Designing a Multi-Epitope Subunit Vaccine against VP1 Major Coat Protein of JC Polyomavirus.

The JC polyomavirus virus (JCPyV) affects more than 80% of the human population in their early life stage. It mainly affects immunocompromised individuals where virus replication in oligodendrocytes and astrocytes may lead to fatal progressive multifocal encephalopathy (PML). Virus protein 1 (VP1) is one of the major structural proteins of the viral capsid, responsible for keeping the virus alive in the gastrointestinal and urinary tracts. VP1 is often targeted for antiviral drug and vaccine development. Similarly, this study implied immune-informatics and molecular modeling methods to design a multi-epitope subunit vaccine targeting JCPyV. The VP1 protein epitopic sequences, which are highly conserved, were used to build the vaccine. This designed vaccine includes two adjuvants, five HTL epitopes, five CTL epitopes, and two BCL epitopes to stimulate cellular, humoral, and innate immune responses against the JCPyV. Furthermore, molecular dynamics simulation (100 ns) studies were used to examine the interaction and stability of the vaccine protein with TLR4. Trajectory analysis showed that the vaccine and TLR4 receptor form a stable complex. Overall, this study may contribute to the path of vaccine development against JCPyV.

Identification and characterization of novel RdRp and Nsp15 inhibitors for SARS-COV2 using computational approach.

The World Health Organization has declared COVID-19 as a global health emergency. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and highlights an urgent need for therapeutics. Here, we have employed a series of computer-aided drug repurposing campaign to discover inhibitors of RNA dependent RNA polymerase (RdRp) and Nsp15/EndoU. Subsequently, MD simulation has been performed to observe dynamic behavior of identified leads at the active site of RdRp and Nsp15. We successfully identified novel lead molecule such as Alectinib for RdRp while Naldemedine and Ergotamine for NSP15. These lead molecules were accommodated in the active site of the enzyme and stabilized by the networks of the hydrogen bond, pi type and hydrophobic interaction with key residues of either target. Interestingly, identified compounds show molecular mimicry in terms of molecular interactions with key residues of RdRp and Nsp15 essential for catalysis and substrate interaction. Previously, Alectinib, Naldemedine and Ergotamine were used as drug in different diseases might be repurposed against selected protein targets of COVID19. Finally, we propose that the identified inhibitors represent a novel lead molecule to design a more effective inhibitor to stop the progress of pathogen.Communicated by Ramaswamy H. Sarma.

Identification and characterization of chlorine-resistant bacteria from water distribution sites of Mumbai.

Chlorination is the oldest and widely practiced method for disinfection of potable water across the globe but some microorganisms survive the chlorine treatment and become resistant. In this study, chlorine-resistant bacteria were isolated from 36 reservoirs of the Municipal Corporation of Greater Mumbai. Water was collected in winter, summer and rainy season. The 8 isolates (out of 89) found to be resistant to 20 ppm of chlorine were identified and belong to the Acinetobacter and Serratia sp. The antibiotic resistance profile showed that the isolates were resistant to a broad spectrum of antibiotics which is of concern. Biofilm production was also observed in most of the isolates. Presence of chlorine-resistant bacteria in drinking water is an alarming situation which needs further analysis especially to understand the further characteristics of these isolates for their antibiotic resistance.

Development and screening of byproduct for its secondary metabolites, antioxidant and anti-diabetic potential from anthracnose-infected fruits of pomegranate: a sustainable approach.

The main focus of the present study was to analyze the antioxidant and anti-diabetic potential of fermentative byproduct, developed from anthracnose-infected pomegranate fruits. The analysis of fermented juice showed a reduction in total phenolic content, total flavanoid content, anthocyanins, and antioxidant potential over the time in 6 months as compared to fresh juice, while total protein and alcohol percent (11%) were increased. Measurements of antioxidant activity by DPPH, ABTS, superoxide radical scavenging activity, and reducing power assays were highly correlated to total phenolic content, with corresponding R 2 values as r DPPH = 0.88, r ABTS = 0.90, r SRS = 0.67, r RPA = 0.80. High-performance liquid chromatography clearly revealed that the increment of antioxidant activity is associated with the release of gallic acid, vanillin, and ferulic acid. LC-MS analysis identified 1263 metabolites in fresh juice, 1580 metabolites after 1 month of fermentation, and 1063 metabolites after 6 months of fermentation. Most of the detected metabolites are linked with antioxidant, anti-diabetic, phenolics, flavanoids, cardiac glycosides, anticancer, and anti-vomiting activity. Mainly, naphthofluorescein, CAY10599, CAY10506, aminofluoropropionic acid, and 8-azaadenosine anti-diabetic compounds were found in fresh juice and fermented juice. Administration of fresh juice and fermented juice for 1 month helped in the reduction of blood plasma glucose level from 112.6 to 94.73 mg/dL before food and 142.43 to 133.20 mg/dL after food as compared to prescribed medicine. The sensory attributes of fermented juice were well appreciated for taste, after taste, and flavor. Further research is necessary to improve the quality and stability of metabolites during storage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02629-z.

