Life Sciences

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The processing of polyprotein(s) to form structural and non-structural components remains an enigma due to the non-existence of an efficient and robust Hepatitis E Virus (HEV) culture system. We used the BacMam approach to construct an HEV replication model in which the HEV genome was cloned in the BacMam vector under the CMV promoter. The recombinant BacMam was used to infect Huh7 cells to transfer the HEV genome. HEV replication was authenticated by the presence of RNAs of both the polarity (+) and (−) and formation of hybrid RNA, a replication intermediate. The presence of genes for Papain-like Cysteine Protease (PCP), methyltransferase (MeT), RNA dependent RNA polymerase (RdRp), and ORF2 was confirmed by PCR amplification. Further, the infectious nature of the culture system was established as evidenced by the cross-infection of uninfected cells using the cell lysate from the infected cells. The HEV replication model was validated by detection of the ORF1 (Open Reading Frame1) encoded proteins, identified by Western blotting and Immunofluorescence by using epitope-specific antibodies against each protein. Consequently, discrete bands of 18, 35, 37, and 56 kDa corresponding to PCP, MeT, RdRp, and ORF2, respectively, were seen. Besides demonstrating the presence of non-structural enzymes of HEV along with ORF2, activity of a key enzyme, HEV-methyltransferase has also been observed. A 20\% decrease in the replicative forms of RNA could be seen in presence of 100 μM Ribavirin after 48 h of treatment. The inhibition gradually increased from 0 to 24 to 48 h post-treatment. Summarily, infectious HEV culture system has been established, which could demonstrate the presence of HEV replicative RNA forms, the structural and non-structural proteins and the methyltransferase in its active form. The system may also be used to study the mechanism of action of Ribavirin in inhibiting HEV replication and develop a therapy. © Copyright © 2020 Kumar, Hooda, Khanna, Patel and Sehgal.

(SoNS) School of Natural Sciences

Fulltext

Frontiers in Microbiology

Development of BacMam Induced Hepatitis E Virus Replication Model in Hepatoma Cells to Study the Polyprotein Processing

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein. © 2020 Elsevier Ltd

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Journal of Molecular Biology

GROEL/ES Buffers Entropic Traps in Folding Pathway during Evolution of a Model Substrate

DNA methylation governs gene regulation in plants in response to environmental conditions. Here, we analyzed role of DNA methylation under desiccation and salinity stresses in three (IR64, stress-sensitive; Nagina 22, drought-tolerant and Pokkali, salinity-tolerant) rice cultivars via bisulphite sequencing. Methylation in CG context within gene body and methylation in CHH context in distal promoter regions were positively correlated with gene expression. Hypomethylation in Nagina 22 and hypermethylation in Pokkali in response to desiccation and salinity stresses, respectively, were correlated with higher expression of few abiotic stress response related genes. Most of the differentially methylated and differentially expressed genes (DMR-DEGs) were cultivar-specific, suggesting an important role of DNA methylation in abiotic stress responses in rice in cultivar-specific manner. DMR-DEGs harboring differentially methylated cytosines due to DNA polymorphisms between the sensitive and tolerant cultivars in their promoter regions and/or coding regions were identified, suggesting the role of epialleles in abiotic stress responses. © 2020 Elsevier Inc.

Genomics

Bisulphite sequencing reveals dynamic DNA methylation under desiccation and salinity stresses in rice cultivars

Glutathione S-transferases (GSTs) are multifunctional proteins that help in oxidative stress metabolism and detoxification of xenobiotic compounds. Studies pertaining to GST gene family have been undertaken in various plant species, however no information is available with respect to GST genes in chickpea. In the current study, we identified a total of 51 GST encoding genes in chickpea (CaGST) genome. Phylogenetic analysis revealed that GST gene family can be divided into eleven distinct classes. Tau and phi were the major classes in chickpea and one third of the CaGST genes represented segmental duplication and purifying selection was common among these genes. Expression of many CaGST genes, in particular, members of tau class were found to be upregulated under abiotic stress conditions. In addition, CaGST genes displayed differential expression patterns across diverse organs/tissues, suggesting their roles in developmental processes. Many CaGST genes showed opposite expression pattern in small- and large-seeded chickpea cultivars during seed development. Higher expression of CaGST genes in small-seeded cultivar at maturation stages of seed development suggested their important role in seed development and seed size/weight determination in chickpea. Overall, these results provide a comprehensive information on GST gene family members in chickpea and is expected to provide a rational platform to explore versatile role of these genes in semi-arid legume crops. © 2020, Springer Nature B.V.

Molecular Biology Reports

Genome-wide analysis of glutathione S-transferase gene family in chickpea suggests its role during seed development and abiotic stress

Oryza coarctata is a wild relative of rice that has adapted to diverse ecological environments, including high salinity and submergence. Thus, it can provide an important resource for discovering candidate genes/factors involved in tolerance to these stresses. Here, we report a draft genome assembly of 573 Mb comprised of 8877 scaffolds with N50 length of 205 kb. We predicted a total of 50,562 protein-coding genes, of which a significant fraction was found to be involved in secondary metabolite biosynthesis and hormone signal transduction pathways. Several salinity and submergence stress-responsive protein-coding and long noncoding RNAs involved in diverse biological processes were identified using RNA-sequencing data. Based on small RNA sequencing, we identified 168 unique miRNAs and 3219 target transcripts (coding and noncoding) involved in several biological processes, including abiotic stress responses. Further, whole genome bisulphite sequencing data analysis revealed at least 19\%–48\% methylcytosines in different sequence contexts and the influence of methylation status on gene expression. The genome assembly along with other datasets have been made publicly available at http://ccbb.jnu.ac.in/ory-coar. Altogether, we provide a comprehensive genomic resource for understanding the regulation of salinity and submergence stress responses and identification of candidate genes/factors involved for functional genomics studies. © 2020 Scandinavian Plant Physiology Society

