Multiple Infection and Microdiversity among Helicobacter pylori Isolates in a Single Host in India

Helicobacter pylori is one of the most diverse bacterial species that chronically infects more than 70% of Indian population. Interestingly, data showing microdiversity of the H. pylori strains within a particular gastric niche remained scarce. To understand the extent of genetic diversity among H. pylori strains within a given host, 30 patients with gastro-duodenal problems were subjected to endoscopy and from each patient 10 single colonies were isolated. Characterization of each of these 10 single colonies by DNA fingerprinting as well as genotyping of several important genetic markers viz. cagA, vacA, iceA, vapD, cag PAI empty site, IS605, RFLP and two other genetic segments within cag PAI revealed that all of the 30 patients were infected with more than one strain and sometimes strains with 5 to 6 types of genetic variants. Analyses of certain genetic loci showed the microdiversity among the colonies from single patient, which may be due to the recombination events during long-term carriage of the pathogen. These results suggest that most of the patients have acquired H. pylori due to repeated exposure to this pathogen with different genetic make-up, which may increase the possibility of super infections. Genetic exchanges between these unrelated H. pylori strains may support certain H. pylori variant to grow better in a given host than the parental strain and thereby increasing the possibility for the severity of the infection.     

The SH3 domain-containing adaptor HIP-55 mediates c-Jun N-terminal kinase activation in T cell receptor signaling

HIP-55 (hematopoietic progenitor kinase 1 (HPK1)-interacting protein of 55 kDa, also called SH3P7 and mAbp1) is a novel SH3 domain-containing protein. HIP-55 binds to actin filaments both in vitro and in vivo. HIP-55 activates HPK1 and c-Jun N-terminal kinase (JNK), which are two important lymphocyte signaling molecules. Until now, the regulation and function of HIP-55 in T cell receptor (TCR) signaling were unknown. We found that HIP-55 was recruited to glycolipid-enriched microdomains upon TCR stimulation, which indicates that HIP-55 is regulated by TCR signaling. HIP-55 interacted with ZAP-70, a critical protein-tyrosine kinase in TCR signaling, and this interaction was induced by TCR signaling. ZAP-70 phosphorylated HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo, and the HIP-55 mutant (Y334F/Y344F) was not tyrosine-phosphorylated in stimulated T cells. To study its function in T cell activation, HIP-55-deficient Jurkat T cells were established using the RNA interference approach. In the HIP-55-deficient cells, TCR (but not UV)-stimulated JNK activation was decreased. Furthermore, the activation of HPK1, a known JNK upstream activator and HIP-55-interacting protein, was also decreased in the HIP-55-deficient cells. Our data reveal the regulation of HIP-55 during TCR signaling, and using a genetic approach, we demonstrate for the first time that HIP-55 plays a functional role in TCR signaling.     

Blood-sampling collection prior to surgery may have a significant influence upon biomarker concentrations measured

Background

Biomarkers can be subtle tools to aid the diagnosis, prognosis and monitoring of therapy and disease progression. The validation of biomarkers is a cumbersome process involving many steps. Serum samples from lung cancer patients were collected in the framework of a larger study for evaluation of biomarkers for early detection of lung cancer. The analysis of biomarker levels measured revealed a noticeable difference in certain biomarker values that exhibited a dependence of the time point and setting of the sampling. Biomarker concentrations differed significantly if taken before or after the induction of anesthesia and if sampled via venipuncture or arterial catheter.

Methods

To investigate this observation, blood samples from 13 patients were drawn 1–2 days prior to surgery (T1), on the same day by venipuncture (T2) and after induction of anesthesia via arterial catheter (T3). The biomarkers Squamous Cell Carcinoma antigen (CanAG SCC EIA, Fujirebio Diagnostics, Malvern, USA), Carcinoembrionic Antigen (CEA), and CYFRA 21-1 (Roche Diagnostics GmbH, Mannheim, Germany) were analyzed.