Biotransformation of xenobiotics by hairy roots.

The exponential industrial growth we see today rides on the back of large scale production of chemicals, explosives and pharmaceutical products. However, the effluents getting released from their manufacturing units are greatly compromising the sustainability of our environment. With greater awareness of the imperative for environmental clean-up, a promising approach that is attracting increasing research interests is biodegradation of xenobiotics. In this approach, biotransformation has proven to be one of the most effective tools. While many different model frameworks have been used to study different aspects of biotransformation, hairy roots (HRs) have been found to be exceptionally valuable. HR cultures are preferred over other in-vitro model systems due to their biochemical stability and hormone-autotrophy. In addition, the multi-enzyme biosynthetic potential of HRs which is similar to the parent plant and their relatively low-cost cultural requirements further characterize their suitability for biotransformation. The recent progress observed in scale-up of HR cultures and understanding of functional genomics has opened up new dimensions providing valuable insights for industrial application. This review article summarizes the potential of HR cultures in the biotransformation of xenobiotics, their limitations in the application on a large scale and current strategies to alleviate them. Advancement in bioreactors engineering enabling large scale cultivation and modern gene technologies improving biotransformation efficiency promises to extend laboratory results to industrial applications.

Optimization of protein extraction and proteomic studies in Cenchrus polystachion (L.) Schult.

Apomicts have been studied at their genetic levels, but there are no any direct evidence of its mechanism. In order to understand the mechanism involved, a close relative of Pennisetum, Cenchrus polystachion, an apomictic species was explored for more insights into protein expression in reproductive structures. Optimization of protein extraction was studied with the leaf tissue and optimized protocol was extrapolated to other five tissues. The phenol-based protein extraction emerged as the best method for plant leaf tissue providing a better protein yield, separation of bands, removal of non-protein components like polyphenolic compounds and nucleic acids. The proteome analysis of leaf, stigma, immature ovary, seed, anther sac and pollen tissues of Cenchrus polystachion were carried out identifying a total of 135407 proteins against the Poaceae database from UNIPROT/TrEMBL. The target candidate proteins found in all the tissues were identified and mainly comprised of Actin Protein, PIP, Starch Synthase, ATP Synthase, Glutathione S Transferase, Dehydroascorbate reductase, Ascorbate peroxidase and heat shock proteins. Visualization and descriptive statistics conveyed all the necessary information to understand the differential expression of proteins in Cenchrus polystachion. This study forms a base to understand the role of tissue specific expressed proteins in an apomictic plant.

Biotransformation of chromium by root nodule bacteria Sinorhizobium sp. SAR1.

The present study aims to address the problem of chromium (Cr) toxicity by providing important insights into the mechanisms involved in its bioremediation. Among the 22 Rhizobium and Sinorhizobium isolates obtained from Sesbania sesban root nodules, Sinorhizobium sp. SAR1 (JX174035.1) tolerated the maximum Cr concentration (1mM) and hence was used for further studies. The excess secretion of extra polymeric substances, as seen from scanning electron micrographs, could be a probable mechanism of adaptation to the Cr stress. The Energy dispersive X-ray spectroscopy data did not show any peaks of Cr. The biosorption studies done on the isolate gave maximum adsorption capacity as 285.71mg/g. The isotherm studies showed a better fit to Langmuir isotherm. The Weber and Morris plot established that the phenomenon of adsorption was governed by film diffusion mechanism. The FTIR analysis suggested the role of cell wall components and extracellular polymeric substances in Cr adsorption to the biomass of Sinorhizobium. On the basis of these results a compiled mechanism of Cr (VI) adsorption and its biotransformation into Cr (III) by Sinorhizobium sp. SAR1 is explained. This work outlines a comprehensive detail for the exact phenomenon of Cr biotransformation by Sinorhizobium sp. SAR1. These results may further help in developing and enhancing effective bioremediation approaches.

Cancer Stem Cells: Acquisition, Characteristics, Therapeutic Implications, Targeting Strategies and Future Prospects.