Physiologia Plantarum

Draft genome and transcriptome analyses of halophyte rice Oryza coarctata provide resources for salinity and submergence stress response factors

In recent years, rapid advancement has been done in generation of genomic resources for the important legume crop chickpea. Here, we provide an update on important advancements made on availability of genomic resources for this crop. The availability of reference genome and transcriptome sequences, and resequencing of several accessions have enabled the discovery of gene space and molecular markers in chickpea. These resources have helped in elucidating evolutionary relationship and identification of quantitative trait loci for important agronomic traits. Gene expression in different tissues/organs during development and under abiotic/biotic stresses has been interrogated. In addition, single-base resolution DNA methylation patterns in different organs have been analyzed to understand gene regulation. Overall, we provide a consolidated overview of available genomic resources of chickpea that may help in fulfilling the promises for improvement of this important crop. © Springer Science+Business Media, LLC, part of Springer Nature 2020.

Methods in Molecular Biology

Updates on genomic resources in chickpea for crop improvement

Kunitz-type domains are ubiquitously found in natural systems as serine protease inhibitors or animal toxins in venomous animals. Kunitz motif is a cysteine-rich peptide chain of ~ 60 amino acid residues with alpha and beta fold, stabilized by three conserved disulfide bridges. An extensive dataset of amino acid variations is found on sequence analysis of various Kunitz peptides. Kunitz peptides show diverse biological activities like inhibition of proteases of other classes and/or adopting a new function of blocking or modulating the ion channels. Based on the amino acid residues at the functional site of various Kunitz-type inhibitors, it is inferred that this ‘flexibility within the structural rigidity’ is responsible for multiple biological activities. Accelerated evolution of functional sites in response to the co-evolving molecular targets of the hosts of venomous animals or parasites, gene sharing, and gene duplication have been discussed as the most likely mechanisms responsible for the functional heterogeneity of Kunitz-domain inhibitors. © 2020, Springer Science+Business Media, LLC, part of Springer Nature.

Journal of Molecular Evolution

Evolutionary Aspects of the Structural Convergence and Functional Diversification of Kunitz-Domain Inhibitors

Blood transfusion therapy involves multiple steps to ensure selection of safe blood component for transfusion. This includes testing for infectious markers, full ABO compatibility, free from any clinically significant red cell antibodies and acceptable donor's red cell survival rates without destruction of recipient's red cells. The red cell antibodies present in healthy blood donors can cause severe haemolytic transfusion reaction, especially in massive blood transfusion recipients and paediatric patients. Hence, screening of red cell antibodies in donor blood is important to provide compatible blood products and to avoid haemolytic transfusion reactions in susceptible patient population. This study was planned to assess prevalence, aetiology and type of unexpected red cell antibodies in a large number of whole blood donor population in north India. Methods: This three-year prospective observational study included blood donor samples for antibody screening from January 2015 to December 2017. A total of 166,803 healthy blood donors including 156,128 (93.6%) males and 10,675 (6.4%) females were screened. Results: The prevalence of red cell antibodies was 0.17 per cent in our donor population. Of the total 286 donors with red cell antibodies, 248 (86.7%) had alloantibodies, 30 (10.5%) had autoantibodies and eight donors (2.8%) showed positive antibody screening with inconclusive results. Interpretation and conclusions: Alloimmunization to red cell antigens is a challenging task for current transfusion practices. The antibody screening in blood donors may improve the quality and safety of blood transfusion in the recipients. It also reduces the risk of complications from incompatible blood transfusions. © 2020 Indian Journal of Medical Research, published by Wolters Kluwer - Medknow for Director-General, Indian Council of Medical Research.

Indian Journal of Medical Research

Prevalence of red blood cell antibodies in whole blood donors: A single-centre experience in north India

With growing urbanization cities are increasingly suffering due to the abundance of built-in spaces, concrete, mortar and tarmac, urban heat islands, increasing frequency of extreme heat events and climate change. The sustainable development goal 11, emphasizes on developing green infrastructure which could minimize the environmental footprint of the cities. In this paper, we discuss the role of green infrastructure in carbon sequestration, mitigating atmospheric pollution, moderating temperature and mitigating climate change, and management of storm water. We also discuss biodiversity of green infrastructure and the latter as habitat of organisms, and provide an overview of economic value of trees in the urban areas. © 2020 Indian National Science Academy. All rights reserved.

Proceedings of the Indian National Science Academy

Green infrastructure of cities: An overview

Antimicrobial drug resistance is one of the most critical problems that plagued the human race in modern times. Discovery of novel antibiotics is important to counter this threat. Accordingly, herein we have reported the discovery of substituted benzimidazole class of molecules with antimicrobial property (specifically against Staphylococcus aureus). They were initially identified through a random screening and a novel catalytic synthetic strategy was utilized to access them. in vitro screening and phenotypic profiling revealed the antimicrobial nature. De novo transcriptome and gene analyses predicted the putative targets. This work provides a solid foundation for developing the benzimidazoles as a target specific antimicrobial preclinical candidate. © 2019 Wiley Periodicals, Inc.