Results

SCC showed a very strong effect in relation to the sampling time and procedure. While the first two points in time (T1; T2) were highly comparable (median fold-change: 0.84; p = 0.7354; correlation ρ = 0.883), patients showed a significant increase (median fold-change: 4.96; p = 0.0017; correlation ρ = -0.036) in concentration when comparing T1 with the sample time subsequent to anesthesia induction (T3). A much weaker increase was found for CYFRA 21-1 at T3 (median fold-change: 1.40; p = 0.0479). The concentration of CEA showed a very small, but systematic decrease (median fold-change: 0.72; p = 0.0039).

Conclusions

In this study we show the unexpectedly marked influence of blood withdrawal timing (before vs. after anesthesia) and procedure (venous versus arterial vessel puncture) has on the concentration of the protein biomarker SCC and to a less extent upon CYFRA21-1. The potential causes for these effects remain to be elucidated in subsequent studies, however these findings highlight the importance of a standardized, controlled blood collection protocol for biomarker detection.     

Improving catalytic function by ProSAR-driven enzyme evolution

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process approximately 4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.     

Research Design and Statistical Methods in Indian Medical Journals: A Retrospective Survey

Good quality medical research generally requires not only an expertise in the chosen medical field of interest but also a sound knowledge of statistical methodology. The number of medical research articles which have been published in Indian medical journals has increased quite substantially in the past decade. The aim of this study was to collate all evidence on study design quality and statistical analyses used in selected leading Indian medical journals. Ten (10) leading Indian medical journals were selected based on impact factors and all original research articles published in 2003 (N = 588) and 2013 (N = 774) were categorized and reviewed. A validated checklist on study design, statistical analyses, results presentation, and interpretation was used for review and evaluation of the articles. Main outcomes considered in the present study were – study design types and their frequencies, error/defects proportion in study design, statistical analyses, and implementation of CONSORT checklist in RCT (randomized clinical trials). From 2003 to 2013: The proportion of erroneous statistical analyses did not decrease (χ²=0.592, Φ=0.027, p=0.4418), 25% (80/320) in 2003 compared to 22.6% (111/490) in 2013. Compared with 2003, significant improvement was seen in 2013; the proportion of papers using statistical tests increased significantly (χ²=26.96, Φ=0.16, p<0.0001) from 42.5% (250/588) to 56.7 % (439/774). The overall proportion of errors in study design decreased significantly (χ²=16.783, Φ=0.12 p<0.0001), 41.3% (243/588) compared to 30.6% (237/774). In 2013, randomized clinical trials designs has remained very low (7.3%, 43/588) with majority showing some errors (41 papers, 95.3%). Majority of the published studies were retrospective in nature both in 2003 [79.1% (465/588)] and in 2013 [78.2% (605/774)]. Major decreases in error proportions were observed in both results presentation (χ²=24.477, Φ=0.17, p<0.0001), 82.2% (263/320) compared to 66.3% (325/490) and interpretation (χ²=25.616, Φ=0.173, p<0.0001), 32.5% (104/320) compared to 17.1% (84/490), though some serious ones were still present. Indian medical research seems to have made no major progress regarding using correct statistical analyses, but error/defects in study designs have decreased significantly. Randomized clinical trials are quite rarely published and have high proportion of methodological problems.     

Claudin-19 Mutations and Clinical Phenotype in Spanish Patients with Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive tubular disorder characterized by excessive renal magnesium and calcium excretion and chronic kidney failure. This rare disease is caused by mutations in the CLDN16 and CLDN19 genes. These genes encode the tight junction proteins claudin-16 and claudin-19, respectively, which regulate the paracellular ion reabsortion in the kidney. Patients with mutations in the CLDN19 gene also present severe visual impairment. Our goals in this study were to examine the clinical characteristics of a large cohort of Spanish patients with this disorder and to identify the disease causing mutations. We included a total of 31 patients belonging to 27 unrelated families and studied renal and ocular manifestations. We then analyzed by direct DNA sequencing the coding regions of CLDN16 and CLDN19 genes in these patients. Bioinformatic tools were used to predict the consequences of mutations. Clinical evaluation showed ocular defects in 87% of patients, including mainly myopia, nystagmus and macular colobomata. Twenty two percent of patients underwent renal transplantation and impaired renal function was observed in another 61% of patients. Results of the genetic analysis revealed CLDN19 mutations in all patients confirming the clinical diagnosis. The majority of patients exhibited the previously described p.G20D mutation. Haplotype analysis using three microsatellite markers showed a founder effect for this recurrent mutation in our cohort. We also identified four new pathogenic mutations in CLDN19, p.G122R, p.I41T, p.G75C and p.G75S. A strategy based on microsequencing was designed to facilitate the genetic diagnosis of this disease. Our data indicate that patients with CLDN19 mutations have a high risk of progression to chronic renal disease.     