Since last two decades, the major cancer research has focused on understanding the characteristic properties and mechanism of formation of Cancer stem cells (CSCs), due to their ability to initiate tumor growth, self-renewal property and multi-drug resistance. The discovery of the mechanism of acquisition of stem-like properties by carcinoma cells via epithelial-mesenchymal transition (EMT) has paved a way towards a deeper understanding of CSCs and presented a possible avenue for the development of therapeutic strategies. In spite of years of research, various challenges, such as identification of CSC subpopulation, lack of appropriate experimental models, targeting cancer cells and CSCs specifically without harming normal cells, are being faced while dealing with CSCs. Here, we discuss the biology and characteristics of CSCs, mode of acquisition of stemness (via EMT) and development of multi-drug resistance, the role of tumor niche, the process of dissemination and metastasis, therapeutic implications of CSCs and necessity of targeting them. We emphasise various strategies being developed to specifically target CSCs, including those targeting biomarkers, key pathways and microenvironment. Finally, we focus on the challenges that need to be subdued and propose the aspects that need to be addressed in future studies in order to broaden the understanding of CSCs and develop novel strategies to eradicate them in clinical applications. Graphical Abstract Cancer Stem Cells(CSCs) have gained much attention in the last few decades due to their ability to initiate tumor growth and, self-renewal property and multi-drug resistance. Here, we represent the CSC model of cancer, Characteristics of CSCs, acquisition of stemness and metastatic dissemination of cancer, Therapeutic implications of CSCs and Various strategies being employed to target and eradicate CSCs.

Identification and Structural Elucidation of New Process Impurity in Testosterone.

The main objective of this study is identification, isolation and characterization of potential impurity of testosterone. Testosterone was analyzed as per European pharmacopeial method for related substances by high-performance liquid chromatography (HPLC). One new unknown impurity was found along with other pharmacopeial impurities at a level more than 0.10%. This unknown impurity was isolated by preparative HPLC using Symmetry C18 column. The isolated impurity was spiked in sample containing impurities and was found to have the same retention time as that of this unknown impurity. This isolated impurity was fully characterized by different spectroscopic techniques such as LC-MS/MS, 1H, 13C NMR and DEPT as per the requirements of the ICH guidelines and was characterized as bisnoralcohol impurity.

Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments.

Pearl millet [Cenchrus americanus (L.) Morrone] is a staple food for more than 90 million farmers in arid and semi-arid regions of sub-Saharan Africa, India and South Asia. We report the ∼1.79 Gb draft whole genome sequence of reference genotype Tift 23D2B1-P1-P5, which contains an estimated 38,579 genes. We highlight the substantial enrichment for wax biosynthesis genes, which may contribute to heat and drought tolerance in this crop. We resequenced and analyzed 994 pearl millet lines, enabling insights into population structure, genetic diversity and domestication. We use these resequencing data to establish marker trait associations for genomic selection, to define heterotic pools, and to predict hybrid performance. We believe that these resources should empower researchers and breeders to improve this important staple crop.

In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia Pallens, an important medicinal plant.

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog's medium containing 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog's medium with respect to media containing 1 mg/L IBA or 1 mg/L NAA. Different parameters such as type of bacterial strains, OD600 of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying ß-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15 mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83 % transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/L) in selection media. T0 transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.

Comprehensive tissue-specific proteome analysis of drought stress responses in Pennisetum glaucum (L.) R. Br. (Pearl millet).

UNLABELLED: Pearl millet is the fifth most important cereal crop worldwide and cultivated especially by small holder farmers in arid and semi-arid regions because of its drought and salt tolerance. The molecular mechanisms of drought stress tolerance in Pennisetum remain elusive. We have used a shotgun proteomics approach to investigate protein signatures from different tissues under drought and control conditions. Drought stressed plants showed significant changes in stomatal conductance and increased root growth compared to the control plants. Root, leaf and seed tissues were harvested and 2281 proteins were identified and quantified in total. Leaf tissue showed the largest number of significant changes (120), followed by roots (25) and seeds (10). Increased levels of root proteins involved in cell wall-, lipid-, secondary- and signaling metabolism and the concomitantly observed increased root length point to an impaired shoot-root communication under drought stress. The harvest index (HI) showed a significant reduction under drought stress. Proteins with a high correlation to the HI were identified using sparse partial least square (sPLS) analysis. Considering the importance of Pearl millet as a stress tolerant food crop, this study provides a first reference data set for future investigations of the underlying molecular mechanisms. BIOLOGICAL SIGNIFICANCE: Drought stress is the most limiting factor for plant growth and crop production worldwide. At the same time drought susceptible cereal crops are among the largest producers worldwide. In contrast, Pearl millet is a drought and salt tolerant cereal crop especially used in arid and semi-arid regions by small farmers. The multifactorial molecular mechanisms of this unique drought tolerance are not known. Here, we employ shotgun proteomics for a first characterization of the Pearl millet drought stress proteome. The experimental setup and the data set generated from this study reveal comprehensive physiological and proteomic responses of the drought stressed Pearl millet plants. Our study reveals statistically significant tissue-specific protein signatures during the adaptation to drought conditions. Thus, the work provides a first reference study of the drought stress proteome and related drought responsive proteins (DRP's) in Pearl millet.

