Biomolecular characterization of Antheraea mylitta cocoonase: A secreted protease.

Cocoonase is a protease secreted during the emergence of silk moths. In the present study cocoonase of Antheraea mylitta was collected, purified and secondary structure was determined using circular dichroism (CD) spectroscopy which revealed the presence of α-helix 4.3%, ß-sheet 55%, turn 8% and random coil 32.7%. The thermal stability of cocoonase was studied using CD spectroscopy while the thermal property was observed using Differential Scanning Calorimetry (DSC). Furthermore, MALDI-TOF peptide mass fingerprinting (PMF) was performed for similar protein identification using the MASCOT server. Using casein as the substrate, the kinetic constants Km and Vmax were 13 × 103 mg/ml and 15.09 × 10-2 µg/mg.s1 respectively. The specific activity of cocoonase was observed to be maximum at temperature 40 °C, pH-8.0. The effect of heavy metals Hg2+, Cd2+, Co2+, Pb2+ showed inhibitory activity at higher concentrations, while few metals like Mn2+, Fe3+ enhanced the activity while the effect of Ca2+ was not much on the activity. Soybean trypsin inhibitor and PMSF showed an inhibitory effect on the activity of cocoonase. Additionally, antioxidant scavenging and fibrinolytic properties were also observed. Furthermore, the imperative information generated through the present study will serve to explore cocoonase for its prospective pharmaceutical applications.

Karanjin, A Promising Bioactive Compound Possessing Anti-cancer Activity against Experimental Model of Non-small Cell Lung Cancer Cells.

AIMS: The aim of this study is to isolate the Millettia pinnata (Karanj) leaf extract for pure compound with anticancer properties and to study the molecular target of the isolates in non-small cell lung cancer cell lines. BACKGROUND: In our earlier research Millettia pinnata leaf extract has demonstrated potential anticancer activities. Thus, in pursuit of the bioactive compounds, the most potential active extract from our previous study was purified. Furthermore, the anticancer properties of the isolated compound karanjin was studied and aimed for apoptosis and restraining growth. METHODS: A novel method was developed through column chromatography for isolation and purification of the compound karanjin from leaf chloroform extract. The purified component was then characterised using FTIR, mass spectrometry, and NMR. An MTT-based cytotoxicity assay was used to analyse cell cytotoxicity, whereas fluorescence staining was used for apoptosis and reactive oxygen species inhibition quantification. Furthermore, the real-time PCR assay was used to determine the molecular mechanism of action in cells causing cytotoxicity induced by karanjin dosing. RESULTS: The anticancer activity of karanjin in A549 cell line exhibited prominent activity revealing IC50 value of 4.85 µM. Conferring the predicted molecular pathway study, karanjin restrains the proliferation of cancer cells through apoptosis, which is controlled by extrinsic pathway proteins FAS/FADD/Caspases 8/3/9. Downregulation of KRAS and dependent gene expression also stopped cell proliferation. CONCLUSION: Karanjin has been identified as a compound with potential effect in non-small cell lung cancer cells. Molecular mechanism for apoptosis and inhibition of reactive oxygen species induced through H2O2 were observed, concluding karanjin have medicinal and antioxidant properties.

Identification of cocoonase and cocoonase like protein using polyclonal antibody of Antheraea mylitta cocoonase.

BACKGROUND: Cocoonase is a proteolytic enzyme released by silk moths during pupal adult emergence. Without damaging the silk fibroin, this enzyme dissolves the shell of the tasar cocoon by exclusively targeting the protein sericin. Prior to this study, there was no available antibody against Antheraea mylitta cocoonase to identify or screen out similar variants or cocoonase like protein. RESULTS: In the present study, naturally secreted A. mylitta cocoonase was purified and used to immunize New Zealand white rabbits. The developed polyclonal antibody of cocoonase was purified and its specific interaction with cocoonase was determined using Indirect ELISA. The confirmation of its specificity and immuno-reactivity was evaluated by western blot using native cocoonase of tasar silkworm A. mylitta. The efficacy and specificity of the polyclonal antibody were further verified and confirmed by western blot which was performed to detect ten different ecotypes of A. mylitta cocoonase. CONCLUSION: The developed antibody successfully detected the cocoonase of different ecotypes. Thus, in future this antibody can serve as one of the molecular detection method for cocoonase and cocoonase-like proteins.

