An ncRNA transcriptomics-based approach to design siRNA molecules against SARS-CoV-2 double membrane vesicle formation and accessory genes.

BACKGROUND: The corona virus SARS-CoV-2 is the causative agent of recent most global pandemic. Its genome encodes various proteins categorized as non-structural, accessory, and structural proteins. The non-structural proteins, NSP1-16, are located within the ORF1ab. The NSP3, 4, and 6 together are involved in formation of double membrane vesicle (DMV) in host Golgi apparatus. These vesicles provide anchorage to viral replicative complexes, thus assist replication inside the host cell. While the accessory genes coded by ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b, 9c, and 10 contribute in cell entry, immunoevasion, and pathological progression. METHODS: This in silico study is focused on designing sequence specific siRNA molecules as a tool for silencing the non-structural and accessory genes of the virus. The gene sequences of NSP3, 4, and 6 along with ORF3a, 6, 7a, 8, and 10 were retrieved for conservation, phylogenetic, and sequence logo analyses. siRNA candidates were predicted using siDirect 2.0 targeting these genes. The GC content, melting temperatures, and various validation scores were calculated. Secondary structures of the guide strands and siRNA-target duplexes were predicted. Finally, tertiary structures were predicted and subjected to structural validations. RESULTS: This study revealed that NSP3, 4, and 6 and accessory genes ORF3a, 6, 7a, 8, and 10 have high levels of conservation across globally circulating SARS-CoV-2 strains. A total of 71 siRNA molecules were predicted against the selected genes. Following rigorous screening including binary validations and minimum free energies, final siRNAs with high therapeutic potential were identified, including 7, 2, and 1 against NSP3, NSP4, and NSP6, as well as 3, 1, 2, and 1 targeting ORF3a, ORF7a, ORF8, and ORF10, respectively. CONCLUSION: Our novel in silico pipeline integrates effective methods from previous studies to predict and validate siRNA molecules, having the potential to inhibit viral replication pathway in vitro. In total, this study identified 17 highly specific siRNA molecules targeting NSP3, 4, and 6 and accessory genes ORF3a, 7a, 8, and 10 of SARS-CoV-2, which might be used as an additional antiviral treatment option especially in the cases of life-threatening urgencies.

Structural Analysis and Epitope Prediction of S2 Domain of SARS-CoV-2, Conservation Analysis Among Major Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. There are four structural proteins of the virus: spike, envelope, membrane, and nucleocapsid proteins. Various vaccines were designed and are effectively being used against the spike protein of the virus. However, several vaccine-related complications have been reported worldwide. Assuming that the structural integrity of the whole protein might be contributing to these complications, this study was performed to design epitopes using the S2 domain of the spike protein, which could trigger a strong immune response. We have also predicted antigenic and allergenic properties of the selected epitopes. A total of 49 B cell epitopes passing antigenicity and other assessment filters were found using three methods. Among them, RDLICAQ had the highest antigenicity score (1.1443). However, only one cytotoxic T lymphocyte epitope, RSFIEDLLF, passed the essential filters with an antigenicity score of 0.5782 to show an appropriate immune response for T cells, while among 21 helper T cell lymphocyte epitopes that were filtered, FAMQMAYRFNGIGVT showed the highest (1.3688) antigenicity score. Conservation analysis revealed that the S2 domain is significantly conserved, thus making it an ideal candidate for vaccine development. We have also designed a vaccine construct based on the best suiting components found during the whole study. This construct and S2 domain solely can be future subjects of interest or might be included in a subunit cocktail formulation for attaining unabridged immunogenicity.

Constitutive expression of Asparaginase in Gossypium hirsutum triggers insecticidal activity against Bemisia tabaci.

Whitefly infestation of cotton crop imparts enormous damage to cotton yield by severely affecting plant health, vigour and transmitting Cotton Leaf Curl Virus (CLCuV). Genetic modification of cotton helps to overcome both the direct whitefly infestation as well as CLCuV based cotton yield losses. We have constitutively overexpressed asparaginase (ZmASN) gene in Gossypium hirsutum to overcome the cotton yield losses imparted by whitefly infestation. We achieved 2.54% transformation efficiency in CIM-482 by Agrobacterium-mediated shoot apex transformation method. The relative qRT-PCR revealed 40-fold higher transcripts of asparaginase in transgenic cotton line vs. non-transgenic cotton lines. Metabolic analysis showed higher contents of aspartic acid and glutamic acid in seeds and phloem sap of the transgenic cotton lines. Phenotypically, the transgenic cotton lines showed vigorous growth and height, greater number of bolls, and yield. Among six representative transgenic cotton lines, line 14 had higher photosynthetic rate, stomatal conductance, smooth fiber surface, increased fiber convolutions (SEM analysis) and 95% whitefly mortality as compared to non-transgenic cotton line. The gene integration analysis by fluorescence in situ hybridization showed single copy gene integration at chromosome number 1. Collectively, asparaginase gene demonstrated potential to control whitefly infestation, post-infestation damages and improve cotton plant health and yield: a pre-requisite for farmer's community.

Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression.

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the ßC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vß, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vß transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vß-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vß-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

HCV-induced regulatory alterations of IL-1ß, IL-6, TNF-α, and IFN-ϒ operative, leading liver en-route to non-alcoholic steatohepatitis.

