Neha Singla

Metagenomics: Concept, methodology, ecological inference and recent advances

Microorganisms constitute two third of the Earths biological diversity. As many as 99% of the microorganisms present in certain environments cannot be cultured by standard techniques. Culture‐independent methods are required to understand the genetic diversity, population structure and ecological roles of the majority of organisms. Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity. New screening methods have been designed to select specific functional genes within metagenomic libraries to detect novel biocatalysts as well as bioactive molecules applicable to mankind. To study the complete gene or operon clusters, various vectors including cosmid, fosmid or bacterial artificial chromosomes are being developed. Bioinformatics tools and databases have added much to the study of microbial diversity. This review describes the various methodologies and tools developed to understand the biology of uncultured microbes including bacteria, archaea and viruses through metagenomic analysis.

Protective role of zinc during aluminum-induced hepatotoxicity

The study was carried out to assess the role of zinc (Zn) in mitigating the biochemical alterations induced by aluminum (Al) in rat liver. Rats were divided into four groups: normal control, Al treated (AlCl3, 100 mg/kg b.wt./day), Zn treated (ZnSO4, 227 mg/L drinking water), and combined Al + Zn treated. Al and zinc treatments were given for a total duration of 2 months. Al treatment caused a significant increase in the activity of alkaline phosphatase (ALP), but decreased aspartate aminotransferase (AST) and alanine aminotranferase (ALT) activities, which showed the reverse trend following Zn supplementation. Levels of lipid peroxidation (LPx) and activities of catalase and glutathione‐S‐transferase (GST) were significantly decreased following Al treatment, which, however, were increased significantly in Zn co‐treated rats. Further Al exposure showed a significant increase in reduced glutathione (GSH) content as well as activities, of superoxide dismutase (SOD) and glutathione reductase (GR). However, Zn supplementation to Al‐treated rats brought down the raised levels of reduced (GSH) and SOD to within normal limits, but caused no effect on GR activity. Furthermore, Al treatment also resulted in alterations in liver histoarchitecture with disruption of hepatic cords and increased vacuolization, which were close to normal following Zn supplementation. The present study reveals that Zn is effective in attenuating the liver damage inflicted by Al toxicity.

Regulatory role of zinc during aluminium‐induced altered carbohydrate metabolism in rat brain

Aluminium is considered an environmental neurotoxicant and causes many neurological disorders, whereas zinc is vital for many biological functions. The present study was carried out to investigate the role of Zn, if any, in mitigating the adverse effects inflicted by Al on carbohydrate metabolism in rat brain. Male Sprague‐Dawley rats weighing 140–160 g were divided into four different groups: normal control, Al‐treated (100 mg/kg b.w./day in drinking water via oral gavage), Zn‐treated (227mg/liter in drinking water), and combined Al‐ and Zn‐treated rats. All the treatments were continued for 2 months, and their effects on carbohydrate‐metabolizing enzymes were studied. Additionally, expressions of the proteins glycogen synthase kinase‐3 (GSK3) and protein phosphatase (PP1), which help in regulating carbohydrate energy metabolism, were also studied. Al treatment resulted in increased activities of the glucose‐6‐phosphatase (G6P), glucose‐6‐isomerase (G6I), and lactate dehydrogenase (LDH), whereas the activities of hexokinase and succinate dehydrogenase (SDH) and glycogen content were decreased. Moreover, no significant change was observed in the biochemical parameters upon Zn supplementation alone. However, Zn supplementation to Al‐treated rats was able to reduce significantly the Al‐induced increased activities of G6P, G6I, and LDH, but it elevated the levels of hexokinase, SDH, and glycogen. Furthermore, Al treatment increased the protein expression of GSK3 and decreased the PP1 expression, which were found to be reversed upon Zn administration. Hence, Zn is effective in regulating theAl‐induced alterations in carbohydrate metabolism.

