Isolation, Molecular Identification and Amino Acid Profiling of Single-Cell-Protein-Producing Phototrophic Bacteria Isolated from Oil-Contaminated Soil Samples.

In the current study, soil samples were gathered from different places where petrol and diesel filling stations were located for isolation of photosynthetic bacteria under anaerobic conditions using the paraffin wax-overlay pour plate method with Biebl and Pfennig's medium. The three isolated strains were named Rhodopseudomonas palustris SMR 001 (Mallapur), Rhodopseudomonas palustris NR MPPR (Nacahram) and Rhodopseudomonas faecalis N Raju MPPR (Karolbagh). The morphologies of the bacteria were examined with a scanning electron microscope (SEM). The phylogenetic relationship between R. palustris strains was examined by means of 16S rRNA gene sequence analysis using NCBI-BLAST search and a phylogenetic tree. The sequenced data for R. palustris were deposited with the National Centre for Biotechnology Research (NCBI). The total amino acids produced by the isolated bacteria were determined by HPLC. A total of 14 amino acids and their derivatives were produced by the R. palustris SMR 001 strain. Among these, carnosine was found in the highest concentration (8553.2 ng/mL), followed by isoleucine (1818.044 ng/mL) and anserine (109.5 ng/mL), while R. palustris NR MPPR was found to produce 12 amino acids. Thirteen amino acids and their derivatives were found to be produced from R. faecalis N Raju MPPR, for which the concentration of carnosine (21601.056 ng/mL) was found to be the highest, followed by isoleucine (2032.6 ng/mL) and anserine (227.4 ng/mL). These microbes can be explored for the scaling up of the process, along with biohydrogen and single cell protein production.

Antimicrobial Activities of Salacia oblonga Wall Leaf and Root Extracts Against Different Bacterial Strains and Fungal Isolates.

Antibiotic resistance and the hazardous nature of synthetic drugs is threatening issue in the health sector. The alternative for this problem is to focus on plants that attribute to various compounds that exhibit therapeutic properties. Therefore, the study aims to evaluate the antimicrobial efficacy of Salacia oblonga leaf and root extracts against tested human pathogens. The S. oblonga extracts showed a significant zone of inhibition against bacteria and fungi. The leaf and root extracts of S. oblonga are prepared using low polar to high polar solvents in the Soxhlet apparatus and tested on the selected bacterial and fungal strains. Agar well diffusion and broth dilution methods evaluate antibacterial activity, antifungal activity, and Minimum Inhibitory Concentration (MIC) of extracts. Among the extracts tested, the ethyl acetate extract of root showed more antimicrobial activity against the tested bacterial and fungal strains. The most susceptible bacterial and fungal species against ethyl acetate extract are Micrococcus luteus, Mycobacterium tuberculosis, Microsporum canis, Trichophyton interdigitale, and Microsporum gypseum. The MIC for bacteria ranged from 13.0 to > 200 µg/ml, whereas for fungi, the MIC ranged from 25.9 to > 200 µg/ml. Ethyl acetate extract of root with 100 µg/ml concentration showed 29.1 mm and 28.7 mm zone of inhibition against bacterial strains M. luteus and M. tuberculosis, respectively. The ethyl acetate extract of root with a 100 µg/ml concentration showed 15.8, 15.2, and 15.6 mm zone of inhibition against fungal isolates M. canis, T. interdigitale, and M. gypseum, respectively. The activity of root and leaf extracts increased in a concentration-dependent manner, and further, the compounds isolated from the crude extracts of leaf and root showed antimicrobial activity. Structural elucidation of isolated compounds Lambertic acid and Ferruginol was done using NMR spectroscopy. Reports indicate that Lambertic acid was isolated previously, but the isolation of hydroxy Ferruginol from S. oblonga leaf extract was reported unprecedented.

Mycobacterium tuberculosis DosR regulon gene Rv2004c contributes to streptomycin resistance and intracellular survival.

