to change for at least 16 weeks after FMT, suggesting that full microbiota recovery from RCDI may take much longer than expected based on the disappearance of diarrheal symptoms immediately after FMT.     

Sex Role Segregation and Mixing among Men Who Have Sex with Men: Implications for Biomedical HIV Prevention Interventions

Objective

Men who have sex with men (MSM) practice role segregation – insertive or receptive only sex positions instead of a versatile role - in several international settings where candidate biomedical HIV prevention interventions (e.g., circumcision, anal microbicide) will be tested. The effects of these position-specific interventions on HIV incidence are modeled.

Materials and Methods

We developed a deterministic compartmental model to predict HIV incidence among Indian MSM using data from 2003–2010. The model’s sex mixing matrix was derived from network data of Indian MSM (n = 4604). Our model captures changing distribution of sex roles over time. We modeled microbicide and circumcision efficacy on trials with heterosexuals.

Results

Increasing numbers of versatile MSM resulted in little change in HIV incidence over 20 years. Anal microbicides and circumcision would decrease the HIV prevalence at 10 years from 15.6% to 12.9% and 12.7% respectively. Anal microbicides would provide similar protection to circumcision at the population level despite lower modeled efficacy (54% and 60% risk reduction, respectively). Combination of the interventions were additive: in 5 years, the reduction in HIV prevalence of the combination (−3.2%) is almost the sum of their individual reductions in HIV prevalence (−1.8% and −1.7%).

Conclusions

MSM sex role segregation and mixing, unlike changes in the sex role distribution, may be important for evaluating HIV prevention interventions in international settings. Synergies between some position-specific prevention interventions such as circumcision and anal microbicides warrant further study.     

The Anaphase-Promoting Complex Protein 5 (AnapC5) Associates with A20 and Inhibits IL-17-Mediated Signal Transduction

IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7), which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3), a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways.     

Computational prediction and experimental validation of the activator function of C2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone on pancreatic and hepatic hexokinase

Abstract

This study identifies and validates hexokinase type 4 (HK4), an isozyme of hexokinase in the liver and pancreas, as an important target of C2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (βdGT), a xanthone glucoside suggested to have antidiabetic property. In the study, we applied the computational pipeline of molecular docking followed by the molecular dynamics simulations to shortlist potential βdGT protein targets. The analysis of protein dynamics and the binding free energy (ΔG) led us to the identification of HK4 as a key βdGT target, whereby the binding mode and domain dynamics suggested the activator function of βdGT. βdGT bound to the allosteric site of the isozyme ～13?Å away from the substrate (glucose)-binding site. The binding free energy of the ligand-protein complex was energetically feasible (ΔG, –41.61?kcal/mol) and the cleft angle deviation between the two (small and large) domains of HK4 revealed differential HK4 dynamics in response to βdGT binding. 3D structure analysis of the isozyme-ligand complex highlighted the role of Arg63, Glu67 and Lys458 in ligand stabilization and hydrophobic interactions mediated by Tyr214 and Met235. Experimental validation of the results of computational analysis confirmed the activator function of βdGT on HK4. The study has implication in diabetes as βdGT may be used to lower the blood glucose level by activating hepatic and pancreatic hexokinase without the risk of hypoglycemia.

Communicated by Ramaswamy H. Sarma     

Common variant in FUT2 gene is associated with levels of vitamin B12 in Indian population

Vitamin B₁₂ is an essential micronutrient synthesized by microorganisms. Mammals including humans have evolved ways for transport and absorption of this vitamin. Deficiency of vitamin B₁₂ (either due to low intake or polymorphism in genes involved in absorption and intracellular transport of this vitamin) has been associated with various complex diseases. Genome-wide association studies have recently identified several common single nucleotide polymorphisms (SNPs) in fucosyl transferase 2 gene (FUT2) to be associated with levels of vitamin B₁₂—the strongest association was with a non-synonymous SNP rs602662 in this gene. In the present study, we attempted to replicate the association of this SNP (rs602662) in an Indian population since a significant proportion has been reported to have low levels of vitamin B₁₂ in this population. A total of 1146 individuals were genotyped for this SNP using a single base extension method and association with levels of vitamin B₁₂ was assessed in these individuals. Regression analysis was performed to analyze the association considering various confounding factors like for age, sex, diet, hypertension, diabetes mellitus and coronary artery disease status. We found that the SNP rs602662 was significantly associated with the levels of vitamin B₁₂ (p value < 0.0001). We also found that individuals adhering to a vegetarian diet with GG (homozygous major genotype) have significantly lower levels of vitamin B₁₂ in these individuals. Thus, our study reveals that vegetarian diet along with polymorphism in the FUT2 gene may contribute significantly to the high prevalence of vitamin B₁₂ deficiency in India.     

Cardiovascular disease after Escherichia coli O157:H7 gastroenteritis

Background:

Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, which can be devastating in outbreak situations. We studied the risk of cardiovascular disease following such an outbreak in Walkerton, Ontario, in May 2000.

