Phylogenetic Distribution and Prevalence of Genes Encoding Class I Integrons and CTX-M-15 Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Healthy Humans in Chandigarh,India

Escherichia coli is generally considered as a commensal inhabitant of gastrointestinal tract of humans and animals. The aim of this study was to gain insight on the distribution of phylotypes and presence of genes encoding integrons, extended β-lactamases and resistance to other antimicrobials in the commensal E. coli isolates from healthy adults in Chandigarh, India. PCR and DNA sequencing were used for phylogenetic classification, detections of integrase genes, gene cassettes within the integron and extended β-lactamases. The genetic structure of E. coli revealed a non-uniform distribution of isolates among the seven phylogenetic groups with significant representation of group A. Integron-encoded integrases were detected in 25 isolates with class 1 integron-encoded intI1 integrase being in the majority (22 isolates). The gene cassettes identified were those for trimethoprim, streptomycin, spectinomycin and streptothricin. The dfrA12-orfF-aadA2 was the most commonly found gene cassette in intI1 positive isolates. Phenotypic assay for screening the potential ESBL producers suggested 16 isolates to be ESBL producers. PCR detection using gene-specific primers showed that 15 out of these 16 ESBL-producing E. coli harboured the bla _CTX-M-15 gene. Furthermore, molecular studies helped in characterizing the genes responsible for tetracycline, chloramphenicol and sulphonamides resistance. Collectively, our study outlines the intra-species phylogenetic structure and highlights the prevalence of class 1 integron and bla _CTX-M-15 in commensal E. coli isolates of healthy adults in Chandigarh, India. Our findings further reinforce the relevance of commensal E. coli strains on the growing burden of antimicrobial resistance.     

Distinct Domains within APOBEC3G and APOBEC3F Interact with Separate Regions of Human Immunodeficiency Virus Type 1 Vif

Human APOBEC3G (A3G) and APOBEC3F (A3F) inhibit the replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their degradation. Thus, the Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development, as inhibiting these interactions could allow the host defense mechanism to control HIV-1 replication. Recently, it has been reported that amino acids 105 to 156 of A3G are involved in the interaction with Vif; however, to date, the region of A3F involved in Vif binding has not been identified. Using our previously reported Vif mutants that are capable of binding to only A3G (3G binder) or only A3F (3F binder), in conjunction with a series of A3G-A3F chimeras, we have now mapped the APOBEC3-Vif interaction domains. We found that the A3G domain that interacts with the Vif YRHHY region is located between amino acids 126 and 132 of A3G, which is consistent with the conclusions reported in previous studies. The A3F domain that interacts with the Vif DRMR region did not occur in the homologous domain but instead was located between amino acids 283 and 300 of A3F. These studies are the first to identify the A3F domain that interacts with the Vif DRMR region and show that distinct domains of A3G and A3F interact with different Vif regions. Pharmacological inhibition of either or both of these Vif-A3 interactions should prevent the degradation of the APOBEC3 proteins and could be used as a therapy against HIV-1.     

Structural studies on mannose-selective glycoprotein receptors using molecular modeling techniques

Glycoproteins play important roles in various cellular events and their presence in appropriate locations in proper active conformations is essential for many biochemical functions. Recent evidences suggest that some glycoproteins may require sorting receptors for efficient exit from the endoplasmic reticulum. These receptors need the presence of calcium or other metal ions for their native activity. The three-dimensional structure of such a receptor, p58/ERGIC-53, has been recently solved by x-ray crystallography, which is a mannose-selective lectin and contains two Ca²⁺ ions. Homology search in the sequence databases indicates a large number of proteins which bear varying degrees of homology in a wide spectrum of species with this receptor. In this study we have systematically searched for such genes which are potential candidates for acting as mannose-mediated glycoprotein receptors in various species as initially inferred from their amino acid sequence homology. Structures of a number of proteins have been predicted using knowledge-based homology modeling, and their ability to act as the glycoprotein receptor has been explored by examining the nature of sugar-binding site. Tetramer of mannose was docked in the binding pockets of the modeled structures followed by energy minimization and molecular dynamics to obtain most probable structures of the complexes. Properties of these modeled complexes were studied to examine the nature of physicochemical forces involved in the complex formation and compared with p58/ERGIC-53-mannose complex.     

