Pyramiding ascorbate-glutathione pathway in Lycopersicum esculentum confers tolerance to drought and salinity stress.

KEY MESSAGE: Stacking Glutathione-Ascorbate pathway genes (PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR) under stress inducible promoter RD29A imparts significant tolerance to drought and salinity stress in Solanum lycopersicum. Although the exposure of plants to different environmental stresses results in overproduction of reactive oxygen species (ROS), many plants have developed some unique systems to alleviate the ROS production and mitigate its deleterious effect. One of the key pathways that gets activated in plants is ascorbate glutathione (AsA-GSH) pathway. To demonstrate the effect of this pathway in tomato, we developed the AsA-GSH overexpression lines by stacking the genes of the AsA-GSH pathway genes isolated from Pennisetum glaucoma (Pg) including PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR under stress inducible promoter RD29A. The overexpression lines have an improved germination and seedling growth with concomitant elevation in the survival rate. The exposure of transgenic seedlings to varying stress regiments exhibited escalation in the antioxidant enzyme activity and lesser membrane damage as reflected by decreased electrolytic leakage and little accumulation of malondialdehyde and H2O2. Furthermore, the transgenic lines accumulated high levels of osmoprotectants with increase in the relative water content. The increased photosynthetic activity and enhanced gaseous exchange parameters further confirmed the enhanced tolerance of AsA-GSH overexpression lines. We concluded that pyramiding of AsA-GSH pathway genes is an effective strategy for developing stress resistant crops.

Fine-tuning the transcriptional regulatory model of adaptation response to phosphate stress in maize (Zea mays L.).

The post green revolution agriculture is based on generous application of fertilizers and high-yielding genotypes that are suited for such high input regimes. Cereals, like maize (Zea mays L.) are capable of utilizing less than 20% of the applied inorganic phosphate (Pi) - a non-renewable fertilizer resource. A greater understanding of the molecular mechanisms underlying the acquisition, transportation and utilization of Pi may lead to engineering genotypes with high phosphorus use efficiency. In this study, we carried out functional domain similarity analysis, promoter analysis and comparative transcriptional expression profiling of 12 selected Pi responsive genes in the Pi stress tolerant maize inbred line HKI-163 under sufficient and deficient Pi conditions. Pi starvation led to significant increase in root length; marked proliferation of root hairs and lesser number of crown roots. Eleven genes were significantly up or down regulated in Pi deficient condition. The putative acid phosphatase, ZmACP5 expression was up regulated by 162.81 and 74.40 fold in root and leaf tissues, respectively. The RNase, ZmRNS1 showed 115 fold up regulation in roots under Pi deprivation. Among the two putative high affinity Pi transporters ZmPht1;4 was found specific to root, whereas ZmPht2 was found to be up regulated in both root and leaf tissues. The genes involved in Pi homeostasis pathway (ZmSIZ1, SPX1 and Pho2) were up regulated in root and leaf. In light of the expression profiling of selected regulatory genes, an updated model of transcriptional regulation under Pi starvation in maize has been presented. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01155-x.

Biomarker study of the biological parameter and neurotransmitter levels in autistics.

Autism is a prevalent developmental disorder that combines repetitive behaviours, social deficits and language abnormalities. The present study aims to assess the autistic subjects using DSM IV-TR criteria followed with the analysis of neurotransmitters, biochemical parameters, oxidative stress and its ions in two groups of autistic subjects (group I < 12 years; group II ≥ 12 years). Antioxidants show a variation of 10% increase in controls compared to autistic age < 12 years. The concentration of pyruvate kinase and hexokinase is elevated in controls approximately 60% and 45%, respectively, with the significance of 95 and 99%. Autistic subjects showed marked variation in levels of neurotransmitters, oxidative stress and its related ions. Cumulative assessment of parameters related to biochemical markers and neurotransmitters paves the way for autism-based research, although these observations draw interest in an integrated approach for autism.

Scrutinizing the molecular, biochemical, and cytogenetic attributes in subjects with Rett syndrome (RTT) and their mothers.