Methods:

In this community-based cohort study, we linked data from the Walkerton Health Study (2002–2008) to Ontario’s large healthcare databases. We included 4 groups of adults: 3 groups of Walkerton participants (153 with severe gastroenteritis, 414 with mild gastroenteritis, 331 with no gastroenteritis) and a group of 11 263 residents from the surrounding communities that were unaffected by the outbreak. The primary outcome was a composite of death or first major cardiovascular event (admission to hospital for acute myocardial infarction, stroke or congestive heart failure, or evidence of associated procedures). The secondary outcome was first major cardiovascular event censored for death. Adults were followed for an average of 7.4 years.

Results:

During the study period, 1174 adults (9.7%) died or experienced a major cardiovascular event. Compared with residents of the surrounding communities, the risk of death or cardiovascular event was not elevated among Walkerton participants with severe or mild gastroenteritis (hazard ratio [HR] for severe gastroenteritis 0.74, 95% confidence interval [CI] 0.38–1.43, mild gastroenteritis HR 0.64, 95% CI 0.42–0.98). Compared with Walkerton participants who had no gastroenteritis, risk of death or cardiovascular event was not elevated among participants with severe or mild gastroenteritis.

Interpretation:

There was no increase in the risk of cardiovascular disease in the decade following acute infection during a major E. coli O157:H7 outbreak.Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, causing 63 000 infections each year and 12 major outbreaks since 2006 in the United States alone.^1,2 This strain was most recently implicated in the outbreak involving beef from XL Foods (September 2012), with 17 confirmed cases across Canada.³ A similar enterohemorrhagic strain E. coli O104:H4 was responsible for an outbreak in Germany in May 2011, causing 3792 cases of gastroenteritis and 43 deaths.^4,5Most patients fully recover from acute gastroenteritis caused by E. coli. However, such an illness may predispose patients to long-term disease. Shiga toxin is produced by E. coli O157:H7; this toxin damages the microvasculature of the kidneys leading to hypertension^6–13 and directly damages the systemic vasculature.^14–16 Infected people may progress from a state of acute inflammation of the vasculature to subclinical chronic inflammation, which could promote atherosclerosis.^17–20In Walkerton, Ontario, in May 2000, heavy rains transported bovine fecal matter into the town’s well, contaminating the inadequately chlorinated municipal water supply with E. coli O157:H7.²¹ Over 2300 people developed acute gastroenteritis, and 7 people died.²² The unique circumstances of this outbreak provided a rare opportunity to study the natural history following exposure to this pathogen in a single cohort.²³ Other outbreaks have been geographically dispersed, making it difficult to track cases.^24,25In Walkerton, affected individuals were followed annually in a clinic to assess their long-term outcomes (Walkerton Health Study, 2002–2008). We previously reported that adults who experienced acute gastroenteritis during the outbreak had a higher than expected incidence of hypertension, chronic kidney disease and self-reported cardiovascular disease in follow-up.²³ However, 46% of participants were lost to follow-up by the end of the study, and there were limitations associated with the assessment of cardiovascular disease by participant recall. Thus, we conducted an expanded and extended follow-up study, linking the Walkerton study data to Ontario’s health care databases. Our objective was to more accurately determine the 10-year risk of major cardiovascular events after exposure to E. coli O157:H7.     

Identification of mycobacterial lectins from genomic data

Sixty‐four sequences containing lectin domains with homologs of known three‐dimensional structure were identified through a search of mycobacterial genomes. They appear to belong to the β‐prism II, the C‐type, the Microcystis virdis (MV), and the β‐trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI‐PLC, and the β‐grasp domains, respectively while mycobacterial β‐trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three‐dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and nonpathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one or the other of the four lectin domains can be gleaned through the examination of homologs proteins, although the structure of none of them is available. Their biological role is also yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Novel insights into the dynamics behavior of glucagon-like peptide-1 receptor with its small molecule agonists

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a well-known target of therapeutics industries for the treatment of various metabolic diseases like type 2 diabetes and obesity. The structural–functional relationships of small molecule agonists and GLP-1R are yet to be understood. Therefore, an attempt was made on structurally known GLP-1R agonists (Compound 1, Compound 2, Compound A, Compound B, and (S)-8) to study their interaction with the extracellular domain of GLP-1R. In this study, we explored the dynamics, intrinsic stability, and binding mechanisms of these molecules through computational modeling, docking, molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) binding free energy estimation. Molecular docking study depicted that hydrophobic interaction (pi–pi stacking) plays a crucial role in maintaining the stability of the complex, which was also supported by intermolecular analysis from MD simulation study. Principal component analysis suggested that the terminal ends along with the turns/loops connecting adjacent helix and strands exhibit a comparatively higher movement of main chain atoms in most of the complexes. MM/PBSA binding free energy study revealed that non-polar solvation (van der Waals and electrostatic) energy subsidizes significantly to the total binding energy, and the polar solvation energy opposes the binding agonists to GLP-1R. Overall, we provide structural features information about GLP-1R complexes that would be conducive for the discovery of new GLP-1R agonists in the future for the treatment of various metabolic diseases.

Communicated by Ramaswamy H. Sarma