Tuberculosis (TB) is the deadly infectious disease challenging the public health globally and its impact is further aggravated by co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. In this study, we attempted to characterise the Rv2004c encoded protein, a member of DosR regulon, for its role in drug resistance. In silico docking analysis revealed that Rv2004c binds with streptomycin (SM). Phosphotransferase assay demonstrated that Rv2004c possibly mediates SM resistance through the aminoglycoside phosphotransferase activity. Further, E. coli expressing Rv2004c conferred resistance to 100µM of SM in liquid broth cultures indicating a mild aminoglycoside phosphotransferase activity of Rv2004c. Moreover, we investigated the role of MSMEG_3942 (an orthologous gene of Rv2004c) encoded protein in intracellular survival, its effect on in-vitro growth and its expression in different stress conditions by over expressing it in Mycobacterium smegmatis (M. smegmatis). MSMEG_3942 overexpressing recombinant M. smegmatis strains grew faster in acidic medium and also showed higher bacillary counts in infected macrophages when compared to M. smegmatis transformed with vector alone. Our results are likely to contribute to the better understanding of the involvement of Rv2004c in partial drug resistance, intracellular survival and adaptation of bacilli to stress conditions.

Psychological interventions to improve the quality of life in Indian lung cancer patients: A neglected area.

The incidence of lung cancer is very high and evidence suggests that patients experience imbalanced emotional capabilities due to less survival rate compared to other cancers. Direct and indirect psychological interventions are mandatory to improve the outcome of lung cancer treatment. Although such interventions are being practiced in developed nations, the effects of psychological interventions on the treatment outcome in the Indian context are lacking. Since there is a definite correlation between treatment outcome and psychological issues, it is high time that clinicians in developing countries including India adopt practices to enhance the quality of life of lung cancer patients.

Association of eNOS and ACE gene polymorphisms and plasma nitric oxide with risk of non-small cell lung cancer in South India.

INTRODUCTION: The role of ACE and eNOS gene polymorphisms and their association with various cancers were reported. However, their role in the lung cancer is unclear. OBJECTIVES: In this study, we analyzed eNOS and ACE gene polymorphisms and the risk of non-small cell lung cancer (NSCLC) in South Indian population. RESULTS: For the eNOS gene, the homozygous "AA" genotypic frequency was significantly associated with NSCLC with an overall risk of 3.6-fold (P = 0.006, odds ratio = 3.58, 95% confidence interval = 1.66, 7.723). The heterozygous "I/D" genotypic frequency of ACE gene was significantly higher in NSCLC patients when compared to the controls with a 2.29-fold risk for NSCLC. Multiple regression analyses indicated that gender, smoking status, and polymorphisms in eNOS and ACE genes as the strongest predicting factors for an increased susceptibility to NSCLC. CONCLUSIONS: We report for the first time that polymorphisms in the eNOS "A/A" (homozygous mutant) and ACE "I/D" genotypes might contribute to the increased risk of NSCLC in the South Indian population.

Mycobacterium tuberculosis DosR Regulon Gene Rv2004c Encodes a Novel Antigen with Pro-inflammatory Functions and Potential Diagnostic Application for Detection of Latent Tuberculosis.

Approximately 1.7 billion people in the world harbor latent Mycobacterium tuberculosis (Mtb) with a substantial risk of progression to clinical outcome. Containment of these seed beds of Mtb is essential to eliminate tuberculosis completely in high burden settings such as India. Hence, there is an urgent need for the identification of new serological markers for detection or vaccine candidates to prevent latent tuberculosis infection (LTBI). DosR regulon antigens of Mtb might serve as attractive targets for LTBI diagnosis or vaccine development as they are specifically expressed and are upregulated during latent phase. In this study, we investigated the role of Rv2004c, a member of DosR regulon (exclusive to Mtb complex), in host-pathogen interaction and its immunogenic potential in LTBI, active TB, and healthy control cohorts. Rv2004c elicited strong antibody response in individuals with LTBI compared to active TB patients and healthy cohorts. Recombinant Rv2004c induced pro-inflammatory cytokine response in human peripheral blood mononuclear cells and THP-1 cells via NF-κB phosphorylation. Interaction of Rv2004c with toll-like receptor (TLR)-2 was confirmed using HEK-Blue hTLR-2 and pull-down assays. Rv2004c enhanced the surface expression of TLR-2 at mRNA and protein levels in THP-1 cells. Our findings revealed that Rv2004c induces strong humoral and cell mediated immune responses. Given these observations, we propose Rv2004c to be a potential diagnostic marker or an attractive vaccine candidate that can be useful against LTBI.

