Dhananjay V. Raje

Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

BackgroundPseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus.ResultsRepeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanons entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.ConclusionsThe developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

SATB1-Binding Sequences and Alu-Like Motifs Define a Unique Chromatin Context in the Vicinity of Human Immunodeficiency Virus Type 1 Integration Sites

ABSTRACT Retroviral integration has recently been shown to be nonrandom, favoring transcriptionally active regions of chromatin. However, the mechanism for integration site selection by retroviruses is not clear. We show here the occurrence of Alu-like motifs in the sequences flanking the reported viral integration sites that are significantly different from those obtained from the randomly picked sequences from the human genome, suggesting that unique primary sequence features exist in the genomic regions targeted by human immunodeficiency virus type 1 (HIV-1). Additionally, these sequences were preferentially bound by SATB1, the T lineage-restricted chromatin organizer, in vitro and in vivo. Alu repeats make up nearly 10% of the human genome and have been implicated in the regulation of transcription. To specifically isolate sequences flanking the viral integration sites and also harboring both Alu-like repeats and SATB1-binding sites, we combined chromatin immunoprecipitation with sequential PCRs. The cloned sequences flanking HIV-1 integration sites were specifically immunoprecipitated and amplified from the pool of anti-SATB1-immunoprecipitated genomic DNA fragments isolated from HIV-1 NL4.3-infected Jurkat T-cell chromatin. Moreover, many of these sequences were preferentially partitioned in the DNA associated tightly with the nuclear matrix and not in the chromatin loops. Strikingly, many of these regions were disfavored for integration when SATB1 was silenced, providing unequivocal evidence for its role in HIV-1 integration site selection. We propose that definitive sequence features such as the Alu-like motifs and SATB1-binding sites provide a unique chromatin context in vivo which is preferentially targeted by the HIV-1 integration machinery.

Comparative evaluation of risk factors, outcome and biomarker levels in young and old acute ischemic stroke patients

Background Stroke is the third leading cause of death and disability worldwide accounting for 400-800 strokes per 100,000 individuals each year. Purpose In the present study, we compared risk factors, clinical outcome, and prognostic biomarkers NSE, S-100 ßß and ITIH4 levels in young and old acute ischemic stroke (AIS) patients. Methods We compared the risk factors and clinical outcomes in young (n = 38) and old (n = 66) AIS patients admitted to tertiary health care centre in Central India. In addition, we also evaluated NSE, S100ββ & ITIH4 levels in admission and discharge samples of young and old AIS patients with different clinical outcome. Results Hypertension was a major risk factor in 45% of young and 80% of old AIS patients. Hospital outcome was less favorable in young AIS patients with higher dependent rates of 24% as compared to 12% in old AIS patients. Whereas long term outcome at 12 and 18 months after discharge was more favorable in young AIS patients with low dependency rates of 16% and 11% as compared to 41% and 24% in older AIS patients respectively. Similarly, serum NSE, S100ββ and ITIH4 levels showed a distinct pattern of expression at discharge time in AIS patients with improved and dependent outcome in both the age groups. Conclusion Young males with hypertension and smoking habits are at a high risk of AIS while old AIS patients are at a greater risk of worse long term outcome. Serum levels of NSE and S100ββ are independent predictors of outcome in AIS patients. Similarly, it also suggests that serum ITIH4 levels could be used as a potential biomarker for predicting the outcome in AIS patients.

Latent TB Infection Diagnosis in Population Exposed to TB Subjects in Close and Poor Ventilated High TB Endemic Zone in India

Background The present study was designed to investigate the utility of Quantiferon TB gold (QFT-G) and Tuberculin skin test (TST) for diagnosis of latent TB infection (LTBI) in high crowding TB endemic zone of Nagpur, India and their comparison with associated risk factors. Methods Out of 342 eligible participants, QFT-G and TST were performed in 162 participants. Results The prevalence of LTBI observed according to QFT-G and TST was 48% and 42% respectively, with an agreement of 52.47%. QFT-G positivity was associated with age while TST positivity was associated with body mass index (BMI). Duration of exposure emerged as a key risk factor significantly associated with both the tests. Conclusion The prevalence of LTBI was quite high in the studied zone as detected by both the evaluated tests and thus, the combination of both the tests will be best predictive for LTBI in such high TB endemic regions.