Over the course of time, Hepatitis C has become a universal health menace. Its deleterious effects on human liver encompass a lot of physiological, genetic as well as epigenetic alterations. Fatty liver (Hepatic steatosis) is an inflammation having multifactorial ancestries; one of them is HCV (steatohepatitis). HCV boosts several cellular pathways involving up-regulation of a number of cytokines. Current study reviews the regulation of some selective key cytokines during HCV infection, to help generate an improved understanding of their role. These cytokines, IL-1ß, IL-6, TNF-α, and IFN-ϒ, are inflammatory markers of the body. These particular markers along with others help hepatocytes against viral infestation. However, recently, their association has been found in degradation of liver on the trail heading to non-alcoholic steatohepatitis (NASH). Consequently, the disturbance in their equilibrium has been repeatedly reported during HCV infection. Quite a number of findings are affirming their up-regulation. Although these cell markers are stimulated by hepatocytes as their standard protection mechanism, but modern studies have testified the paradoxical nature of this defense line. Nevertheless, direct molecular or epigenetic research is needed to question the actual molecular progressions and directions commanding liver to steatosis, cirrhosis, or eventually HCC (Hepatocellular Carcinoma).

Microarray: gateway to unravel the mystery of abiotic stresses in plants.

Environmental factors, such as drought, salinity, extreme temperature, ozone poisoning, metal toxicity etc., significantly affect crops. To study these factors and to design a possible remedy, biological experimental data concerning these crops requires the quantification of gene expression and comparative analyses at high throughput level. Development of microarrays is the platform to study the differential expression profiling of the targeted genes. This technology can be applied to gene expression studies, ranging from individual genes to whole genome level. It is now possible to perform the quantification of the differential expression of genes on a glass slide in a single experiment. This review documents recently published reports on the use of microarrays for the identification of genes in different plant species playing their role in different cellular networks under abiotic stresses. The regulation pattern of differentially-expressed genes, individually or in group form, may help us to study different pathways and functions at the cellular and molecular level. These studies can provide us with a lot of useful information to unravel the mystery of abiotic stresses in important crop plants.

In-Silico Determination of Insecticidal Potential of Vip3Aa-Cry1Ac Fusion Protein Against Lepidopteran Targets Using Molecular Docking.

Study and research of Bt (Bacillus thuringiensis) transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac) insecticidal protein and vegetative insecticidal protein (Vip3Aa) have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN) and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, and Spodoptera litura) revealed that the Ser290, Ser293, Leu337, Thr340, and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

In silico study for diversing the molecular pathway of pigment formation: an alternative to manual coloring in cotton fibers.

Diversity of colors in flowers and fruits is largely due to anthocyanin pigments. The flavonoid/anthocyanin pathway has been most extensively studied. Dihydroflavonol 4-reductase (DFR) is a vital enzyme of the flavonoid pathway which displays major impact on the formation of anthocyanins, flavan 3-ols and flavonols. The substrate specificity of the DFR was found to play a crucial role in determination of type of anthocyanidins. Altering the flavonoid/anthocyanin pathway through genetic engineering to develop color of our own choice is an exciting subject of future research. In the present study, comparison among four DFR genes (Gossypium hirsutum, Iris × hollandica, Ang. DFRI and DFRII), sequence alignment for homology as well as protein modeling and docking is demonstrated. Estimation of catalytic sites, prediction of substrate preference and protein docking were the key features of this article. For specific substrate uptake, a proline rich region and positions 12 plus 26 along with other positions emphasizing the 26-amino acid residue region (132-157) was tested. Results showed that proline rich region position 12, 26, and 132-157 plays an important role in selective attachment of DFRs with respective substrates. Further, "Expasy ProtParam tool" results showed that Iris × hollandica DFR amino acids (Asn 9: Asp 23) are favorable for reducing DHQ and DHM thus accumulating delphinidin, while Gossypium hirsutum DFR has (Asn 13: Asp 21) hypothesized to consume DHK. Protein docking data showed that amino acid residues in above mentioned positions were just involved in attachment of DFR with substrate and had no role in specific substrate uptake. Advanced bioinformatics analysis has revealed that all above mentioned positions have role in substrate attachment. For substrate specificity, other residues region is involved. It will help in color manipulations in different plant species.

Molecular docking based screening of neem-derived compounds with the NS1 protein of Influenza virus.

UNLABELLED: Different strains of influenza virus are affecting a large number of people worldwide to combat with Influenza virus destruction, numerous synthetic antiviral medicines are available for influenza virus in the market. But still there was a need for the development of drug which will target all the strains of influenza virus. For this purpose conserved residues within the influenza virus NS1 protein have been found by aligning all the available sequences of existing strains from the national center of biotechnology information(NCBI) protein database. The compounds from leaf extracts of neem (Azadirachta indica), previously known to have antiviral properties, were virtually screened to identify side effects free natural drug. Molecular docking identified eight potential compounds (Tetratriacontane, 127-40-2, 6-o-ACETYLNIMBANDIOL, Rutin, Tiplasinin, Hyperoside, ( )- Nimocinolide and Quercitrin) found to have perfect binding with reported conserved residues (R19, R35, S42 and D39) of influenza virus NS1 protein involved in the binding of drugs. From, further analysis 6-o-ACETYLNIMBANDIOL, Rutin and Tiplasinin were found as drug against influenza strains because their binding residues were conserved in all strains. The potential of neem chemical against influenza virus has best been highlighted through this study and it provides direction for further consideration of these products for in-vivo and in-vitro validations. ABBREVIATIONS: NS1 protein - Non Structural 1 protein, NA - Neuraminidase, HA - Hemagglutinin, M - Yersinia enterocolitica 8081, ypk - Yersinia pestis KIM, yps - Yersinia pseudotuberculosis Db1, KEGG - Kyoto Encyclopedia of Genes and Genomes, KO - KEGG Orthology, KOBAS - KO Based Annotation System.