In planta synthesis of silver nanoparticles and its effect on adventitious shoot growth and withanolide production in Withania somnifera (L.) Dunal.

Silver (Ag) is a non-essential heavy metal with substantial environmental toxicity but an excellent promotor for plant organogenesis. It is used as an elicitor for secondary metabolite production and for in planta synthesis of metal nanoparticles (MNPs). In the present study, the Ag accumulation and reduction capability of in vitro shoots of Withania somnifera and the toxicity and elicitation effect of Ag on in vitro shoots were explored. In vitro shoot cultures of W. somnifera were treated with different concentrations of silver nitrate for a specific treatment period. Growth index, withaferin A, elemental and electron microscopy analyses were done on silver-treated in vitro shoots of W. somnifera. 1 mM silver nitrate treatment for 12 days period was found to give increased growth index (1.425 ± 0.05c) and withaferin A (2.568 ± 0.08e mg g-1) content. The concentration of bioaccumulated Ag in 1 mM silver nitrate treated in vitro shoot was found to be 50.8 ppm. The presence of nano-Ag was also found in the leaves of 1 mM silver nitrate-treated in vitro shoots. In summary, this is the first report portraying the bioaccumulation and in planta reduction capability of the in vitro shoot system of W. somnifera, which makes it a potential medicinal plant of commercial value for silver contaminated soils.

Combined machine learning and pharmacophore based virtual screening approaches to screen for antibiofilm inhibitors targeting LasR of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a virulent pathogen affects patients with cystic fibrosis and nosocomial infections. Quorum sensing (QS) mechanism plays a crucial role in causing these ailments by mediating biofilm formation and expressing virulent genes. A novel approach to circumvent this bacterial infection is by hindering its QS network. Targeting LasR of las system serves beneficial as it holds the top position in QS system cascade. Here, we have integrated machine learning, pharmacophore based virtual screening, molecular docking and simulation studies to look for new leads as inhibitors for LasR. Support vector machine (SVM) learning algorithm was used to generate QSAR models from 66 antagonist dataset. The top three models resulted in correlation coefficient (R2) values of 0.67, 0.86, and 0.91, respectively. The correlation coefficient (R2test) values on external test set were found to be 0.62, 0.57, and 0.55, respectively. A four-point pharmacophore model was developed. The pharmacophore hypothesis AAAD_1 was used to screen for potential leads against MolPort database in ZincPharmer. The leads which showed predicted pIC50 value of >8.00 by SVM models were subjected to docking analysis that reranked the compounds based on docking scores. Four top leads namely ZINC3851967 N-[3,5-bis(trifluoromethyl)phenyl]-5-tert-butyl-6-chloropyrazine-2-carboxamide, ZINC4024175 4-Amino-1-[(2R,3S,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-oxopyrimidine-5-carbonitrile, ZINC2125703 N-[(5-Methoxy-4,7-dimethyl-2-oxo-2H-chromen-3-yl)acetyl]-beta-alanine, and ZINC3851966 N-[3,5-Bis(trifluoromethyl)phenyl]5-tert-butylpyrazine-2-carboxamide were selected. These compounds were checked for its stability by performing a molecular dynamics simulation for a period of 100 ns. The ADME properties of the leads were also determined. Hence, the compounds identified in this study can be used as possible leads for developing a novel inhibitor for LasR.Communicated by Ramaswamy H. Sarma.

Quantitative Proteomics Analysis for the Identification of Differential Protein Expression in Calf Muscles between Young and Old SD Rats Using Mass Spectrometry.

Aging is associated with loss of muscle mass and strength that leads to a condition termed sarcopenia. Impaired conditions, morbidity, and malnutrition are the factors of devaluation of muscle fibers in aged animals. Satellite cells play an important role in maintaining muscle homeostasis during tissue regeneration and repair. Proteomic profiling on the skeletal muscle tissues of different age group rats helps to determine the differentially expressed (DE) proteins, which may eventually lead to the development of biomarkers in treating the conditions of sarcopenia. In this study, nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis was implemented in the calf tissues of young and old groups of rats. The mass spectrometry (MS) analysis revealed the presence of 335 differentially expressed proteins between the two different age conditions, among which those based on log-fold change 25 proteins were upregulated and 77 were downregulated. The protein-protein interaction network analysis revealed 18 upregulated proteins with three distinct interconnected networks and 57 downregulated proteins with two networks. Further, gene ontology (GO) enrichment analysis showed the biological process, cellular component, and molecular function of the differential proteins. Pathway enrichment analysis of the DE proteins identified nine significantly enriched pathways with a list of eight significant genes (Cryab, Hspb2, Acat1, Ak1, Adssl1, Anxa5, Gys1, Ogdh, Gc, and Adssl1). Quantification of significant genes by quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the downregulation at the mRNA level. Western blot analysis of their protein expression showed concordant results on two candidate proteins (Ogdh and annexin 5) confirming their differential regulation between the two age groups of rats. Thus, these proteomic approaches on young and aged rats provide insights into the development of protein targets in the treatment of sarcopenia (muscle loss).