Demonstration of sequential adaptation strategy for developing salt tolerance in bacteria for wastewater treatment: A study using Escherichia coli as model

A wastewater isolate identified as Escherichia coli HPC781 was adapted for high salt concentration through sequential transfers in Luria Broth (LB). The cells were grown in LB with 5% sodium chloride (NaCl) and were analyzed for the acquired salt resistance network through gene expression profiles. Microarray studies revealed TCA, glyoxylate shunt and acetyl Co-A metabolism as key nodes for stress combat to arrive at compromised physiology. It also proposed that the cells were receiving signals from salt environment via OmpR-EnvZ two component systems and stress dependent general regulatory protein rpoH and rpoE. The salt adapted culture, when challenged with wastewater having additional 5% salt showed growth. The work represents a tactic to adjust biochemical network towards stress and reveals its applicability via real-time PCR measurement of genes in wastewater. The study proposes that the recycled biomass with an adaptation strategy could be applied for treatment of wastewater with high salt levels.

Distinguishing features of 16S rDNA gene for five dominating bacterial genus observed in bioremediation.

Defining a microbial community and identifying bacteria, at least at the genus level, is a first step in predicting the behavior of a microbial community in bioremediation. In biological treatment systems, the most dominating groups observed are Pseudomonas, Moraxella, Acinetobactor, Burkholderia, and Alcaligenes. Our interest lies in identifying the distinguishing features of these bacterial groups based on their 16S rDNA sequence data, which could be used further for generating genus-specific probes. Accordingly, 20 sequences representing different species from each genus above were retrieved, which constituted a training set. A 16-dimensional feature vector comprised of transition probabilities of nucleotides was considered and each sampled sequence was expressed in terms of these features. A stepwise feature selection method was used to identify features that are distinct across the species of these five groups. Wilks lambda selection criterion was used and resulted in a subset with six distinguishing features. The discriminating efficacy of this subset was tested through multiple group discriminant analysis. Two linear composites, as a function of these features, could discriminate the test set of forty-five sequences from these groups with 95% accuracy, thereby ascertaining the relevance of the identified features. The geometric representation of feature correlation in the reduced discriminant space demonstrated the dominance of identified features in specific groups. These features independently or in combination could be used to generate genus-specific patterns to design probes, so as to develop a tracking tool for the selected group of bacteria.

Prediction of Outcome in Diabetic Acute Ischemic Stroke Patients: A Hospital-Based Pilot Study Report

Background: Demographic and clinical characteristics are known to influence the outcome in acute ischemic stroke (AIS) patients. Purpose: This study is aimed at evaluating short- and long-term outcomes in diabetic AIS patients. In addition, the study also evaluates the impact of diabetes on the performance of indigenously reported biomarker, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and known biomarkers, neuron-specific enolase (NSE) and glial-derived S-100 beta beta protein (S-100ββ). Methods: This study was performed on 29 diabetes and 75 non-diabetes AIS patients. Outcome of AIS patients was analyzed by using modified Rankin scale at discharge, then at 12 and 18 months after discharge. Based on the obtained scores, patients were classified as improved group (scales 1-3) and dependent/expired group (scales 3-6). Blood samples were collected during admission and at discharge/expired time. Levels of NSE, S100ββ, and ITIH4 were analyzed in all samples. Results: On discharge, frequencies of dependent/expired outcome were 4/29 (14%) and 19/75 (17%) in diabetic and non-diabetic AIS patients. However, follow-up outcome at 12 and 18 months showed higher dependent/expired cases of 43 and 41% among diabetic AIS patients compared to 27 and 21% in non-diabetic patients. Multivariate analysis revealed that diabetes is an independent risk factor for dependent/expired outcome in AIS patients (OR 0.484 (at discharge); 1.307 (at 12 months) and 1.675 (at 18 months)). NSE, S100ββ, and ITIH4 showed a differential expression in both the outcome groups of AIS patients, irrespective of diabetes. Conclusion: Diabetes increases the risk of dependent/expired outcome in AIS patients. Also, serum NSE, S100ββ, and ITIH4 are independent biomarkers for prognosis of outcome in AIS patients, irrespective of diabetes.