Mycobacterial Dormancy Systems and Host Responses in Tuberculosis.

Tuberculosis (TB) caused by the intracellular pathogen, Mycobacterium tuberculosis (Mtb), claims more than 1.5 million lives worldwide annually. Despite promulgation of multipronged strategies to prevent and control TB, there is no significant downfall occurring in the number of new cases, and adding to this is the relapse of the disease due to the emergence of antibiotic resistance and the ability of Mtb to remain dormant after primary infection. The pathology of Mtb is complex and largely attributed to immune-evading strategies that this pathogen adopts to establish primary infection, its persistence in the host, and reactivation of pathogenicity under favorable conditions. In this review, we present various biochemical, immunological, and genetic strategies unleashed by Mtb inside the host for its survival. The bacterium enables itself to establish a niche by evading immune recognition via resorting to masking, establishment of dormancy by manipulating immune receptor responses, altering innate immune cell fate, enhancing granuloma formation, and developing antibiotic tolerance. Besides these, the regulatory entities, such as DosR and its regulon, encompassing various putative effector proteins play a vital role in maintaining the dormant nature of this pathogen. Further, reactivation of Mtb allows relapse of the disease and is favored by the genes of the Rtf family and the conditions that suppress the immune system of the host. Identification of target genes and characterizing the function of their respective antigens involved in primary infection, dormancy, and reactivation would likely provide vital clues to design novel drugs and/or vaccines for the control of dormant TB.

Quality of Life, Psychological Interventions and Treatment Outcome in Tuberculosis Patients: The Indian Scenario.

Objective: Psychological distress is being recognized in individuals affected with many diseases since it affects quality of life (QOF) and has gained importance in the clinical settings. Psychological interventions and their effect on the treatment outcome have yielded encouraging results in many diseased conditions. Tuberculosis (TB) ranks as a deadly disease resulting in millions of deaths worldwide. However, the effect of TB on the psychological status of patients and interventions to improve treatment outcome is neglected, especially in underdeveloped and developing countries. Methods: Systematic review of research papers that published on the QOF in TB and the effect of psychological interventions on treatment outcome were conducted. Results: Tuberculosis patients experience high levels of stress and decreased QOF. In the Indian scenario, TB patients undergo immense psychological stress similar to what is reported in other locations. Psychological interventions renewed hope on life and adherence to medication and treatment outcomes. Such psychological interventions are not practiced in Indian clinical settings. Conclusion: There is an urgent need for both governmental and non-governmental organizations to devise strategies to include psychological interventions mandatory during TB treatments. In the absence of such interventions, the fight against TB in India will remain incomplete.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany.

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A-H (sized 6.2-73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Association of TNFα-308, IFNγ+874, and IL10-1082 gene polymorphisms and the risk of non-small cell lung cancer in the population of the South Indian state of Telangana.

BACKGROUND: Cytokine-mediated inflammation is important in the pathogenesis of non-small cell lung cancer (NSCLC). Genetic polymorphisms in cytokine genes and their association with lung cancer in the Indian population have not been reported. METHODS: For the first time, we analyzed genetic polymorphisms of TNFα -308, IFNγ +874, and IL10 -1082 genes in 246 NSCLC patients and 250 healthy controls in the South Indian population from Telangana using ARMS PCR. RESULTS: IFNγ+874 A/T and IL10-1082 G/G gene polymorphisms were found to be significantly associated with NSCLC with 1.56- and 1.68-fold disease risk, respectively. There was no association between the risk of NSCLC and TNFα-308 polymorphism. Gene polymorphisms stratified according to smoking revealed that IFNγ+874 A/T polymorphisms in smokers increased the disease risk by 2.91 fold. IL10-1082 G/G polymorphisms showed 2-fold increased risk among patients who were smokers when compared to the controls. However, there was no association between TNFα-308, IFNγ+874, and IL10-1082 gene polymorphism and the stage of the NSCLC patients. The overall risk associated with the combination of these polymorphisms indicated that the TNFα-308 G/A + IFNγ+874 A/T + IL10-1082 G/G genotype increased the risk by 1.5 fold. CONCLUSIONS: The results of our study indicate an association between cytokine gene polymorphisms and the risk of NSCLC in an Indian population.