Marker aided introgression of opaque 2 (o2) allele improving lysine and tryptophan in maize (Zea mays L.).

Maize is the predominant food source for the world population, but lack of lysine and tryptophan in maize endosperm cannot fulfill the nutritional requirements of humans. Hence, the improvement of lysine and tryptophan content is the ultimate goal of maize biofortification programs. In the present study, the marker-assisted backcross (MABC) breeding strategy was used to enhance the lysine and tryptophan content of the elite maize inbred line UMI1230 by introgressing opaque 2 (o2) gene from the VQL1. During the transfer of the gene into UMI1230, SSR marker umc1066 tightly associated with o2 used for foreground selection. Background recovery was estimated using 168 SSR markers. Phenotype screening for morphological traits was adopted to choose plants parallel to UMI1230. As a result, four BC2F3 improved lines (DBT5-1-14/25-5/25-8/25-8/25, DBT5-1-14/25-5/25-8/25-7/25, DBT5-1-14/25-5/25-8/25-10/25 and DBT5-1-14/25-5/25-8/25-12/25) with o2 were developed. The improved line's background genome recovery varied between 90.60 and 94.80%. Also, the improved lines had better agronomic performance along with increased lysine (0.311-0.331%) and tryptophan (0.040-0.048%) contents. In summary, the MABC breeding strategy has successfully improved the levels of lysine and tryptophan in UMI1230 without affecting agronomic performance. The improved line's hold great potential as donors in biofortification programs in maize.

Compound Prunetin Induces Cell Death in Gastric Cancer Cell with Potent Anti-Proliferative Properties: In Vitro Assay, Molecular Docking, Dynamics, and ADMET Studies.

Gastric cancer is the common type of malignancy positioned at second in mortality rate causing burden worldwide with increasing treatment options. Prunetin (PRU) is an O-methylated flavonoid that belongs to the group of isoflavone executing beneficial activities. In the present study, we investigated the anti-proliferative and cell death effect of the compound PRU in AGS gastric cancer cell line. The in vitro cytotoxic potential of PRU was evaluated and significant proliferation was observed. We identified that the mechanism of cell death was due to necroptosis through double staining and was confirmed by co-treatment with inhibitor necrostatin (Nec-1). We further elucidated the mechanism of action of necroptosis via receptor interacting protein kinase 3 (RIPK3) protein expression and it has been attributed by ROS generation through JNK activation. Furthermore, through computational analysis by molecular docking and dynamics simulation, the efficiency of compound prunetin against RIPK3 binding was validated. In addition, we also briefed the pharmacokinetic properties of the compound by in silico ADMET analysis.

Withanolides against TLR4-Activated Innate Inflammatory Signalling Pathways: A Comparative Computational and Experimental Study.

Innate inflammations are dominant causes of poor health and high mortality. The pathogen-associated molecular pattern and lipopolysaccharide (LPS) are sensed by immune cells through activation of toll-like receptor 4 leading to mitogen-activated protein kinases (MAPKs) and NF-κB activations. Controlled MAPK and Nf-κB inhibitors have been proposed as potential antiinflammatory drugs. Withania somnifera is an important medicinal herb with known antiinflammatory activity. In this study, the selected Withania somnifera extracts and withanolides were analysed on LPS-induced macrophages comparatively. Molecular docking analysis revealed withaferin A, withanone and withanolide A as effective withanolides against inflammatory target molecules. In experiments, withaferin A and withanone treatment had prominent suppressions on LPS-induced expression of pro-inflammatory cytokines in bone marrow-derived macrophages. Withaferin A regulated all the major four pathways (MAPKs and NF-κB) involved in innate inflammations. Similarly among the Withania extracts analysed, the in vitro propagated leaf and field grown root extracts containing high withaferin A content suppressed the inflammatory molecules through NF-κB and MAPK pathways. Withaferin A was found to be best in suppressing the activated inflammatory pathways among all the analysed withanolides. Therefore, withaferin A and extracts with high withaferin A content can be used as promising drug candidates against innate inflammations. Copyright © 2016 John Wiley & Sons, Ltd.

Free radical scavenging activity and comparative metabolic profiling of in vitro cultured and field grown Withania somnifera roots.

