Functional Characterization of the Tau Class Glutathione-S-Transferases Gene (SbGSTU) Promoter of Salicornia brachiata under Salinity and Osmotic Stress

Reactive oxygen or nitrogen species are generated in the plant cell during the extreme stress condition, which produces toxic compounds after reacting with the organic molecules. The glutathione-S-transferase (GST) enzymes play a significant role to detoxify these toxins and help in excretion or sequestration of them. In the present study, we have cloned 1023 bp long promoter region of tau class GST from an extreme halophyte Salicornia brachiata and functionally characterized using the transgenic approach in tobacco. Computational analysis revealed the presence of abiotic stress responsive cis-elements like ABRE, MYB, MYC, GATA, GT1 etc., phytohormones, pathogen and wound responsive motifs. Three 5’-deletion constructs of 730 (GP2), 509 (GP3) and 348 bp (GP4) were made from 1023 (GP1) promoter fragment and used for tobacco transformation. The single event transgenic plants showed notable GUS reporter protein expression in the leaf tissues of control as well as treated plants. The expression level of the GUS gradually decreases from GP1 to GP4 in leaf tissues, whereas the highest level of expression was detected with the GP2 construct in root and stem under control condition. The GUS expression was found higher in leaves and stems of salinity or osmotic stress treated transgenic plants than that of the control plants, but, lower in roots. An efficient expression level of GUS in transgenic plants suggests that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). And this property, make it as a potential candidate to be used as an alternative promoter for crop genetic engineering.     

Identification of potential tumour-associated carbonic anhydrase isozyme IX inhibitors: atom-based 3D-QSAR modelling,pharmacophore-based virtual screening and molecular docking studies

Abstract

Tumour hypoxia results in dramatic changes in the gene expression, proliferation and survival of tumour cells. The tumour cells shift towards anaerobic glycolysis which results in change of pH in their microenvironment. In response to this stress, over expression of carbonic anhydrase IX (CA IX) genes is observed in many solid tumours. So, selective inhibition of CA IX can be a promising target for anti-cancer drugs. In this work in silico tools like atom-based 3D-QSAR modelling, pharmacophore-based virtual screening and molecular docking were used to identify potential CA IX inhibitors. Based on the training set used in the QSAR model, twenty pharmacophore models were generated. Out of these, HHHR_1, AHHR_1, DHHHR_1, AHHHR_1 model was used to screen a database of 1,50,000 compounds retrieved from ZINC 15 database. R² and Q² was 0.9864 and 0.8799, respectively, for the developed QSAR model. 163 compounds showed a phase screen score above 2.4 in which ZINC02260669 was the highest ranked (screen score, 2.852058) compound in all the four models. Built QSAR model was used to predict the activity of all these 163 compounds and ZINC72370966 showed the highest predicted activity with pKi value of 7.649. These compounds were docked against CA IX (human) protein (PDB ID 5FL6) and molecular docking results showed favourable binding interactions for the best ten identified hits. This work gives design insights and some potential scaffolds which can be developed as CA IX inhibitors.

Communicated by Ramaswamy H. Sarma     

Molecular in silico structure and recombination analysis of betasatellite in Calotropis procera associated with begomovirus

The uncharacterised betasatellite of begomovirus associated with Calotropis procera was characterised by using molecular and in silico tools and techniques. Attempts to identify the presence of a DNA-β in the infected C. procera samples, using rolling circular amplification (RCA) followed by restriction digestion, produced a ca. 1.4 kb product, corresponding to that expected for a full-length amplicon from a betasatellite, which was sequenced (accession number HQ631430). During BLASTp, analysis of second reading frame of HQ631430 (HQ631430/2-f) against Protein Databank revealed 35% identity with Tryptophanyl–tRNA synthetase of Giardia lamblia (3FOC). Ramachandran plot of HQ631430/2-f.pdb had only 57.1% residues in the most favoured region while 3FOC.pdb had 94.2% residues in the most favoured region; therefore, only template 3FOC.pdb model could be placed in good quality category. The protein binding function was predicted for HQ631430/2-f as an important functional site of the model with 0.29 confidence level through 3d2GO. The Croton yellow vein mosaic betasatellite (GU111995 CroYVMB) serve as major parent and Croton yellow vein mosaic betasatellite-Panipat 8 (HM143908 PaLCuVM) as minor parent for HQ631430. Perhaps this is the first report of recombination in Croton yellow vein mosaic betasatellite (HQ631430).     

