Overexpression of <Emphasis Type="Italic">MuHSP70</Emphasis> gene from <Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> confers multiple abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H₂O₂, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.     

Daesiho-Tang Is an Effective Herbal Formulation in Attenuation of Obesity in Mice through Alteration of Gene Expression and Modulation of Intestinal Microbiota

BackgroundObesity has become a major global health challenge due to its increasing prevalence, and the associated health risk. It is the main cause of various metabolic diseases including diabetes, hypertension, cardiovascular disease, stroke and certain forms of cancer.

Methods and Results

In the present study we evaluated the anti-obesity property of Daesiho-tang (DSHT), an herbal medicine, using high fat diet (HFD)-induced obese mice as a model. Our results showed that DSHT ameliorated body weight gain, decreased total body fat, regulated expression of leptin and adiponectin genes of adipose tissue and exerted an anti-diabetic effect by attenuating fasting glucose level and serum insulin level in HFD-fed animals. In addition, DSHT-treatment significantly reduced total cholesterol (TC), triglycerides (TG) and increased high density lipoprotein-cholesterol (HDL), glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) levels in serum and reduced deposition of fat droplets in liver. DSHT treatment resulted in significantly increased relative abundance of bacteria including Bacteroidetes, Bacteroidetes/Firmicutes ratio, Akkermansia Bifidobacterium., Lactobacillus, and decreased the level of Firmicutes. Using RT2 profiler PCR array, 39 (46%) genes were found to be differentially expressed in HFD-fed mice compared to normal control. However, normal gene expressions were restored in 36 (92%) genes of HFD-fed mice, when co-exposed to DSHT.

Conclusion/Major Findings

The results of this study demonstrated that DSHT is an effective herbal formulation in attenuation of obesity in HFD-fed mice through alteration of gene expressions and modulation of intestinal microbiota.     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Investigation on the role of p53 codon 72 polymorphism and interactions with tobacco, betel quid, and alcohol in susceptibility to cancers in a high-risk population from North East India

The association of TP53 codon 72 polymorphism with cancer susceptibility remains uncertain and varies with ethnicity. Northeast India represents a geographically, culturally, and ethnically isolated population. The area reports high rate of tobacco usage in a variety of ways of consumption, compared with the rest of Indian population. A total of 411 cancer patients (161 lung, 134 gastric, and 116 oral) and 282 normal controls from the ethnic population were analyzed for p53 codon 72 polymorphism by polymerase chain reaction-restriction fragment length polymorphism. No significant difference in genotypic distribution of p53 between cases and controls was observed. Results suggested betel quid chewing as a major risk factor for all the three cancers (odds ratio [OR]=3.54, confidence interval [CI]=2.01-6.25, p<0.001; OR=1.74, CI=1.04-2.92, p=0.03; and OR=1.85, CI=1.02-3.33, p=0.04 for lung, gastric, and oral cancers, respectively). Tobacco smoking was associated with risk of lung and oral cancers (OR=1.88, CI=1.11-3.19, p=0.01 and OR=1.68, CI=1.00-2.81, p=0.04). Interactions between p53 genotypes and risk factors were analyzed to look for gene-environment interactions. Interaction of smoking and p53 genotype was significant only for oral cancer. Interactions of betel quid with p53 genotypes in lung cancer showed significant increase for all the three genotypes, indicating a major role of betel quid (OR=5.90, CI=1.67-20.81, p=0.006; OR=5.44, CI=1.67-17.75, p=0.005; and OR=5.84, CI=1.70-19.97, p=0.005 for Arg/Arg, Arg/Pro, and Pro/Pro, respectively). In conclusion, high incidence of these cancers in northeast India might be an outcome of risk habits; further, tissue- and carcinogen-specific risk modification by p53 gene is probable.     

Glucose deprivation induced upregulation of phosphoenolpyruvate carboxykinase modulates virulence in Leishmania donovani

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP⁺ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.     

Pheromone peptide cOB1 from native Enterococcus faecalis forms amyloid‐like structures: A new paradigm for peptide pheromones

Pheromone peptides are an important component of bacterial quorum‐sensing system. The pheromone peptide cOB1 (VAVLVLGA) of native commensal Enterococcus faecalis has also been identified as an antimicrobial peptide (AMP) and reported to kill the prototype clinical isolate strain of E. faecalis V583. In this study, the pheromone peptide cOB1 has shown to form amyloid‐like structures, a characteristic which is never reported for a pheromone peptide so far. With in silico analysis, the peptide was predicted to be highly amyloidogenic. Further, under experimental conditions, cOB1 formed aggregates displaying characteristics of amyloid structures such as bathochromic shift in Congo red absorbance, enhancement in thioflavin T fluorescence, and fibrillar morphology under transmission electron microscopy. This novel property of pheromone peptide cOB1 may have some direct effects on the binding of the pheromone to the receptor cells and subsequent conjugative transfer, making this observation more important for the therapeutics, dealing with the generation of virulent and multidrug‐resistant pathogenic strains.     

Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613

Conditions for the laboratory-scale production of acetoin plus diacetyl by Enterobacter Cloacae ATCC 27613 were studied. Thirty-five g acetoin plus diacetyl/50 g sucrose were obtained when fermentation was carried out in 2. 5 liter medium containing 12.5 g peptone and 12. 5 g yeast extract, at pH 7.0, in a 5 liter conical flask on a shaker (240rpm) at 28–30°C for 48 hr. Recovery of pure diacetyl was 85% of the total plus diacetyl.     

Comparative Study of Antibiofilm Activity of Copper Oxide and Iron Oxide Nanoparticles Against Multidrug Resistant Biofilm Forming Uropathogens

The effectiveness of the metal oxide nanoparticles viz. CuO and Fe₂O₃ as antibacterial agents against multidrug resistant biofilm forming bacteria was evaluated. CuO nanoparticles were also experimented for antibiofilm and time kill assay. The CuO displayed maximum antibacterial activity with zone of inhibition of (22 ± 1) mm against methicillin resistant Staphylococcus aureus (MRSA) followed by Escherichia coli (18 ± 1) mm. The Fe₂O₃ showed the zone of inhibition against MRSA of (14 ± 1) mm followed by E. coli (12 ± 1) mm. CuO proved to be more toxic than Fe₂O₃ nanoparticles showing significantly high antibacterial activity and found to possess dose dependent antibiofilm properties.