A putative nitroreductase from the DosR regulon of Mycobacterium tuberculosis induces pro-inflammatory cytokine expression via TLR2 signaling pathway.

Tuberculosis caused by Mycobacterium tuberculosis is a global encumbrance and it is estimated that nearly one third population of the world acts as a reservoir for this pathogen without any symptoms. In this study, we attempted to characterise one of the genes of DosR regulon, Rv3131, a FMN binding nitroreductase domain containing protein, for its ability to alter cytokine profile, an essential feature of M. tuberculosis latency. Recombinant Rv3131 stimulated pro-inflammatory cytokines in THP-1 cells and human peripheral blood mononuclear cells in a time and dose dependent manner. In silico analyses using docking and simulations indicated that Rv3131 could strongly interact with TLR2 via a non-covalent bonding which was further confirmed using cell based colorimetric assay. In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively. Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κß. Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway. Therefore, it can be surmised that cytokine secretion induced by Rv3131 might contribute to establishment of M. tuberculosis in the granulomas.

Association of CYP1A1, GSTM1 and GSTT1 gene polymorphisms with risk of non-small cell lung cancer in Andhra Pradesh region of South India.

BACKGROUND: Lung cancer is one of the most preventable causes of death globally both in developed and developing countries. Although it is well established that smokers develop lung cancer, there are some smokers who are free from the disease risk. The predisposition to lung cancer is attributed to genetic polymorphisms in xenobiotic metabolizing genes. Reports on assessment of xenobiotic metabolizing genes like Cytochrome P 450 1A1 (CYP1A1), Glutathione -S -transferase M1 (GSTM1) and T1 (GSTT1) polymorphisms from India are meagre, and reports from Andhra Pradesh are lacking. METHODS AND RESULTS: Assessment of polymorphisms in CYP1A1, GSTM1 and GSTT1 in NSCLC patients and healthy individuals specific to population of Andhra Pradesh, a South Indian state was attempted by multiplex PCR and RFLP, and this is the first study which tried to correlate oxidative stress with the polymorphisms in xenobiotic metabolizing genes. Results showed that CYP1A1 m1 'CC' genotype was significantly associated with lung cancer susceptibility with a 2.3-fold risk, CYP1A1 m2 'AG' gene polymorphisms with 8.8-fold risk and GSTT1 (-/-) genotype demonstrated a twofold risk of disease susceptibility. CONCLUSIONS: A combined role of genetic polymorphisms and smoking status can be attributed for the cause of lung cancer. Further, the association between oxidative stress and genetic polymorphisms showed a correlation between GSTT1 and super oxide dismutase activity; CYP1A1 m1, m2 and GSTT1 with glutathione peroxidase activity; CYP1A1 m1 and GSTM1 with melondialdehyde levels; and CYP1A1 m1 and GSTT1 with 8-oxo-7,8-dihydro-2'-deoxyguanosine. A higher risk of lung cancer seems to be associated with combined gene polymorphisms of phase I and phase II enzymes than that ascribed to single gene polymorphism.

Genetic instability in peripheral lymphocytes as biological marker for non-small cell lung cancer patients in the South Indian state of Andhra Pradesh.

OBJECTIVES: This study aims, first, at evaluating the DNA and chromosomal damage in non-small cell lung cancer (NSCLC) patients from the South Indian state of Andhra Pradesh, and then at correlating these results with possible confounding factors that might potentially play a role in causing genetic damage. METHODS: The study included 246 NSCLC patients (177 men and 69 women) and 250 healthy controls (180 men and 70 women) for the analysis of DNA and chromosomal damage using the comet assay and micronucleus test. RESULTS: Both DNA and chromosomal damage were found to be increased in NSCLC patients compared to healthy controls, and the extent of the damage was higher in males than female patients. The smoking status had a profound effect on the extent of DNA and chromosomal damage in NSCLC patients. The degree of genetic damage correlated with the stage of the disease. However, the histological status had no effect on the extent of DNA and chromosomal damage among NSCLC patients. CONCLUSIONS: We here report, for the first time, that the NSCLC patients selected form the Andhra Pradesh population had increased DNA damage and higher mean micronucleus frequencies in peripheral lymphocytes, indicating a strong background level of genetic instability.