Mutational analysis of flavonol synthase of M. pinnata towards enhancement of binding affinity: a computational approach.

Millettia pinnata is an important medicinal plant that has been used as a treatment of various diseases due to presence of wide range of pharmacological properties. The plant contains quercetin, kaempferol, karanjin, pongaglabrone, kanjone, kanugin, gammatin, pongaglabol, and other bioflavonoids. Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, and antidiabetic activities etc. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols, i.e. biosynthesis of kaempferol from dihydrokaempferol. The current work examined the binding affinity-based approach to improve the enzyme catalytic activity using computational methods. Sequential site-directed mutagenesis was used to create four mutants with the goal to increase hydrogen bonds and further improving the ligand (dihydrokaempferol) binding efficiency. Simulations were done to monitor the stability of the mutants followed by molecular docking to confirm interactions with ligand. For structure validation, various dynamic analysis like RMSD, RMSF, ROG, SASA, H-bond, PCA, DCCM, and FEL were performed, which predicts the stability of wild-type (WT) proteins and mutants. The Mutant_2 and Mutant_3 showed maximum H-bonding and better stability than other mutants and WT that proved higher affinity suggesting improved catalysis. Mutant_2 and Mutant_3 exhibited binding affinities of -7.6 and -8.2 kcal/mol, respectively for the ligand. The outcome of present study will provide significant improvement in synthesis of kaempferol and other plant-based flavonoids.Communicated by Ramaswamy H. Sarma.

Molecular Beacon Probe (MBP)-Based Real-Time PCR.

In the advancement of molecular biology techniques, several probe-based techniques, like molecular beacon probe (MBP) assay, TaqMan probe, and minor groove binder (MGB) probe assay, have been reported to identify specific sequences through real-time polymerase chain reaction (PCR). All probe-based methods are more sensitive than the conventional PCR for the detection and quantification of target genes. MBP is a hydrolysis probe that emits fluorescence when getting the specific sequences on the gene. Here, we describe the application of MBP for the identification of the motif sequences present in the promoters of differentially expressed genes.

The molecular docking and molecular dynamics study of flavonol synthase and flavonoid 3'-monooxygenase enzymes involved for the enrichment of kaempferol.

Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, antidiabetic activities etc. It has been reported in many vegetables, fruits, herbs and medicinal plants. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols. Whereas flavonoid 3'-monooxygenase (F3'H, EC 1.14.14.82) catalyses the hydroxylation of dihydroflavonol, and flavonol. FLS is involved in the synthesis of the kaempferol whereas F3'H causes degradation of kaempferol. The present study aimed to analyse the binding affinity, stability and activating activity of enzyme FLS as well as inhibitory activity of enzyme F3'H involved in the enrichment of the kaempferol using the in-silico approaches. Computational study for physico-chemical properties, conserved domain identification, 3-D structure prediction and its validation, conservation analysis, molecular docking followed by molecular dynamics analysis of FLS and F3'H, protein-activator (FLS-LIG Complex) and protein-inhibitor (F3'H-LIG Complex) complexes have been performed. Other structural analyses like root mean square fluctuation (RMSF), root mean square deviation (RMSD), surface area solvent accessibility (SASA), radius of gyration (Rg), hydrogen bond analysis, principal component analysis (PCA), Poisson-Boltzmann analysis (MM_PBSA) and the dynamic cross correlation map (DCCM) analysis to explore the structural, functional and thermodynamic stability of the proteins and the complexes were also studied. The molecular docking result showed that FLS binds strongly with the activator ascorbate (CID _54670067) while F3'H binds with the inhibitor ketoconazole (CID_456201). The most powerful inhibitor (ketoconazole for F3'H) and activator (ascorbate for FLS) is determined by computing the thermodynamic binding free energy through MM_PBSA analysis. The current work provides wide-ranging structural and functional information about FLS and F3'H enzymes showing detailed molecular mechanism of kaempferol biosynthesis and its degradation and hence kaempferol enrichment. Finding of the present work opens up new possibilities for future research towards enrichment of kaempferol by using activator (ascorbate) for FLS and inhibitor (ketoconazole) for F3'H as well as for its large-scale production using in vitro approaches.Communicated by Ramaswamy H. Sarma.

Protein-protein docking and molecular dynamics studies of sericin and cocoonase of silkworm: an insight for cocoon softening.