Heart Rate Variability During Sleep Following the Practice of Cyclic Meditation and Supine Rest

Day time activities are known to influence the sleep on the following night. Cyclic meditation (CM) has recurring cycles. Previously, the low frequency (LF) power and the ratio between low frequency and high frequency (LF/HF ratio) of the heart rate variability (HRV) decreased during and after CM but not after a comparable period of supine rest (SR). In the present study, on thirty male volunteers, CM was practiced twice in the day and after this the HRV was recorded (1) while awake and (2) during 6 h of sleep (based on EEG, EMG and EGG recordings). This was similarly recorded for the night’s sleep following the day time practice of SR. Participants were randomly assigned to the two sessions and all of them practiced both CM and SR on different days. During the night following day time CM practice there were the following changes; a decrease in heart rate, LF power (n.u.), LF/HF ratio, and an increase in the number of pairs of Normal to Normal RR intervals differing by more than 50 ms divided by total number of all NN intervals (pNN50) (P < 0.05, in all cases, comparing sleep following CM compared with sleep following SR). No change was seen on the night following SR. Hence yoga practice during the day appears to shift sympatho-vagal balance in favor of parasympathetic dominance during sleep on the following night.     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Procedure for the Preparation of Milligram Quantities of Adenovirus Messenger Ribonucleic Acid

Late after adenovirus 2 infection (18 hr), nearly all newly synthesized polysomal messenger ribonucleic acid (mRNA) is viral specified. Large amounts of adenovirus mRNA have been purified by utilizing membrane filtration at high ionic strength. With this procedure, molecules that contain polyadenylic acid [poly (A)] tracts are bound selectively, and ribosomal RNA can be separated from the viral mRNA which contains poly(A). Polysomal RNA synthesized 18 hr after infection was labeled in the presence of 0.02 mug of actinomycin D per ml and extracted at pH 9.0. This RNA annealed 40% to 3 mug of adenovirus 2 deoxyribonucleic acid; the RNA selected by membrane filtration bound 80% under the same conditions. The RNA eluted from membrane filters was 80 to 90% greater than 18S and contained species migrating as 31, 27, and 24S. Binding of polysomal RNA to individual membrane filters was linear, using as much as 300 mug of RNA per membrane. A 1.1-mg amount of viral RNA was prepared from 17.7 mg of polysomal RNA that had been purified by extraction at pH 9.0.     

Influence of suspended particulate matter on nutrient biogeochemistry of a tropical shallow lagoon,Chilika, India

The behaviour of suspended particulate matter (SPM), salinity profile, dissolved nutrients, total (T.Chl-a) and size fractionated chlorophyll-a (F.Chl-a) were studied seasonally at Chilika Lagoon, east coast of India, during 2008–2009. The study showed large spatio-temporal variations among these parameters. The concentration of dissolved inorganic nitrogen and inorganic phosphate were found to be maximum during the monsoon, followed by post- and pre-monsoon, although the mean N:P ratios, which indicate the relative availability of N with respect to P, were 9.13 ± 3.09, 16.57 ± 11.53 and 5.47 ± 3.13, respectively. It was evident from the results that during pre-monsoon and postmonsoon, the lagoon exhibits nitrogen limitation. Mean T.Chl-a biomass in the lagoon showed distinct seasonality with maximum values during the pre-monsoon (23.12 ± 9.75 mg m^?3) followed by monsoon and post-monsoon. Irrespective of seasons, maximum T.Chl-a was found in the northern part of the lagoon. SPM concentrations during the monsoon were relatively higher in the freshwater dominated zones compared to seawater dominated areas, indicating its riverine sources. The correlation between SPM and various dissolved nutrients (p < 0.05) suggests its influence on the physico-chemical conditions at varying levels. It is summarized that seasonal variation of SPM and nutrients contributed by rivers, wind induced re-suspension events and in situ  regeneration processes play a crucial role in the lagoon biogeochemical cycle.     

Stochastic risk assessment of water quality using advection dispersion equation and Bayesian approximation: A case study

The influence of various heterogeneous parameters, stochastic uncertain factors, and pollutant particles from the industrial effluents in the water system is investigated using advection dispersion equation (ADE) and the Bayesian approximation. Here, the decay coefficient is decomposed into the exact part and the deviation part. The coefficient is used to find out the errors and deviation in decay during the flow of pollutants. Two Bayesian models are developed to analyze the posterior distributions and to find out the Bayes factor for the stochastic covariance estimation. The Bayesian calibration focused the characteristics of both on mechanistic and statistical approximation. The efficiency and accuracy of the developed models are checked from the results obtained on the basis of the confidence interval. Markov chain Monte Carlo simulation is used to acquire the convergence point of parameters for the posterior estimation. The stochastic covariance or white noise represents the effect of random factors on the river system. The analysis revealed that the rate of decay is dependent upon the duration and distance traveled by the pollutants. The collaboration of ADE and Bayesian approximation encourage the water-quality management and environmental modeling.