Assessment of 8-oxo-7, 8-dihydro-2'-deoxyguanosine and malondialdehyde levels as oxidative stress markers and antioxidant status in non-small cell lung cancer.

OBJECTIVE: The present investigation was taken up to evaluate the 8-oxo-7,8-dihydro-2'-deoxyguanosine and malondialdehyde as markers of oxidative stress, the levels of antioxidants and the correlations between these oxidative stress markers and antioxidants in lung cancer patients. METHODS: The study included 222 patients (158 men and 64 women, age ranging from 32 to 85 years) and 207 control subjects (153 men and 54 women, aged 30-80 years) for the analysis of urinary excretion of 8-oxodG using an ELISA assay, plasma malondialdehyde using spectrophotometer and red cell Cu-Zn SOD and GPx activities by kit methods. RESULTS: The levels of 8-oxodG and malondialdehyde were significantly higher (p < 0.001) and red cell superoxide dismutase and glutathione peroxidase activities (p < 0.001) were significantly lower in lung cancer patients than in controls. There was a significantly positive correlation between 8-oxodG and malondialdehyde (r=0.912, p < 0.001) and a negative correlation between 8-oxodG and antioxidants. CONCLUSIONS: Our results demonstrate that an increased rate of oxidative stress might play a role in the pathogenesis of lung cancer as evidenced by a failure in the oxidant/antioxidant balance in favour of lipid peroxidation and DNA damage.

Association of CYP1A1 Gene Polymorphism with Ischemic Stroke in South Indian Population.

Stroke is a major cause of morbidity and mortality worldwide. Genetic and environmental factors are involved in the pathogenesis of stroke. Hypertension, diabetes mellitus, and cigarette smoking are the major risk factors, and smoking doubles the risk of ischemic stroke. Smoking cessation decreased the risk for ischemic stroke. CYP1A1 is the phase I metabolizing enzyme which plays a key role in metabolic activation of polycyclic aromatic hydrocarbons which are present in cigarette smoke and considered carcinogenic. So far, the association of CYP1A1 gene polymorphism with stroke has not been investigated in Indian population. So, the study is taken up to evaluate the association of this polymorphism with ischemic stroke in a South Indian population. We genotyped 215 ischemic stroke patients and 162 age-matched controls using polymerase chain reaction-restriction fragment length polymorphism. Statistical analysis showed that CYP1A1 "CC" genotype is associated with five times increased risk of ischemic stroke (odds ratio (OR) = 5.14; 95% confidence interval (95% CI) = 1.14-23.14, p = 0.01), while "TT" (OR = 0.78, 95% CI = 0.51-1.19, p = 0.25) and "TC" (OR = 1.04, 95% CI = 0.67-1.60, p = 0.85) genotypes were nonsignificant with the increased risk of stroke. T and C allele frequencies in stroke were 76.5% and 23.5% as against 81.8% and 18.2% in control group, respectively, thus, suggesting no statistically significant differences in the T (OR = 0.72, 95% CI = 0.50-1.03, p = 0.07) and C (OR = 1.37, 95% CI = 0.96-1.97, p = 0.07) allele frequencies between the two groups. The distribution of CYP1A1 genotypes and allelic frequency within the stroke subtypes showed a significant association of CC genotype only in intracranial large artery atherosclerosis (OR = 5.21, 95% CI = 1.03-26.38, p = 0.02) while other subtypes did not show any association. Further analysis of CYP1A1 genotypes in patients and control subjects with smoking habit also showed a similar trend. Hence, we conclude that the CYP1A1 CC genotype is associated with the increased risk of ischemic stroke.