Molecular pathogenesis of Japanese encephalitis and possible therapeutic strategies.

Japanese encephalitis virus (JEV), a single-stranded, enveloped RNA virus, is a health concern across Asian countries, associated with severe neurological disorders, especially in children. Primarily, pigs, bats, and birds are the natural hosts for JEV, but humans are infected incidentally. JEV requires a few host proteins for its entry and replication inside the mammalian host cell. The endoplasmic reticulum (ER) plays a significant role in JEV genome replication and assembly. During this process, the ER undergoes stress due to its remodelling and accumulation of viral particles and unfolded proteins, leading to an unfolded protein response (UPR). Here, we review the overall strategy used by JEV to infect the host cell and various cytopathic effects caused by JEV infection. We also highlight the role of JEV structural proteins (SPs) and non-structural proteins (NSPs) at various stages of the JEV life cycle that are involved in up- and downregulation of different host proteins and are potentially relevant for developing efficient therapeutic drugs.

Discovery of Bispecific Lead Compounds from Azadirachta indica against ZIKA NS2B-NS3 Protease and NS5 RNA Dependent RNA Polymerase Using Molecular Simulations.

Zika virus (ZIKV) has been characterized as one of many potential pathogens and placed under future epidemic outbreaks by the WHO. However, a lack of potential therapeutics can result in an uncontrolled pandemic as with other human pandemic viruses. Therefore, prioritized effective therapeutics development has been recommended against ZIKV. In this context, the present study adopted a strategy to explore the lead compounds from Azadirachta indica against ZIKV via concurrent inhibition of the NS2B-NS3 protease (ZIKVpro) and NS5 RNA dependent RNA polymerase (ZIKVRdRp) proteins using molecular simulations. Initially, structure-based virtual screening of 44 bioflavonoids reported in Azadirachta indica against the crystal structures of targeted ZIKV proteins resulted in the identification of the top four common bioflavonoids, viz. Rutin, Nicotiflorin, Isoquercitrin, and Hyperoside. These compounds showed substantial docking energy (-7.9 to -11.01 kcal/mol) and intermolecular interactions with essential residues of ZIKVpro (B:His51, B:Asp75, and B:Ser135) and ZIKVRdRp (Asp540, Ile799, and Asp665) by comparison to the reference compounds, O7N inhibitor (ZIKVpro) and Sofosbuvir inhibitor (ZIKVRdRp). Besides, long interval molecular dynamics simulation (500 ns) on the selected docked poses reveals stability of the respective docked poses contributed by intermolecular hydrogen bonds and hydrophobic interactions. The predicted complex stability was further supported by calculated end-point binding free energy using molecular mechanics generalized born surface area (MM/GBSA) method. Consequently, the identified common bioflavonoids are recommended as promising therapeutic inhibitors of ZIKVpro and ZIKVRdRp against ZIKV for further experimental assessment.

In vivo removal of profenofos in agricultural soil and plant growth promoting activity on Vigna radiata by efficient bacterial formulation.

This study evaluated the plant growth and profenofos (PF) removal efficiency of Acinetobacter sp.33F and Comamonas sp. 51 F bacteria as individual strains and in combination F1. Plant growth-promoting activities such as indole 3 acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, phosphate solubilization, ammonia production, and exopolysaccharide (EPS) production were observed in Acinetobacter sp. 33 F and Comamonas sp. 51 F. However, PGP properties observed were higher in Acinetobacter sp. 33 F as compared to the Comamonas sp. 51 F. In pot sand and pot soil studies, the physiological parameters such as sprout length, shoot length, root length, chlorophyll a, chlorophyll b, and carotenoids were higher for combination F1. PF degradation in pot sand and pot soil resulted in highest degradation by combination F1. In pot soil study, soil enzyme activities such as cellulase, dehydrogenase, urease, protease, and phosphate activities and root cross-section area, total stele area and xylem vessel area were recorded higher for the formulation F1. The study demonstrated that the together Acinetobacter sp. 33 F and Comamonas sp. 51 F as formulation has higher plant growth-promoting activities as compared to the individual bacteria.

Cross-Reactivity of Prokaryotic 16S rDNA-Specific Primers to Eukaryotic DNA: Mistaken Microbial Community Profiling in Environmental Samples.

16S ribosomal RNA gene sequences are characteristically used as gold-standard genetic marker for the determination of bacterial and/or archaeal biodiversity, and community profiling of environmental samples. The 16S rRNA amplicon analysis till-date is taken as a standard method for investigation and identification of uncultivable bacteria in microbial diversity studies. The accuracy of these analyses strongly depends upon the choice of primers. It is presumed that these primers do not participate in non-specific amplifications. In the present study, by in silico, PCR and denaturing gradient gel electrophoresis (DGGE) analysis, we have shown that primers do cross-react with eukaryotic DNAs as well, eventually leading to overestimation of microbial biodiversity. We further demonstrated that the overestimation is not only due to cross-reaction with eukaryotic mitochondrial or plastid DNA, but also with eukaryotic chromosomal DNA, that is ubiquitous in environmental samples. We tried to establish methanogenic diversity in municipal solid waste (MSW) leachates and cow dung samples before and after enrichment of the prokaryotic DNA from eukaryotic ones. Results revealed that bands disappeared/get lightened in bacterial 16S rRNA-based DGGE community profiles, after prokaryotic DNA enrichment, but not in mcrA-based community profiles.

Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences.

Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about "methanogenic archaea composition" and "abundance" in the contrasting ecosystems like "landfill" and "marshland" may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

A multi-targeted approach to identify potential flavonoids against three targets in the SARS-CoV-2 life cycle.

