NMR-Based Metabolomics: A New Paradigm to Unravel Defense-Related Metabolites in Insect-Resistant Cotton Variety through Different Multivariate Data Analysis Approaches.

Cotton (Gossypium hirsutum) is an economically important crop and is widely cultivated around the globe. However, the major problem of cotton is its high vulnerability to biotic and abiotic stresses. It has been around three decades since the cotton plant was genetically engineered with genes encoding insecticidal proteins (mainly Cry proteins) with an aim to protect it against insect attack. Several studies have been reported on the impact of these genes on cotton production and fiber quality. However, the metabolites responsible for conferring resistance in genetically modified cotton need to be explored. The current work aims to unveil the key metabolites responsible for insect resistance in Bt cotton and also compare the conventional multivariate analysis methods with deep learning approaches to perform clustering analysis. We aim to unveil the marker compounds which are responsible for inducing insect resistance in cotton plants. For this purpose, we employed 1H-NMR spectroscopy to perform metabolite profiling of Bt and non-Bt cotton varieties, and a total of 42 different metabolites were identified in cotton plants. In cluster analysis, deep learning approaches (linear discriminant analysis (LDA) and neural networks) showed better separation among cotton varieties compared to conventional methods (principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLSDA)). The key metabolites responsible for inter-class separation were terpinolene, α-ketoglutaric acid, aspartic acid, stigmasterol, fructose, maltose, arabinose, xylulose, cinnamic acid, malic acid, valine, nonanoic acid, citrulline, and shikimic acid. The metabolites which regulated differently with the level of significance p < 0.001 amongst different cotton varieties belonged to the tricarboxylic acid cycle (TCA), Shikimic acid, and phenylpropanoid pathways. Our analyses underscore a biosignature of metabolites that might involve in inducing insect resistance in Bt cotton. Moreover, novel evidence from our study could be used in the metabolic engineering of these biological pathways to improve the resilience of Bt cotton against insect/pest attacks. Lastly, our findings are also in complete support of employing deep machine learning algorithms as a useful tool in metabolomics studies.

Co-pyrolysis of sugarcane bagasse and polystyrene with ex-situ catalytic bed of metal oxides/HZSM-5 with focus on liquid yield.

Catalytic co-pyrolysis of sugarcane bagasse (SCB) and polystyrene (PS) was conducted in a fixed bed reactor over microporous HZSM-5, mesoporous metal oxides (MgO, CaO) and their blends to examine the effect on pyrolytic liquid yields and quality. Though the catalyst addition decreased the liquid yield, improvement in mono-aromatic hydrocarbon yield with the least content of oxygenates was achieved in the catalytic trials. Results revealed that HZSM-5 showed maximum conversion efficiency of acids, furans and phenols acting as hydrocarbon source for aromatic production. Basic MgO, with acidic HZSM-5, was found to conduce better catalytic performance yielding improved oil quality compared to HZSM-5:CaO catalyst. Mass ratio of 1:3 HZSM-5:MgO exhibited most eminent synergistic effect with maximum (56.8 wt%) mono-aromatic hydrocarbon (MAH) yield and lowest (20.8 wt%) poly-aromatic hydrocarbon (PAH) content. Additionally, increased calorific value and density upgradation comparable to standard diesel fuel quality were observed in the presence of dual catalyst layout.

Arsenic processing of yeast isolates IIB-As1 & IIB-As2 and production of glutathione under stress conditions.

Four arsenic resistant yeast were isolated from the industrial wastewater. Two strains IIB-As1 and IIB-As2 identified as Candida tropicalis and Saccharomyces cerevisiae, respectively. IIB-As1 and IIB-As2 showed maximum arsenic resistance. IIB-As1 showed maximum growth at 35 °C whereas it was 30 °C for IIB-As2. The yeast isolate showed typical growth curves, but arsenic extended the lag phase. Glutathione plays an important role in metal tolerance. In the present study, As increased the level glutathione and non-protein thiols in yeast isolates. Removal of As from supernatant was analyzed using the atomic absorption spectrophotometer. They removed arsenic from the medium after 72 h of incubation. Both yeast strains efficiently removed arsenic from the industrial effluent when used individually or in consortia.

Thermodynamic and saccharification analysis of cloned GH12 endo-1,4-ß-glucanase from Thermotoga petrophila in a mesophilic host.