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4‐KD, miRNA156‐OE, MYB4‐OE, COMT‐KD and FPGS‐KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second‐ versus the first‐year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second‐year growth of transgenics targeted for wall modification, GAUT4‐KD, MYB4‐OE, COMT‐KD and FPGS‐KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next‐generation bio‐feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.     

Crystal Structure of the Leucine Aminopeptidase from Pseudomonas putida Reveals the Molecular Basis for its Enantioselectivity and Broad Substrate Specificity

The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure-function relationships, the enzyme was studied by X-ray crystallography. Crystal structures of native ppLAP at pH 9.5 and pH 5.2, and in complex with the inhibitor bestatin, show that the overall folding and hexameric organization of ppLAP are very similar to those of the closely related di-zinc leucine aminopeptidases (LAPs) from bovine lens and Escherichia coli. At pH 9.5, the active site contains two metal ions, one identified as Mn²⁺ or Zn²⁺ (site 1), and the other as Zn²⁺ (site 2). By using a metal-dependent activity assay it was shown that site 1 in heterologously expressed ppLAP is occupied mainly by Mn²⁺. Moreover, it was shown that Mn²⁺ has a significant activation effect when bound to site 1 of ppLAP. At pH 5.2, the active site of ppLAP is highly disordered and the two metal ions are absent, most probably due to full protonation of one of the metal-interacting residues, Lys267, explaining why ppLAP is inactive at low pH. A structural comparison of the ppLAP-bestatin complex with inhibitor-bound complexes of bovine lens LAP, along with substrate modelling, gave clear and new insights into its substrate specificity and high level of enantioselectivity.     

An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers

Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75?%) was observed with BAP at 3?mg?l^?1. Shoots were multiplied for 30?d on fresh medium with similar composition. A total of 67?% of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81?cm) with BAP (3?mg?l^?1) alone. Maximum rooting response (87?%) was observed with naphthaleneacetic acid at 0.5?mg?l^?1. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.     

Bioactivity,mechanism of action,and cytotoxicity of copper-based nanoparticles: A review

Nanotechnology is an emerging branch of science, which has potential to solve many problems in different fields. The union of nanotechnology with other fields of sciences including physics, chemistry, and biology has brought the concept of synthesis of nanoparticles from their respective metals. Till date, many types of nanoparticles have been synthesized and being used in different fields for various applications. Moreover, copper nanoparticles attract biologists because of their significant and broad-spectrum bioactivity. Due to the large surface area to volume ratio, copper nanoparticles have been used as potential antimicrobial agent in many biomedical applications. But the excess use of any metal nanoparticles increase the chance of toxicity to humans, other living beings, and environment. In this article, we have critically reviewed the bioactivities and cytotoxicity of copper nanoparticles. We have also focused on possible mechanism involved in its interaction with microbes.     

Unravelling the Carbon and Sulphur Metabolism in Coastal Soil Ecosystems Using Comparative Cultivation-Independent Genome-Level Characterisation of Microbial Communities

Bacterial autotrophy contributes significantly to the overall carbon balance, which stabilises atmospheric CO₂ concentration and decelerates global warming. Little attention has been paid to different modes of carbon/sulphur metabolism mediated by autotrophic bacterial communities in terrestrial soil ecosystems. We studied these pathways by analysing the distribution and abundance of the diagnostic metabolic marker genes cbbM, apsA and soxB, which encode for ribulose-1,5-bisphosphate carboxylase/oxygenase, adenosine phosphosulphate reductase and sulphate thiohydrolase, respectively, among different contrasting soil types. Additionally, the abundance of community members was assessed by quantifying the gene copy numbers for 16S rRNA, cbbL, cbbM, apsA and soxB. Distinct compositional differences were observed among the clone libraries, which revealed a dominance of phylotypes associated with carbon and sulphur cycling, such as Gammaproteobacteria (Thiohalomonas, Allochromatium, Chromatium, Thiomicrospira) and Alphaproteobacteria (Rhodopseudomonas, Rhodovulum, Paracoccus). The rhizosphere soil was devoid of sulphur metabolism, as the soxB and apsA genes were not observed in the rhizosphere metagenome, which suggests the absence or inadequate representation of sulphur-oxidising bacteria. We hypothesise that the novel Gammaproteobacteria sulphur oxidisers might be actively involved in sulphur oxidation and inorganic carbon fixation, particularly in barren saline soil ecosystems, suggesting their significant putative ecological role and contribution to the soil carbon pool.