The Werner and Bloom syndrome proteins help resolve replication blockage by converting (regressed) holliday junctions to functional replication forks

Cells cope with blockage of replication fork progression in a manner that allows DNA synthesis to be completed and genomic instability minimized. Models for resolution of blocked replication involve fork regression to form Holliday junction structures. The human RecQ helicases WRN and BLM (deficient in Werner and Bloom syndromes, respectively) are critical for maintaining genomic stability and thought to function in accurate resolution of replication blockage. Consistent with this notion, WRN and BLM localize to sites of blocked replication after certain DNA-damaging treatments and exhibit enhanced activity on replication and recombination intermediates. Here we examine the actions of WRN and BLM on a special Holliday junction substrate reflective of a regressed replication fork. Our results demonstrate that, in reactions requiring ATP hydrolysis, both WRN and BLM convert this Holliday junction substrate primarily to a four-stranded replication fork structure, suggesting they target the Holliday junction to initiate branch migration. In agreement, the Holliday junction binding protein RuvA inhibits the WRN- and BLM-mediated conversion reactions. Importantly, this conversion product is suitable for replication with its leading daughter strand readily extended by DNA polymerases. Furthermore, binding to and conversion of this Holliday junction are optimal at low MgCl(2) concentrations, suggesting that WRN and BLM preferentially act on the square planar (open) conformation of Holliday junctions. Our findings suggest that, subsequent to fork regression events, WRN and/or BLM could re-establish functional replication forks to help overcome fork blockage. Such a function is highly consistent with phenotypes associated with WRN- and BLM-deficient cells.     

META-GSA: Combining Findings from Gene-Set Analyses across Several Genome-Wide Association Studies

Introduction

Gene-set analysis (GSA) methods are used as complementary approaches to genome-wide association studies (GWASs). The single marker association estimates of a predefined set of genes are either contrasted with those of all remaining genes or with a null non-associated background. To pool the p-values from several GSAs, it is important to take into account the concordance of the observed patterns resulting from single marker association point estimates across any given gene set. Here we propose an enhanced version of Fisher’s inverse χ²-method META-GSA, however weighting each study to account for imperfect correlation between association patterns.

Simulation and Power

We investigated the performance of META-GSA by simulating GWASs with 500 cases and 500 controls at 100 diallelic markers in 20 different scenarios, simulating different relative risks between 1 and 1.5 in gene sets of 10 genes. Wilcoxon’s rank sum test was applied as GSA for each study. We found that META-GSA has greater power to discover truly associated gene sets than simple pooling of the p-values, by e.g. 59% versus 37%, when the true relative risk for 5 of 10 genes was assume to be 1.5. Under the null hypothesis of no difference in the true association pattern between the gene set of interest and the set of remaining genes, the results of both approaches are almost uncorrelated. We recommend not relying on p-values alone when combining the results of independent GSAs.

Application

We applied META-GSA to pool the results of four case-control GWASs of lung cancer risk (Central European Study and Toronto/Lunenfeld-Tanenbaum Research Institute Study; German Lung Cancer Study and MD Anderson Cancer Center Study), which had already been analyzed separately with four different GSA methods (EASE; SLAT, mSUMSTAT and GenGen). This application revealed the pathway GO0015291 “transmembrane transporter activity” as significantly enriched with associated genes (GSA-method: EASE, p = 0.0315 corrected for multiple testing). Similar results were found for GO0015464 “acetylcholine receptor activity” but only when not corrected for multiple testing (all GSA-methods applied; p≈0.02).