Cocoonase is known to digest the sericin protein that encapsulates the silkworm cocoon's fibroin protein. Silk fibroin and sericin are two types of proteins that make up silk, and accounts for around 20-30% of the overall cocoon weight. The aim of the study was to see the protein-protein interaction (PPI) and molecular dynamic study of sericin, cocoonase and protein-protein docked complex of silkworm by computational approaches. Here motif analysis, phylogenetic analysis, principal component analysis, root-mean-square deviation (RMSD), root mean square fluctuation, radius of gyration, structural and functional study of cocoonase and sericin as well as molecular docking study were carried out. The 33 amino acid residues of cocoonase shows interaction with 38 aa residues of sericin involving 4 disulphide bonds, 22 hydrogen bonds and 319 non-bonded contacts. The confirmational stability and flexibility of both the proteins as well as protein-protein complex were achieved at 70 ns of MD simulation study. RMSD-based data indicated that cocoonase is more stable than sericin and complex, and complex has a greater fluctuation with more compact (higher Rg) value than cocoonase and sericin, inferring higher conformational stability and flexibility of protein-protein complex than cocoonase and sericin. This study provides a new dimension for PPI study by computational approaches.Communicated by Ramaswamy H. Sarma.

CRISPR/Cas technology: Opportunities for phytopathogenic viruses detection.

Detection and monitoring of viruses are essential for healthy plants and prosperity. Recent development in CRISPR/Cas system in diagnosis has open an avenue well suited for pathogen detection. Variety of CRISPR associated proteins are being discovered, suggesting array of application and detection strategies in diagnosis. Phytopathogenic viruses are diverse with respect to their nucleic acid compositions, which presents a challenge in developing a single device applicable for almost all viruses. The review describes about the efficient use of CRISPR/Cas Technology in diagnosis, such as SHERLOCK, DETECTR and SATORI. These methods are different in their characteristic to identify specific nucleic acids and processing the detectable signals. These technologies are in their infancy and lot of scope is there to develop commercial kits. Plant tissue culture-based industries, climate control green houses, indoor cultivation facilities etc. has been considered as few examples. This review will be beneficial for researchers seeking to develop detection mechanism based on CRISPR/Cas technology. The outcome in the form of cost-effective detection of viruses will be boon for agro-based industries, which are facing challenges through virus contamination.

Evaluating the role of trypsin in silk degumming: An in silico approach.

The trypsin being universal enzyme forming family of proteases catalyzes the hydrolysis of proteins into amino acids and regenerates the serine hydroxyl an active site. The trypsin enzyme from D. saccharalis, uses sericin as its preferred substrate. Presence of catalytic triad (serine, aspartic acid and histidine) at the substrate binding site of this enzyme is very important for the catalytic activity. In the current study, the interacting mechanism between the substrate sericin protein and enzyme trypsin protein were explored by integrating various computational approaches including physico-chemical properties, biophysical properties, dynamics, gene ontology, molecular docking, protein - protein interactions, binding free energy calculation and structural motifs were studied. The evolutionary study performed by MEGA X showed that trypsin protein sequence (ALE15212.1) is closely related to cocoonase protein sequence (ADG26770.1) from Antheraea pernyi. 3-D models of trypsin and sericin proteins were predicted using I-TASSER and further validated by PROCHECK, and ProSAweb softwares. The predicted trypsin structure model was assigned E.C. no. 3.4.21.4 which refers hydrolytic mechanism. Gene Ontology predicted by QuickGO showed that trypsin has serine hydrolase activity (GO: 00017171), and part of proteolysis (GO: 0006508) as well as protein metabolic process (GO:0019538) actvity. Molecular docking studies between trypsin and sericin proteins were conducted by the HADDOCK 2.4 having best docked protein complex with Z-score - 1.9. 2D and 3D protein-protein interaction was performed with LIGPLOT+ and HAWKDOCK, PDBsum, respectively. The amino acid residues interacting across proteins interface are sericin_chain A representing "Ser133, Tyr214, Thr188, Thr243, Ser225, Ser151, Ser156, His294, Arg293, Gly296″ and trypsin_chain B "Lys120, Tyr246, Asn119, Glu239, Ser62, Tyr194, Ile197, Ser171, Tyr169, Gly170″. Based on our results trypsin shows similarity with cocoonase and presumably trypsin can be used as an alternative source in cocoon degumming.