Association of mannose-binding lectin gene polymorphism with tuberculosis susceptibility and sputum conversion time

Mannose‐binding lectin (MBL) plays an important role in innate immunity. The effect of low MBL levels producing variants of MBL2 gene on tuberculosis (TB) has been controversial with some studies reporting it to confer protection against the disease, whereas others estimating a susceptibility relation. Other than conducting a case–control study to evaluate the role of MBL A/B polymorphism on TB, we conducted a longitudinal study to check whether this MBL variant can influence the host response to Mycobacterium tuberculosis infection. A total of 357 TB patients (286 pulmonary TB, 71 extrapulmonary (EP) TB) and 392 healthy controls belonging to same ethnicity were included in the study. We found the mutant allele ‘B’ allele confers a protective role against TB in our study population. This effect was absent in EP patients. On stratification on the basis of sex, the protective role of the ‘B’ allele was found to be limited to females only and males reported no significant difference. No effect of MBL A/B polymorphism on sputum conversion time was reported. We conclude that MBL ‘B’ allele is associated with protection against TB, but no influence was found on sputum conversion rate.

Zinc protection against aluminium induced altered lipid profile and membrane integrity

The aim of the present study was to investigate the effects of Zinc (Zn) supplementation on lipid profile and fluidity of cerebrum and cerebellum membranes of rats treated with aluminium (Al). Sprague dawley male rats were divided into four different treatment groups viz: Control, aluminium treated, zinc treated and aluminium+zinc treated. Aluminium (AlCl3) was administered orally at a dose of 100mg/kgb.wt./day (dissolved in drinking water). Zinc as zinc sulphate was supplemented to rats at a dose of 227mg/l in drinking water. A significant decrease in the levels of total lipids, glycolipids, phospholipids, cholesterol and gangliosides contents were observed in both the cerebrum and cerebellum following Al exposure, which were found to be significantly increased following Zn supplementation. On the contrary, Al treatment caused a significant increase in the formation of conjugated dienes, which were observed to be reduced on Zn co-treatment. Further, Al treatment significantly elevated the fluorescence polarization, anisotropy and order parameter, which however were normalized upon Zn co-administration. Hence, the present study depicts the potential of Zn in moderating the changes caused by Al on membrane composition and fluidity in rat brain.

Role of TLR4 C>1196T (Thr399Ile) and TLR4 A>896G (Asp299Gly) polymorphisms in a North Indian population with asthma: a case-control study.

Toll‐like receptor 4 (TLR4) is the most important TLR among the pattern recognition receptors which recognizes lipopolysaccharide of gram‐negative bacteria. They identify a highly conserved structure of microbes called pathogen‐associated molecular patterns and activate immune and inflammatory responses that have been shown to be involved in the pathogenesis of asthma. The role of TLR4 gene polymorphisms in asthma was detected in a total of 964 individuals, including 483 healthy controls and 481 asthma patients from a North Indian population. The genotyping was carried out using polymerase chain reaction–restriction fragment length polymorphism method. Statistical analysis revealed that the heterozygous genotype as well as the mutant (T) allele of the TLR4 C>1196T (Thr399Ile) polymorphism shows resistance towards asthma with OR = 0.70, 95% CI (0.49–0.99), P corrected value = 0.046 and OR = 0.72, 95% CI (0.52–0.98), P corrected value = 0.039, respectively. However, no association was found between the TLR4 A>896G (Asp299Gly) polymorphism and asthma patients (P > 0.05). This is the first study conducted in India conferring TLR4 (Thr399Ile) polymorphism resistance towards asthma, while lack of association was found between TLR4 (Asp299Gly) polymorphism and asthma in the studied North Indian population.