Enhanced production of an anti-malarial compound artesunate by hairy root cultures and phytochemical analysis of Artemisia pallens Wall.

Artemisinin and its derivatives are still one of the most effective drugs for the treatment of malaria. Artemisia pallens commonly known as Dhavanam, is an aromatic herb belonging to the family Asteraceae. Increasing the artemisinin content of A. pallens by genetic engineering would improve the availability of this much needed drug. In the present study, Agrobacterium rhizogenes (strain NCIM 5140) mediated genetic transformation of Artemisia pallens were carried out for hairy root induction. The effect of different media (Half MS, MS, MS along with BAP 0.5 mg/l and MS along with Kinetin 0.5 mg/l) and type of explants (leaf and stem) on hairy root induction and culture were also studied. Maximum transformation efficiency (70.0 %) was observed in case of stem explants when it was co-cultivated with Agrobacterium rhizogenes and kept on half strength MS media. Artesunate is a derivative of artemisinin, was quantified using HPLC from dried aerial extract and hairy roots. The content of artesunate in hairy roots was increased up to twofold as compared to aerial part of Artemisia pallens. The maximum amount of artesunate found in hairy roots was 5.62 ± 0.16 µg/g of dry weight. Apart from artesunate the other phytochemicals like alkaloids, polyphenols, and flavonoids are important because they impart the medicinal properties in this plant. Therefore, we have also quantified total alkaloids, flavonoids and polyphenolic content in the aerial part of the plants. The total alkaloids and flavonoids content were found 1.72 ± 0.00 mg/g dry weight in aqueous extract and 3.8 ± 0.00 mg/g in methanolic extract in terms of colchicine and rutin equivalents, respectively. Similarly, total phenolic content is 3.70 ± 0.01 mg/g in ethanolic extract in terms of tannic acid equivalent.

Characterization of Indian beers: chemical composition and antioxidant potential.

Chemical composition, antioxidant potential and corresponding lipid preoxidation of Indian commercial beers were evaluated. The presence of polyphenolic compounds such as tannic acid, gallic acid, catechol, vanillin, caffeic acid, quercetin, p-coumaric acid and rutin was quantified using LC-MS while the organic acids including tartaric, malic, acetic, citric and succinic acids were analysed using HPLC. Beer sample B8 had the greatest concentration of phenolic and flavonoid components (0.620 ± 0.084 mg/mL and 0.379 ± 0.020 mg/mL respectively) among the beer samples studied. The DPPH radical scavenging activity was observed in the range of 68.34 ± 0.85 % to 89.90 ± 0.71 % and ABTS radical cation scavenging activity was in the range of 59.75 ± 0.20 % to 76.22 ± 0.50 %. Percent protection in lipid peroxidation was quantified to be maximum (54.45 ± 3.39 %) in sample B5. Total phenolic content positively correlates with antioxidant assays, DPPH and ABTS (r = 0.35 and r = 0.58 respectively) with p < 0.001 and also with lipid peroxidation (r = 0.04) with p < 0.001. Negative correlation was observed between total flavonoid content with ABTS and lipid peroxidation (r = -0.1 and r = -0.05) respectively. The process of brewing warrants additional research to determine how the concentration of selected phenolic compounds can be increased.

Differential Expression of Antioxidant Enzymes During Degradation of Azo Dye Reactive black 8 in Hairy roots of Physalis minima L.

The enzymes involved in the protection of plant metabolism in presence of azo dye was characterized by studying activities of the role of antioxidant enzymes in the hairy roots (HRs) of Physalis minima L. during degradation of an azo dye, Reactive Black 8 (RB8). When the HRs were exposed to RB8 (30 mg L(-1)), a  nine fold increase in SOD activity was observed after 24 h, while 22 and 50 fold increase in activity was observed for POX and APX respectively after 72 h, whereas there was no significant change in activity of CAT. The activation of different antioxidant enzymes at different time intervals under dye stress suggests the synchronized functioning of antioxidant machinery to protect the HRs from oxidative damage. FTIR analysis confirmed the degradation of dye and the non-toxic nature of metabolites formed after dye degradation was confirmed by phytotoxicity study.