In silico structural and functional characterization of Antheraea mylitta cocoonase.

BACKGROUND: Cocoonase is a serine protease present in sericigenous insects and majorly involved in dissolving of sericin protein allowing moth to escape. Cocoon structure is made up of sericin protein which holds fibroin filaments together. Cocoonase enzyme hydrolyzes sericin protein without harming the fibroin. However, until date, no detailed characterization of cocoonase enzyme and its presence in wild silk moth Antheraea mylitta has been carried out. Therefore, current study aimed for detailed characterization of amplified cocoonase enzyme, secondary and tertiary structure prediction, sequence and structural alignment, phylogenetic analysis, and computational validation. Several computational tools such as ProtParam, Iterative Threading Assembly Refinement (I-TASSER), PROCHECK, SAVES v6.0, TM-align, Molecular Evolutionary Genetics Analysis (MEGA) X, and Figtree were employed for characterization of cocoonase protein. RESULTS: The present study elucidates about the isolation of RNA, cDNA preparation, PCR amplification, and in silico characterization of cocoonase from Antheraea mylitta. Here, total RNA was isolated from head region of A. mylitta, and gene-specific primers were designed using Primer3 followed by PCR-based amplification and sequencing. The newly constructed 377-bp length sequence of cocoonase was subjected to in silico characterization. In silico study of A. mylitta cocoonase showed 26% similarity to A. pernyi strain Qing-6 cocoonase using Blastp and belongs to member of chymotrypsin-like serine protease superfamily. From phylogenetic study, it was found that A. mylitta cocoonase sequence is closely related to A. pernyi cocoonase sequence. CONCLUSIONS: The present study revealed about the detailed in silico characterization of cocoonase gene and encoded protein obtained from A. mylitta head region. The results obtained infer the presence of cocoonase enzyme in the wild silkworm A. mylitta and can be used for cocoon degumming which will be a valuable and cost-effective strategy in silk industry.

ACC deaminase producing rhizobacterium Enterobacter cloacae ZNP-4 enhance abiotic stress tolerance in wheat plant.

Plant growth promoting rhizobacterium (PGPR) designated as ZNP-4, isolated from the rhizosphere of Ziziphus nummularia, was identified as Enterobacter cloacae following 16S rRNA sequence analysis. The isolated strain exhibited various plant growth promoting (PGP) traits. The 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) activity was evaluated under diverse physiological conditions that could be useful for minimizing the abiotic stress-induced inhibitory effects on wheat plants. The strain showed resistance to salt (NaCl) and metal (ZnSO4) stress. The effect of E. cloacae ZNP-4 on the augmentation of plant growth was studied under salinity stress of 150 mM (T1 treatment) & 200 mM (T2 treatment) NaCl. The inoculation of strain ZNP-4 significantly improved the various growth parameters of wheat plant such as shoot length (41%), root length (31%), fresh weight (28%), dry weight (29%), photosynthetic pigments chlorophyll a (62%) and chlorophyll b (34%). Additionally, the strain was found to be efficient for minimizing the imposed Zn stress in terms of improving plant growth, biomass and photosynthetic pigments in pots containing different levels of metal stress of 150 mg kg-1 (treatment T1) and 250 mg kg-1 (treatment T2). Isolate ZNP-4 also improved the proline content and decreased malondialdehyde (MDA) level under both salinity and metal stress, therefore maintaining the membrane integrity. Furthermore, bacterial inoculation increased the activities of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX). The positive effects of PGPR occurred concurrently with the decrease in abiotic stress-induced reactive oxygen species (ROS) molecules such as hydrogen peroxide (H2O2) and superoxide (O2-) contents. Overall, the observed results indicate that use of bacteria with such beneficial traits could be used as bio-fertilizers for many crops growing under stress conditions.

Computational models for prediction of protein-protein interaction in rice and Magnaporthe grisea.