The advent and persistence of the Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS-CoV-2)-induced Coronavirus Disease (COVID-19) pandemic since December 2019 has created the largest public health emergency in over a century. Despite the administration of multiple vaccines across the globe, there continues to be a lack of approved efficacious non-prophylactic interventions for the disease. Flavonoids are a class of phytochemicals with historically established antiviral, anti-inflammatory and antioxidative properties that are effective against cancers, type 2 diabetes mellitus, and even other human coronaviruses. To identify the most promising bioactive flavonoids against the SARS-CoV-2, this article screened a virtual library of 46 bioactive flavonoids against three promising targets in the SARS-CoV-2 life cycle: human TMPRSS2 protein, 3CLpro, and PLpro. By examining the effects of glycosylation and other structural-activity relationships, the presence of sugar moiety in flavonoids significantly reduces its binding energy. It increases the solubility of flavonoids leading to reduced toxicity and higher bioavailability. Through protein-ligand contact profiling, it was concluded that naringin formed more hydrogen bonds with TMPRSS2 and 3CLpro. In contrast, hesperidin formed a more significant number of hydrogen bonds with PLpro. These observations were complimented by the 100 ns molecular dynamics simulation and binding free energy analysis, which showed a considerable stability of docked bioflavonoids in the active site of SARS-CoV-2 target proteins. Finally, the binding affinity and stability of the selected docked complexes were compared with the reference ligands (camostat for TMPRSS2, GC376 for 3CLpro, and GRL0617 for PLpro) that strongly inhibit their respective SARS-COV-2 targets. Overall analysis revealed that the selected flavonoids could be potential therapeutic agents against SARS-CoV-2. Naringin showed better affinity and stability for TMPRSS2 and 3CLpro, whereas hesperidin showed a better binding relationship and stability for PLpro.

Comparative study on the degradation of dibutyl phthalate by two newly isolated Pseudomonas sp. V21b and Comamonas sp. 51F.

Dibutyl phthalate is (DBP) the top priority toxicant responsible for carcinogenicity, teratogenicity and endocrine disruption. This study demonstrates the DBP degradation capability of the two newly isolated bacteria from municipal solid waste leachate samples. The isolated bacteria were designated as Pseudomonas sp. V21b and Comamonas sp. 51F after scanning electron microscopy, transmission electron microscopy, Gram-staining, antibiotic sensitivity tests, biochemical characterization, 16S-rRNA gene identification and phylogenetic studies. They were able to grow on DBP, benzyl butyl phthalate, monobutyl phthalate, diisodecyl phthalate, dioctyl phthalate, and protocatechuate. It was observed that Pseudomonas sp. V21b was more efficient in DBP degradation when compared with Comamonas sp. 51F. It degraded 57% and 76% of the initial DBP in minimal salt medium and in DBP contaminated samples respectively. Kinetics for the effects of DBP concentration on Pseudomonas sp. V21b and Comamonas sp. 51F growth was also evaluated. Stoichiometry for DBP degradation and biomass formation were compared for both the isolates. Two major metabolites diethyl phthalate and monobutyl phthalates were identified using GC-MS in the extracts. Key genes were amplified from the genomes of Pseudomonas sp. V21b and Comamonas sp. 51F. DBP degradation pathway was also proposed.

Biodegradation of endocrine disruptor dibutyl phthalate (DBP) by a newly isolated Methylobacillus sp. V29b and the DBP degradation pathway.

Bacteria of the genus Methylobacillus are methanotrophs, a metabolic feature that is widespread in the phylum Proteobacteria. The study demonstrates the isolation and characterization of a newly isolated Methylobacillus sp. V29b. which grows on methanol, protocatechuate, monobutyl phthalate, dibutyl phthalate, diethyl phthalate, benzyl butyl phthalate, dioctyl phthalate and diisodecyl phthalate. Methylobacillus sp. V29b was characterized with scanning electron microscopy, transmission electron microscopy, Gram staining, antibiotics sensitivity tests and biochemical characterization. It degrades 70 % of the initial DBP in minimal salt medium and 65 % of the initial DBP in samples contaminated with DBP. DBP biodegradation kinetics was explained by the Monod growth inhibition model. Values for maximum specific growth rate (µ max) and half-velocity constant (K s) are 0.07 h-1 and 998.2 mg/l, respectively. Stoichiometry for DBP degradation was calculated for Methylobacillus sp. V29b. Four metabolic intermediates, dibutyl phthalate (DBP), monobutyl phthalate, phthalic acid and pyrocatechol, were identified. Based on the metabolic intermediates identified, a chemical pathway for DBP degradation was proposed. Six genes for phthalic acid degradation were identified from the genome of Methylobacillus sp. V29b.

Determination of Physico-chemical Parameters for Prediction of MSW Leachate Transport through Vadose Zone by Breakthrough Curve in a Realistic Undisturbed Soil Column.

The studies were carried out to find out transport parameters to predict leachate transport and contaminant plume profile underneath a municipal solid waste(MSW) dump. For this, both theoretical and practical avenues were explored. Batch experiments with disturbed soil were carried out with a tracer dye Brilliant Blue FCF to obtain the adsorption isotherm of the tracer. Column experiments with undisturbed realistic soil column were carried out to determine physico-chemical transport parameters using the tracer dye. MSW leachate transport were simulated and sensitivity analysis for MSW leachate transport was carried out for different Darcy velocity for pulse input.For determination of mass-transfer coefficient between the immobilized and mobilized water phase, we usedthe concept of number of transfer units (NTUs) and height of transfer units (HTUs) frequently used by Chemical Engineers. The obtained value of mass transfer coefficient calculated by this method was in excellent agreement with the value obtained from inverse calculation of parameter values by a standard software package (CXTFIT).