Hairy root induction and phytoremediation of textile dye, Reactive green 19A-HE4BD, in a halophyte, Sesuvium portulacastrum (L.) L.

In this study, we report phytoremediation of textile dyes using hairy roots derived through Agrobacterium rhizogenes (NCIM 5140) infection of in vitro leaf and stem explants of a halophyte Sesuvium portulacastrum (L.) L. Leaf explants showed higher frequency of hairy root induction (70%) than stem explants (30%), and maximum number of roots (leaf 42.3 ± 2.4 and stem 50.3 ± 1.7). Transformed nature of hairy roots was ascertained by amplifying 970 bp region of T-DNA of Ri plasmid. Hairy roots were screened for phytoremediation of various textile dyes and results showed that HRs were able to degrade Reactive green 19A HE4BD upto 98% within 5 days of incubation. Spectrophotometric analysis showed decrease in dye concentration while HPLC and FTIR analysis confirmed its degradation. Seed germination assay demonstrated non-toxic nature of the extracted metabolites. This is the first report on induction of hairy root culture in Sesuvium portulacastrum and phytoremediation of textile dyes.

Computer-aided drug designing.

Computer-aided drug designing has emerged as a cost-effective and rapid tool for the discovery of newer therapeutic agents. Several algorithms have been developed to analyze protein structure and function, to identify interacting ligands, active site residues, and to study protein-ligand interactions, which can eventually lead to the identification of new drugs. In silico drug designing involves identification of the target protein which is responsible for the development of the disease under study. The three-dimensional structure of the protein can be predicted using homology modeling, while molecular docking is applied to study the interaction of a drug molecule with the protein. The best orientation of the ligand-protein docked structure which has overall minimum energy needs to be obtained. In silico methods can be used to identify potential drugs for various diseases. Thus, computer-aided drug designing has become an indispensible and integral part of the drug discovery process.

A comparative study of karyomorphology among three populations of Garcinia indica (Clusiaceae) (Thomas-Dupetite) Choisy.

Garcinia indica is a tree species of the family Clusiaceae. This species is endemically distributed only in Ratnagiri and Sindhudurga district of Maharashtra. Plants collected in particularly Dive Agar and Sawantwadi show morphological variations. Mitotic chromosomes from three different population from the Konkan region were studied using propionic orcein stain. Chromosome numbers 2n = 54 is reported for all the populations. Three populations differed from each other in some aspects. Karyotype analysis showed that Deorukh population from Ratnagiri region showed one pair of satellite and one pair of secondary constrictions while Otawane population from Sawantwadi showed one satellite chromosome but differ in pattern of secondary constriction. Third, Diveagar population from Raigad district showed three pairs of satellite chromosomes and one pair of secondary constriction. So, it can be concluded that, plants from Bapat Garden of Dive Agar and Otawane region can be considered as cytotypes.

Biochemical characterization of laccase from hairy root culture of Brassica juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes.

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg(-1) of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg(-1) specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0-6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and L: -cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.

Degradation analysis of Reactive Red 198 by hairy roots of Tagetes patula L. (Marigold).

Tagetes patula L. (Marigold) hairy roots were selected among few hairy root cultures from other plants tested for the decolorization of Reactive Red 198. Hairy roots of Tagetes were able to remove dye concentrations up to 110 mg L(-l) and could be successively used at least for five consecutive decolorization cycles. The hairy roots of Tagetes decolorized six different dyes, viz. Golden Yellow HER, Methyl Orange, Orange M2RL, Navy Blue HE2R, Reactive Red M5B and Reactive Red 198. Significant induction of the activity of biotransformation enzymes indicated their crucial role in the dye metabolism. UV-vis spectroscopy, HPLC and FTIR spectroscopy analyses confirmed the degradation of Reactive Red 198. A possible pathway for the biodegradation of Reactive Red 198 has been proposed with the help of GC-MS and metabolites identified as 2-aminonaphthol, p-aminovinylsulfone ethyl disulfate and 1-aminotriazine, 3-pyridine sulfonic acid. The phytotoxicity study demonstrated the non-toxic nature of the extracted metabolites. The use of such hairy root cultures with a high ability for bioremediation of dyes is discussed.