Introduction: Plant-microbe interactions play a vital role in the development of strategies to manage pathogen-induced destructive diseases that cause enormous crop losses every year. Rice blast is one of the severe diseases to rice Oryza sativa (O. sativa) due to Magnaporthe grisea (M. grisea) fungus. Protein-protein interaction (PPI) between rice and fungus plays a key role in causing rice blast disease. Methods: In this paper, four genomic information-based models such as (i) the interolog, (ii) the domain, (iii) the gene ontology, and (iv) the phylogenetic-based model are developed for predicting the interaction between O. sativa and M. grisea in a whole-genome scale. Results and Discussion: A total of 59,430 interacting pairs between 1,801 rice proteins and 135 blast fungus proteins are obtained from the four models. Furthermore, a machine learning model is developed to assess the predicted interactions. Using composition-based amino acid composition (AAC) and conjoint triad (CT) features, an accuracy of 88% and 89% is achieved, respectively. When tested on the experimental dataset, the CT feature provides the highest accuracy of 95%. Furthermore, the specificity of the model is verified with other pathogen-host datasets where less accuracy is obtained, which confirmed that the model is specific to O. sativa and M. grisea. Understanding the molecular processes behind rice resistance to blast fungus begins with the identification of PPIs, and these predicted PPIs will be useful for drug design in the plant science community.

Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental.

BACKGROUND: Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. RESULTS: Predicted normalized B-factors of cocoonase and sericin with respect to α and ß regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25-26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. CONCLUSIONS: Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

Gene network modules associated with abiotic stress response in tolerant rice genotypes identified by transcriptome meta-analysis.

Abiotic stress tolerance is a complex trait regulated by multiple genes and gene networks in plants. A range of abiotic stresses are known to limit rice productivity. Meta-transcriptomics has emerged as a powerful approach to decipher stress-associated molecular network in model crops. However, retaining specificity of gene expression in tolerant and susceptible genotypes during meta-transcriptome analysis is important for understanding genotype-dependent stress tolerance mechanisms. Addressing this aspect, we describe here "abiotic stress tolerant" (ASTR) genes and networks specifically and differentially expressing in tolerant rice genotypes in response to different abiotic stress conditions. We identified 6,956 ASTR genes, key hub regulatory genes, transcription factors, and functional modules having significant association with abiotic stress-related ontologies and cis-motifs. Out of the 6956 ASTR genes, 73 were co-located within the boundary of previously identified abiotic stress trait-related quantitative trait loci. Functional annotation of 14 uncharacterized ASTR genes is proposed using multiple computational methods. Around 65% of the top ASTR genes were found to be differentially expressed in at least one of the tolerant genotypes under different stress conditions (cold, salt, drought, or heat) from publicly available RNAseq data comparison. The candidate ASTR genes specifically associated with tolerance could be utilized for engineering rice and possibly other crops for broad-spectrum tolerance to abiotic stresses.

Anticancer activity of plant leaves extract collected from a tribal region of India.

The goal of this research was to explore the preliminary anticancer properties of five plants namely Calotropis procera, Moringa oleifera, Millettia pinnata, Basela alba and Euphorbia neriifolia available in Jharkhand which is used for the medicinal purpose by local tribes. In the present study, plant leaves from five species were collected, dried and extracted with solvents of increasing polarity, followed by assessment of their cytotoxicity in A549 non-small-cell lung cancer cells. In the antimicrobial assay, the methanol extract of the M. pinnata leaves exhibited comparatively higher zone of inhibition of 0.7 ± 0.20 cm against a Salmonella typhi culture than the other extracts. M. pinnata leaves extract also displayed the maximum percentage inhibition in the DPPH, 83.97 ± 0.01 FRAP, 193.14 ± 3.01 mM assays. Furthermore, the cytotoxicity of the chloroform (37.45 ± 1.04) and ethyl acetate extracts (34.20 ± 0.81) of M. pinnata against A549 cells was found relatively higher with respect to another extract. In contrast, a study with the L132 normal epithelial lung cell line revealed less toxicity from the chloroform extract (0.33 ± 0.19) compared to the ethyl acetate extract (6.65 ± 0.59). Based on these findings, phytochemical investigation on chloroform and ethyl acetate extract of M. pinnata was performed using UPLC-ESI-MS/MS analysis revealing the presence of ß-sitosterol, lanceolatin B, karanjin, and stigmasterol. Congruently, a complete phytochemical and cytotoxic investigation of the M. pinnata extract constituents might infer the potency of this extract/s as anticancer, antioxidant and antimicrobial agents.

Method development for optimised green synthesis of gold nanoparticles from Millettia pinnata and their activity in non-small cell lung cancer cell lines.