Association of the wild-type A/A genotype of MBL2 codon 54 with asthma in a North Indian population

Background: High serumMBL level as well as polymorphisms in the mannose-binding lectin 2 (MBL2) gene resulting in MBL deficiency are involved in the mechanism of a number of non-infectious diseases such as asthma, conferring either risk or protection in different population studies. MBL being the first reactant of the MBL pathway is also a major determinant of the fate of the anaphylatoxins such as C3a and C5a, which are also pro-inflammatory mediators. The MBL2 gene polymorphisms thus control the serum levels of MBL as well as C3a and C5a. Objective: This is the first case-control study conducted in India, investigating the role of MBL2 codon 54 A/B polymorphism in asthma pathogenesis. Methods: A case-control study was performed with a total of 992 adult subjects, including 410 adult asthmatics and 582 healthy controls from regions of North India. The MBL2 codon 54 A/B polymorphism was genotyped by PCR-RFLP. Results: Statistical analysis for the codon 54 polymorphism revealed that the wild (A) allele was significantly associated with asthma with OR = 1.9, 95% CI (1.4–2.4), and p < 0.001. Conclusion: The MBL2 codon 54 A/B polymorphism is significantly associated with asthma and its phenotypic traits as the wild (A/A) genotype confers a significant risk towards the disease in the studied North Indian population.

Modulation of 14C‐labeled glucose metabolism by zinc during aluminium induced neurodegeneration

Aluminium (Al) is one of the most prominent metals in the environment and is responsible for causing several neurological disorders, including Alzheimers disease. On the other hand, zinc (Zn) is an essential micronutrient that is involved in regulating brain development and function. The present study investigates the protective potential of Zn in the uptake of 14C‐labeled amino acids and glucose and their turnover in rat brain slices during Al intoxication. Male Sprague Dawley rats (140–160 g) were divided into four different groups: normal control, Al treated (100 mg/kg body weight/day via oral gavage), Zn treated (227 mg/liter in drinking water), and Al + Zn treated. Radiorespirometric assay revealed an increase in glucose turnover after Al exposure that was attenuated after Zn treatment. Furthermore, the uptake of 14C‐labeled glucose was increased after Al treatment but was appreciably decreased upon Zn supplementation. In addition, the uptakes of 14C‐lysine, 14C‐leucine, and 14C‐aspartic acid were also found to be elevated following Al exposure but were decreased after Zn treatment. Al treatment also caused alterations in the neurohistoarchitecture of the brain, which were improved after Zn coadministration. Therefore, the present study suggests that Zn provides protection against Al‐induced neurotoxicity by regulating glucose and amino acid uptake in rats, indicating that Zn could be a potential candidate for the treatment of various neurodegenerative disorders.

Potential of zinc as an antiapoptotic agent during aluminium-induced neurodegeneration

kg/day) or control diet for 16 weeks. Thereafter, mice were transcardially perfused and pial-arteries surgically removed. Arterial proteins were extracted, trypsin-digested, fractionated by strong cation exchange (gel-free-method) and 1D SDS-PAGE (gel-based-method), and analyzed by nanoLC-MS/MS using nanoAcquity UPLC (Waters) and ESI-LTQ Orbitrap (Thermo). Protein identification was performed using Mascot. MatchRx software was used for alignment and quantification across the multiple samples, and peptide intensity validation was performed using MSight. Differentially-expressed proteins were identified at 1.5 and 2 fold-changes, and at p 0.05 and p 0.01. Three biological and two technical replicates were conducted. Results: We identified 6,566 pial-arterial proteins, of which 975 (15%) were differentially-expressed between WT and APP mice at fold-change 1.5 and p 0.05. The altered proteins were associated with biological pathways related to amyloidosis, oxidative stress and vasomotricity, as well as processes over-represented in the AlzGene database. A more stringent criteria ( 2.0-fold, p 0.01) identified 94 robustly differentially-expressed proteins. Of these, 72 were modulated by pioglitazone-treatment, with 61% showing the same expressions levels as WT, and 39% showing partial-normalization. 20% of these proteins had known PPRE elements and/or were known responders to pioglitazone. Conclusions: We demonstrated that increased soluble Ab adversely impacts the cerebrovascular proteome. Pioglitazone-mediated functional rescue of the cerebrovasculature is associated with normalization of the majority of altered proteins in the vessel wall, particularly those related to oxidative stress and inflammation.