Investigating the Phylogeny of Hydrogen Metabolism by Comparative Genomics: Horizontal Gene Transfer

The phylogenetic analysis based on molecular characteristics indicates that lithotrophic metabolism was followed by phototrophy. Hydrogen (H2) metabolism is a signature of such environments. This property is prominent among organisms found in geothermal conditions and in deep aquifers. H2 is generated readily by abiotic mechanisms where the terminal electron acceptor is likely to be the limiting factor. In the post-fossil fuel era, H2 has in fact emerged as a strong contender for future fuel. It is thus important to understand the molecular mechanisms which lead to H2 production and associated biological systems. These can help to comprehend issues such as sustainability, environmental emissions and energy security. Comparative genomic analysis reveals events of horizontal transfer of genes of H2 metabolism among taxonomically diverse organisms. This offers an opportunity to identify those genomes which can be tailored for transforming presently ‘non’-H2 producers into producers. This also suggests that naturally occurring events can be mimicked to provide future fuel H2.

Impact of socioeconomic status and living condition on latent tuberculosis diagnosis among the tribal population of Melghat: A cohort study

Aims: To study socioeconomic status (SES) and living conditions (LC) as risk factors for latent tuberculosis infection (LTBI) and their impact on QuantiFERON-TB gold (QFT-G) and tuberculin skin test (TST) outcome for determining a better diagnostic test for LTBI in the malnourished tribal population of Melghat. Settings and Design: Six hundred sixty nine participants matching the inclusion criteria were recruited from 10 tribal villages of Melghat region, India. Subjects and Methods: Complete information related to various risk factors and test outcome was obtained on 398 participants, which was analyzed as per predefined conceptual framework. Factors were classified based on their relevance either at individual or household level, and subsequently based on the possibility of intervention. Data were partitioned into concordant and discordant sets depending on test agreement. Results: In concordant set, the two tests revealed that LTBI was significantly associated with smoking (adjusted odds ratio [aOR]: 2.64 [95% confidence interval [CI]: 1.03-6.79]), tobacco usage (aOR: 2.74 [95% CI: 1.50-4.99]), and malnourishment (aOR: 1.97 [95% CI: 1.12-3.48]) after basic adjustment. Inclusion of latent variable SES and LC in the model has mediating effect on the association of above factors with LTBI. Further, the association of SES and LC with LTBI in concordant set was unaltered in presence of other cofactors. From discordant set, results of QFT-G corroborated with that of concordant set. Conclusions: Poor SES and LC can be considered as strong risk factors linked with LTBI as compared to malnourishment, which is often targeted in such communities. Further, our study showed QFT-G test as a reliable tool in screening of LTBI in the tribal population of Melghat, India.

Relation of Residues in the Variable Region of 16S rDNA Sequences and Their Relevance to Genus-Specificity

It has been observed that the short nucleotide sequences in a variable region, representing species level diversity in a set of 16S rDNA sequences carries the genus specific signature. In this study our aim is to assess the relationship of residues at different positions and thereby obtain consensus patterns using different statistical tools. If such patterns are found genus-specific then it would facilitate in designing hybridization arrays to target even unexplored species of the same genus in complex samples such as environmental DNA.