Synthesis of azatricyclodiones & octahydro-benzo[f]isoindoles and their antimicrobial evaluation

A series of azatricyclodiones and octahydro-benzo[f]isoindoles have been synthesized by (4+2) Diels-Alder cycloaddition of maleimides with furfuryl amine. Reaction of azatricyclodiones with isocyanates led to the respective ureides. All of the compounds were screened against a number of bacteria and fungi. One of the compounds (2) displayed moderate antitubercular activity while two compounds (2) and (4) inhibited the fungal growth at 25 μg/mL.     

Slow and fast development in Parthenium beetle and its effect on reproductive attributes

The present study was designed to investigate the existence of slow and fast developing individuals and to evaluate their effect on reproductive attributes of Parthenium beetle, Zygogramma bicolorata Pallister (Coleoptera: Chrysomelidae); a biocontrol agent of Parthenium weed. Significantly different rates of development were found in a cohort of the beetle. Under constant rearing conditions, the emergence ratio of slow and fast developers was determined to be 0.47 and 0.53, respectively. Males developed faster than females, and significant differences in development were found among all immature stages and total developmental period. Slow developing females laid higher numbers of eggs with a higher percentage egg viability than fast developing females. This is the first attempt of such a study for the Parthenium beetle, and provides great insight into Parthenium beetle development for evolutionary biologists, improving our understanding of their ecological thresholds. The information could help in the selection of fast developing lines and thus in the mass multiplication of this bioagent.     

Non contiguous-finished genome sequence and description of Enorma massiliensis gen. nov., sp. nov., a new member of the Family Coriobacteriaceae

Enorma massiliensis strain phI^T is the type strain of E. massiliensis gen. nov., sp. nov., the type species of a new genus within the family Coriobacteriaceae, Enorma gen. nov. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. E. massiliensis strain phI^T is a Gram-positive, obligately anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,280,571 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 62.0% and contains 1,901 protein-coding and 51 RNA genes, including 3 rRNA genes.     

Modeling the activity of glutathione as a hydroxyl radical scavenger considering its neutral non-zwitterionic form

Glutathione is an immensely important antioxidant, particularly in the central nervous system. The scavenging mechanism of glutathione towards the OH radical was studied theoretically, considering its neutral, non-zwitterionic form relevant to acidic media. Gibbs free barrier and released energies involved in hydrogen abstraction from the different sites of glutathione by an OH radical were studied at the B3LYP/6-31G(d,p), B3LYP/AUG-cc-pVDZ, M06/AUG-cc-pVDZ, M06-2X/AUG-cc-pVDZ levels of density functional theory. Solvation in bulk aqueous media was also studied at all these levels of theory employing the polarizable continuum model. Our study shows that a hydroxyl radical can abstract a hydrogen atom easily from glutathione. Thus, glutathione is shown to be an efficient scavenger of OH radicals, which is in agreement with the results of previous studies.

Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland

Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na⁺/H⁺ antiporter gene that compartmentalises excess Na⁺ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.     

A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids

Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs) as key components of crucial nutrients, soaps, industrial chemicals, and fuels. However, our ability to control the composition of microbially synthesized FFAs is still limited, particularly, for producing medium-chain fatty acids (MCFAs). This is mainly due to the lack of high-throughput approaches for FFA analysis to engineer enzymes with desirable product specificity. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. In particular, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and a higher m/z peak ratio at 730 and 758 was used as an indication for improved MCFA production. This colony-based method can be performed at a rate of ~2 s per sample, representing a substantial improvement over gas chromatography-MS (typically >30 min per sample) as the gold standard method for FFA detection. To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified nine missense mutations that resulted in improved MCFA production relative to the wild-type strain. Colony-based MALDI-ToF MS screening provides an effective approach for engineering microbial fatty acid compositions in a high-throughput manner.     

Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential

Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 3′ UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 3′ UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t_1/2 (>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t_1/2 (<1 h). MTCH2 read-through–deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normal mitochondrial membrane potential.     

Conserved boundary elements from the Hox complex of mosquito,Anopheles gambiae

The conservation of hox genes as well as their genomic organization across the phyla suggests that this system of anterior–posterior axis formation arose early during evolution and has come under strong selection pressure. Studies in the split Hox cluster of Drosophila have shown that proper expression of hox genes is dependent on chromatin domain boundaries that prevent inappropriate interactions among different types of cis-regulatory elements. To investigate whether boundary function and their role in regulation of hox genes is conserved in insects with intact Hox clusters, we used an algorithm to locate potential boundary elements in the Hox complex of mosquito, Anopheles gambiae. Several potential boundary elements were identified that could be tested for their functional conservation. Comparative analysis revealed that like Drosophila, the bithorax region in A. gambiae contains an extensive array of boundaries and enhancers organized into domains. We analysed a subset of candidate boundary elements and show that they function as enhancer blockers in Drosophila. The functional conservation of boundary elements from mosquito in fly suggests that regulation of hox genes involving chromatin domain boundaries is an evolutionary conserved mechanism and points to an important role of such elements in key developmentally regulated loci.     

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. ¹HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CI_xrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.