Green synthesis of gold nanoparticles (GNPs) has received substantial attention, because nanoparticles are produced in an eco-friendly way using biomolecules present in plant extracts in a single step reaction. This research article highlights GNPs obtained using shade-dried leaf extracts of Millettia pinnata (L.) with aqueous auric chloride (HAuCl4) at ambient temperature. In the present study, GNPs with average particle size 37 nm in size were fabricated. Furthermore, the synthesis method to obtain stable and monodispersed GNPs was advanced by optimising enzyme concentration 100 µg/ml, pH 5.4, substrate concentration 0.45 mM and 12 h time of reaction. The confirmation of GNPs formation and characterisation was followed by UV-vis-absorption spectroscopy, dynamic light scattering (DLS), and zeta potential (ZP) for the analysis of shape, size, and stability, respectively. TEM images and powder XRD revealed the GNPs synthesis of spherical-shaped nanoparticles in the face-centred cubic arrangement. Cytotoxicity of GNPs was studied against A549 lung cancer cells with IC50 14.76 µg/ml and found lower as compared to doxorubicin IC50 11.23 µg/ml but significant enough to be used as a vehicle GNPs produced using green source can be used as significant therapeutic agents and drug delivery carriers.

Identification of miRNA, their targets and miPEPs in peanut (Arachis hypogaea L.).

MicroRNAs (miRNAs) are one of the major cytoplasmic tools employed by the eukaryotes for post-transcriptional gene regulation. These ˜21 nt small non-coding RNA molecules are highly conserved among species which forms a base for identification of new miRNAs. In this study, we used previously known mature miRNAs to search their homologs in Arachis hypogaea ESTs. A total of 50 non-protein coding sequences showing homology with no more than 3 mismatches were folded back to hairpin stem-loop structures using mfold. These predicted structures were passed through strict filtration criteria to obtain 18 miRNAs, all of which were other than those reported in miRBase. Out of 18 miRNAs, 7 were found to be new. These miRNAs belonged to miR156, miR166, miR167, miR319, miR398, miR399, miR482 and miR1507 family. These miRNAs were found to target a total of 118 genes in Arabidopsis. These targets included disease resistant proteins, auxin responsive proteins, squamosa promoter binding like proteins, co-transporter protein, transposable element genes, NAD(P) binding protein and topoisomerase II. KEGG pathway analysis showed potential involvement of these miRNAs in regulating different pathways. Apart from miRNA and their targets, microRNA encoded peptides (miPEPs) for 14 miRNAs were also identified. These findings can be used in the appropriate manipulation of miRNAs and corresponding miPEPs that will be helpful towards the peanut crop improvement.

Identification of GCC-box and TCC-box motifs in the promoters of differentially expressed genes in rice (Oryza sativa L.): Experimental and computational approaches.

The transcription factor selectively binds with the cis-regulatory elements of the promoter and regulates the differential expression of genes. In this study, we aimed to identify and validate the presence of GCC-box and TCC-box motifs in the promoters of upregulated differentially expressed genes (UR-DEGs) and downregulated differentially expressed genes (DR-DEGs) under anoxia using molecular beacon probe (MBP) based real-time PCR. The GCC-box motif was detected in UR-DEGs (DnaJ and 60S ribosomal protein L7 genes), whereas, the TCC-box was detected in DR-DEGs (DnaK and CPuORF11 genes). In addition, the mechanism of interaction of AP2/EREBP family transcription factor (LOC_Os03g22170) with GCC-box promoter motif present in DnaJ gene (LOC_Os06g09560) and 60S ribosomal protein L7 gene (LOC_Os08g42920); and TCC-box promoter motif of DnaK gene (LOC_Os02g48110) and CPuORF11 gene (LOC_Os02g01240) were explored using molecular dynamics (MD) simulations analysis including binding free energy calculations, principal component analyses, and free energy landscapes. The binding free energy analysis revealed that AP2/EREBP model residues such as Arg68, Arg72, Arg83, Lys87, and Arg90 were commonly involved in the formation of hydrogen bonds with GCC and TCC-box promoter motifs, suggesting that these residues are critical for strong interaction. The movement of the entire protein bound to DNA was restricted, confirming the stability of the complex. This study provides comprehensive binding information and a more detailed view of the dynamic interaction between proteins and DNA.