The thermotolerant endo-1,4-ß-glucanase gene, of Thermotoga petrophila RKU-1, was cloned and over-expressed in E. coli strain BL21 CodonPlus. Enzyme was purified to homogeneity, producing a single band on SDS-PAGE corresponding to 38 kDa, by purification steps of heat treatment combined with ion-exchange column chromatography. The purified enzyme was optimally active, with specific activity of 530 Umg(-1) against carboxymethyl cellulose (CMC), at pH 6.0 and 95°C and was also stable upto 8 h at 80°C. The enzyme also showed activity against ß-glucan barley: 303 %, laminarin: 13.7 %, Whatman filter paper: 0.017 % with no activity against starch and Avicel. The recombinant enzyme exhibited Km, Vmax and Kcat of 12.5 mM, 735 µmol mg-1min-1 and 2351.23 s-1, respectively against CMC as a substrate. The stable recombinant enzyme manifested half life (t1/2) of 6.6 min even at temperature as high as 97°C, with free energy of denaturation (ΔG*D), enthalpy of denaturation (ΔH*D), and entropy of denaturation (ΔS*D) of 98.2 kJ mol(-1), 528.9 kJ mol(-1), and 1.17 kJ mol(-1)K(-1), respectively at 97°C. In addition, the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for hydrolysis of CMC substrate by endo-1,4-ß-glucanase were calculated at 95°C as 48.2 kJ mol(-1), 54.6 kJ mol(-1) and -17.4 J mol(-1) K(-1), respectively. The recombinant enzyme saccharified pre-treated wheat straw and bagasse to 3.32 % and 3.2 %, respectively after 6 h incubation at 85°C. Its thermostability, resistance to heavy metal ions and specific activity make this enzyme an interesting candidate for industrial applications.

Cloning, characterization and molecular docking of a highly thermostable ß-1,4-glucosidase from Thermotoga petrophila.

A genomic DNA fragment, encoding a thermotolerant ß-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5 kDa protein (by SDS-PAGE) encoded by 1.341 kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-ß-D-glucopyranoside were 2.8 mM and 42.7 mmol min(-1) mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.

Kinetic and thermodynamic study of cloned thermostable endo-1,4-ß-xylanase from Thermotoga petrophila in mesophilic host.

The 1,044 bp endo-1,4-ß-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and ß-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 µmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(½)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-ß-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.

Screening of pneumococcal pneumonia by amplification of pneumolysin gene in children visiting hospitals in lahore, pakistan.

OBJECTIVE: Streptococcus pneumoniae is a common worldwide potential pathogen causing pneumonia among children and the detection of pneumococcal infections by conventional culturing techniques is cumbersome. The present study describes a comparative analysis of sensitive nested-PCR and bacterial culture in pediatric patients with clinical and radiological indication of S. pneumoniae infection. METHODS: PCR was performed using outer primers to amplify a 348-bp region and inner primers a 208-bp region of the pneumolysin gene. For pneumolysin PCR assay, DNA from peripheral blood and middle ear fluid (MEF) samples was extracted by salting out method. The sensitivity of the assay was evaluated with about 0.06 pg of purified S. pneumoniae genomic DNA. FINDINGS: Among 90 MEF culture negative samples from acute otitis media pediatric patients, 8.8% pneumolysin-PCR positivity was detected, demonstrating the sensitivity and reliability of PCR for rapid pneumonia evaluation. Binomial test of proportionality performed on (SPSS 17) gives P< 0.05 indicating that PCR technique is statistically significant and sensitive in the diagnosis of S. pneumoniae infection. CONCLUSION: The research work evaluated the effectiveness and efficacy of nested-PCR for detecting S. pneumoniae in pediatric patients with clinical and radiological confirmation of bacterial infection. This simplified method permitted quick selection of the patients and played a significant role in preliminary management of pneumococcal infections.

Evaluation of consortia of microorganisms for efficient removal of hexavalent chromium from industrial wastewater.

The Chromium (Cr) uptake ability of Cr-resistant bacterium Bacillus thuringiensis, yeast Candida etschellsii, and a protozoan Stylonychia mytilus, isolated from industrial waste water, was evaluated individually and in different combinations. It was found that the three types of microorganisms grown together in a culture medium could collectively uptake 90% of Cr(6+) from the culture medium as against 82% by bacterium + protozoan or yeast + protozoan combined culture, each. Consortium of bacterium, yeast and ciliates therefore could make much more efficient inoculum for remediation of Cr-contaminated industrial waste water.