Free radical scavenging activity (FRSA), total phenolic content (TPC), and total flavonoid content (TFC) of in vitro cultured and field grown Withania somnifera (Ashwagandha) roots were investigated. Withanolides analysis and comprehensive metabolic profiling between 100% methanol extracts of in vitro and field grown root tissues was performed using high performance thin layer chromatography (HPTLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Significantly higher levels of FRSA, TPC, and TFC were observed in in-vitro cultured roots compared with field grown samples. In addition, 30 day-cultured in vitro root samples (1 MIR) exhibited a significantly higher FRSA (IC50 81.01 µg/mL), TPC (118.91 mg GAE/g), and TFC (32.68 mg CE/g) compared with those in 45 day-cultured samples (1.5 MIR). Total of 29 metabolites were identified in in vitro cultured and field grown roots by GC-MS analysis. The metabolites included alcohols, organic acids, purine, pyrimidine, sugars, and putrescine. Vanillic acid was only observed in the in vitro cultured root samples, and higher level of the vanillic acid was observed in 1 MIR when compared to 1.5 MIR. Therefore, it is suggested that 1 MIR might serve as an alternative to field grown roots for the development of medicinal and functional food products.

Transcriptome analysis reveals in vitro cultured Withania somnifera leaf and root tissues as a promising source for targeted withanolide biosynthesis.

BACKGROUND: The production of metabolites via in vitro culture is promoted by the availability of fully defined metabolic pathways. Withanolides, the major bioactive phytochemicals of Withania somnifera, have been well studied for their pharmacological activities. However, only a few attempts have been made to identify key candidate genes involved in withanolide biosynthesis. Understanding the steps involved in withanolide biosynthesis is essential for metabolic engineering of this plant to increase withanolide production. RESULTS: Transcriptome sequencing was performed on in vitro adventitious root and leaf tissues using the Illumina platform. We obtained a total of 177,156 assembled transcripts with an average unigene length of 1,033 bp. About 13% of the transcripts were unique to in vitro adventitious roots but no unique transcripts were observed in in vitro-grown leaves. A putative withanolide biosynthetic pathway was deduced by mapping the assembled transcripts to the KEGG database, and the expression of candidate withanolide biosynthesis genes -were validated by qRT PCR. The accumulation pattern of withaferin A and withanolide A varied according to the type of tissue and the culture period. Further, we demonstrated that in vitro leaf extracts exhibit anticancer activity against human gastric adenocarcinoma cell lines at sub G1 phase. CONCLUSIONS: We report here a validated large-scale transcriptome data set and the potential biological activity of in vitro cultures of W. somnifera. This study provides important information to enhance tissue-specific expression and accumulation of secondary metabolites, paving the way for industrialization of in vitro cultures of W. somnifera.

Biodecolorization and biodegradation of reactive Levafix Blue E-RA granulate dye by the white rot fungus Irpex lacteus.

The treatment of effluents from textile industry with microorganisms, especially bacteria and fungi, has recently gained attention. The present study was conducted using white rot fungi Irpex lacteus, Trametes hirsuta, Trametes sp., and Lentinula edodes for the decolorization of reactive textile Levafix Blue E-RA granulate dye. I. lacteus resulted in the best decolorization and degradation of the dye within four days. Therefore, more detailed studies were carried out using I. lacteus. The decolorization was evaluated at various concentration, pH values, and temperatures. The activities of laccase, manganese peroxidase, and lignin peroxidase enzymes were estimated to reveal the roles of enzymes in decolorization. The colorless nature of the fungal cells revealed that decolorization occurred through degradation, and confirmed by analysis of the metabolites by UV-visible spectroscopy and High Performance Liquid Chromatography after decolorization. The metabolites were identified by Gas Chromatography-Mass Spectrometry, and functional group analysis was performed by Fourier Transform Infrared Spectroscopy. The degraded dye metabolites were assessed for phytotoxicity using Vigna radiata and Brassica juncea, which demonstrated nontoxic nature of the metabolites formed after degradation of dye.

Comparative root protein profiles of Korean ginseng (Panax ginseng) and Indian ginseng (Withania somnifera).

Ginsenosides and withanolides are the secondary metabolites from Panax ginseng and Withania somnifera, respectively. These compounds have similar biological properties. Two-dimensional electrophoresis (2-DE) analysis was utilized to reveal the protein profile in the roots of both plants, with the aim of clarifying similarly- and differentially-expressed proteins. Total proteins of Korea ginseng (P. ginseng) and Indian ginseng (W. somnifera) roots were separated by 2-DE using a pH 4-7 immobilized pH gradient strip in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The protein spots were visualized by silver staining. Twenty-one P. ginseng proteins and 35 W. somnifera proteins were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; of these, functions were ascribed to 14 and 22 of the P. ginseng and W. somnifera proteins, respectively. Functions mainly included general cell metabolism, defense and secondary metabolism. ATPase and alcohol dehydrogenase proteins were expressed in both plants. The results of this study, to our knowledge, are the first to provide a reference 2-DE map for the W. somnifera root proteome, and will aid in the understanding of the expression and functions of proteins in the roots of Korean ginseng and Indian ginseng.

The soybean F3'H protein is localized to the tonoplast in the seed coat hilum.

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3'-hydroxylase (F3'H) gene (sf3'h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3'h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3'h1 was not detected in To7G. A truncated sf3'h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3'H was detected using microsomal fractions from yeast transformed with sf3'h1 from To7B, but not from To7G. These results indicate that sf3'h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3'h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3'h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3'H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

Bioremediation of the heavy metal complex dye Isolan Dark Blue 2SGL-01 by white rot fungus Irpex lacteus.

The present study was conducted to evaluate the decolorization and degradation of the chromium metal complex dye Isolan Dark Blue 2SGL-01 by Irpex lacteus, a white rot lignolytic fungus. I. lacteus effectively decolorized the sulphonated reactive dye at a high concentration of 250 mg/l over a wide range of pH values of 5-9 and temperatures between 20 and 35°C. Complete (100%) decolorization occurred within 96h, and I. lacteus demonstrated resistance to the metallic dye. UV-vis spectroscopy, HPLC, GC-MS, and FT-IR analyses of the extracted metabolites confirmed that the decolorization process occurred due to degradation of the dye and not merely by adsorption. GC-MS analysis indicated the formation of 1(2H)-naphthalenone, 3,4-dihydro- and 2-naphthalenol as the main metabolite. ICP analysis demonstrated the removal of 13.49% chromium, and phytotoxicity studies using germinated seeds of Vigna radiata and Brassica juncea demonstrated the nontoxic nature of the metabolites formed during the degradation of Isolan Dark Blue 2SGL-01 dye.

Identification and characterization of class I chitinase in Panax ginseng C. A. Meyer.

The role of plant chitinases in protecting plants against a variety of fungal pathogens is well established. In the present study, a cDNA clone containing a class I chitinase (Chi-1) gene, designated as PgChi-1, has been isolated from the oriental medicinal plant Panax ginseng. PgChi-1 is predicted to encode a protein of 34.9 kDa consisting of 323 amino acid residues. PgChi-1 was found to be expressed constitutively in all of the studied organs of ginseng plant. Under various abiotic stress treatments including Cu, H2O2, mannitol, SA, JA, and NaCl, the expression of PgChi-1 in plantlets and hairy roots increased significantly compared to the control. When different parts of root were analyzed, maximum level was observed in taproot. In addition, levels of PgChi-1 expression were compared between healthy root and fungal, bacterial, and nematode infected root. Significant increase of PgChi-1 was noticed in pathogen infected roots than healthy roots. This study revealed that PgChi-1 may protect the P. ginseng under both biotic and abiotic stress conditions.

Generation and analysis of expressed sequence tags from leaf and root of Withania somnifera (Ashwgandha).

The first transcriptomes, expressed sequence tags (ESTs) in a leaf and root from Withania somnifera plant referenced in this report are the first of its kind. A cDNA library was constructed from samples of the 2-months-old, in vitro cultured leaves and roots, which generated 1,047 leaf cDNA and 1,034 root cDNA clones representing 48.5% and 61.5% unique sequences. The ESTs from leaf and root grouped into 239 and 230 clusters representing 22.8% and 22.2% of total sequences. Of these, about 70% encoded proteins found similar (E-value > or =10(-14)) to characterized or annotated proteins from the NCBI non-redundant database and diverse molecular functions and biological processes based on gene ontology (GO) classification. We identified genes with potential role in photosynthesis (cytochrome p-450), pathogenesis (arginine decarboxylase, chitinase) and withanolide biosynthesis (squalene epoxidase, CDP-ME kinase). Highly expressed transcripts, with a particularly high abundance of cytochrome p-450 (0.85% in leaf) were noticed. Pfam analysis revealed the presence of functional domains in selected sequences. W. somnifera is a source of multifarious and beneficial alkaloids referred as withanolides. High levels of withanolides accumulate in mature leaves and roots. Since, the knowledge for synthesis and presence of some of these important biochemical constituent is limited, identification of the genes involved in two different pathways of secondary metabolite synthesis (MVA and MEP), in different tissue will be requisite for articulation of withanolide biosynthesis. This investigation aimed at elucidating the differential gene expression in two vital sites where withanolides essentially found and leaf and root transcriptomes were comparatively analyzed. The comparative analysis of the sequences provides a framework for future research in proteomics and evolutionary genomics in the withanolide biosynthesis.