Rett syndrome (RTT) is a stern dominant progressive neurological development disorder linked with X chromosome ranking second for mental slowdown, exclusively in females after few months of birth with normal development and growth period. Genetically any defects in universally expressed methyl-CpG binding protein 2 (MeCP2) transcription regulator gene are considered as radix for RTT in almost all the previous studies. Our study mainly focuses in unraveling the genetic alterations like identifying MeCP2 gene polymorphisms, chromosomal abnormalities, or X-chromosome inactivation (XCI) as underlying cause of RTT in prototypes sorted through Diagnostic and Statistical Manual of Mental Disorders-Text Revised (DSM IV). In addition, we have examined the probable surrogates of brain function disabilities like serotonin, homocysteine (Hcy), calcium, potassium, and lead from blood in both RTT porotypes and their mothers. In our investigation, we have observed varied amino acid substitution of MeCP2 and varied frequency of skewed XCI in RTT prototype. Our study validates that the demonstration of chromosomal analysis, biochemical analysis, and genomic observations helps in concluding RTT condition and can be helpful in providing appropriate treatment and counseling as well as improve the currently available protocol of diagnosis.

Insights into maize genome editing via CRISPR/Cas9.

Maize is an important crop for billions of people as food, feed, and industrial raw material. It is a prime driver of the global agricultural economy as well as the livelihoods of millions of farmers. Genetic interventions, such as breeding, hybridization and transgenesis have led to increased productivity of this crop in the last 100 years. The technique of genome editing is the latest advancement in genetics. Genome editing can be used for targeted deletions, additions, and corrections in the genome, all aimed at genetic enhancement of crops. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) system is a recent genome editing technique that is considered simple, precise, robust and the most revolutionary. This review summarizes the current state of the art and predicts future directions in the use of the CRISPR/Cas9 tool in maize crop improvement.

Glutathione reductase a unique enzyme: molecular cloning, expression and biochemical characterization from the stress adapted C4 plant, Pennisetum glaucum (L.) R. Br.

The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of ~53.5 kDa. The PgGR peptide exhibits 54-89% sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal- and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.

Enhancing C3 photosynthesis: an outlook on feasible interventions for crop improvement.

Despite the declarations and collective measures taken to eradicate hunger at World Food Summits, food security remains one of the biggest issues that we are faced with. The current scenario could worsen due to the alarming increase in world population, further compounded by adverse climatic conditions, such as increase in atmospheric temperature, unforeseen droughts and decreasing soil moisture, which will decrease crop yield even further. Furthermore, the projected increase in yields of C3 crops as a result of increasing atmospheric CO2 concentrations is much less than anticipated. Thus, there is an urgent need to increase crop productivity beyond existing yield potentials to address the challenge of food security. One of the domains of plant biology that promises hope in overcoming this problem is study of C3 photosynthesis. In this review, we have examined the potential bottlenecks of C3 photosynthesis and the strategies undertaken to overcome them. The targets considered for possible intervention include RuBisCO, RuBisCO activase, Calvin-Benson-Bassham cycle enzymes, CO2 and carbohydrate transport, and light reactions among many others. In addition, other areas which promise scope for improvement of C3 photosynthesis, such as mining natural genetic variations, mathematical modelling for identifying new targets, installing efficient carbon fixation and carbon concentrating mechanisms have been touched upon. Briefly, this review intends to shed light on the recent advances in enhancing C3 photosynthesis for crop improvement.

Phloem-specific expression of the lectin gene from Allium sativum confers resistance to the sap-sucker Nilaparvata lugens.

Rice production is severely hampered by insect pests. Garlic lectin gene (ASAL) holds great promise in conferring protection against chewing (lepidopteran) and sap-sucking (homopteran) insect pests. We have developed transgenic rice lines resistant to sap-sucking brown hopper (Nilaparvata lugens) by ectopic expression of ASAL in their phloem tissues. Molecular analyses of T0 lines confirmed stable integration of transgene. T1 lines (NP 1-2, 4-3, 11-6 & 17-7) showed active transcription and translation of ASAL transgene. ELISA revealed ASAL expression was as high as 0.95% of total soluble protein. Insect bioassays on T2 homozygous lines (NP 18 & 32) revealed significant reduction (~74-83%) in survival rate, development and fecundity of brown hoppers in comparison to wild type. Transgenics exhibited enhanced resistance (1-2 score) against brown hoppers, minimal plant damage and no growth penalty or phenotypic abnormalities.

CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.).

Weed infestation is a significant concern to crop yield loss, globally. The potent broad-spectrum glyphosate (N-phosphomethyl-glycine) has a widely utilized herbicide, acting on the shikimic acid pathway within chloroplast by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This crucial enzyme plays a vital role in aromatic amino acid synthesis. Repurposing of CRISPR/Cas9-mediated gene-editing was the inflection point for generating novel crop germplasm with diverse genetic variations in essential agronomic traits, achieved through the introduction of nucleotide substitutions at target sites within the native genes, and subsequent induction of indels through error-prone non-homologous end-joining DNA repair mechanisms. Here, we describe the development of efficient herbicide-resistant maize lines by using CRISPR/Cas9 mediated site-specific native ZmEPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-mZmEPSPS) with triple amino acid substitution. The novel triple substitution conferred high herbicide tolerance in edited maize plants. Transgene-free progeny harbouring the triple amino acid substitutions revealed agronomic performances similar to that of wild-type plants, suggesting that the GATIPS-mZmEPSPS allele substitutions are crucial for developing elite maize varieties with significantly enhanced glyphosate resistance. Furthermore, the aromatic amino acid contents in edited maize lines were significantly higher than in wild-type plants. The present study describing the introduction of site-specific CRISPR/Cas9- GATIPS mutations in the ZmEPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize.

Function identification and characterization of Oryza sativa ZRT and IRT-like proteins computationally for nutrition and biofortification in rice.

Zinc plays a very critical role and function in all organisms. Its deficiency can cause a serious issue. In Oryza sativa, the ZRT/IRT transporter-like proteins play a role in the zinc metal uptake and transport. Few OsZIPs genes have been validated and characterized for their biological functions and most of OsZIPs are not well physiologically, biochemically and phenotypically characterized. In the current study, they analyzed for their function through subcellular localization, phylogenetic analysis, homology modeling, expression analysis, protein-protein interaction (PPI) network prediction, and prediction of their binding sites. Hierarchical clustering of OsZIP genes based on different anatomical parts and developmental stages also orthologs prediction was identified. The presence of SNPs, SSRs, ESTs, FSTs, MPSS, and SAGE tags were analyzed for useful development of markers. SNPs were identified in all OsZIPs genes and each gene was further classified based on their number and position in the 3'UTR and 5'UTR regions of the gene-specific sequences. Binding clusters and their location on the protein sequences were predicted. We found Changing in residues number and position which were due to partial overlapping and sequence alignment, but they share the same mechanism of binding and transporting Zinc. A wide range of CRISPR Cas9 gRNAs was designed based on single nucleotide polymorphism (SNP) for each OsZIP transporter gene for well-function identification and characterization with genome-wide association studies. Hence this study would provide useful information, understanding, and predicting molecular insights for the future studies that will help for improvement of nutritional quality of rice varieties.Communicated by Ramaswamy H. Sarma.

Pseudomonas fluorescens imparts cadmium stress tolerance in Arabidopsis thaliana via induction of AtPCR2 gene expression.

BACKGROUND: Cadmium is a non-essential, third largest heavy metal contaminant with long retention time that poses environmental hazards. It emanating majorly from industrial processes and phosphate fertilizers. Cadmium is effortlessly assimilated by plants and leads to yield loss. Henceforth, identification of mechanisms to attenuate the heavy metal toxicity in crops is beneficial for enhanced yields. RESULTS: Beneficial soil bacteria have been known to combat both biotic and abiotic stress, thereby promoting plant growth. Amongst them, Pseudomonas fluorescens has been shown to enhance abiotic stress resistance in umpteen crops for instance maize and groundnut. Here, we investigated the role of P. fluorescens in conferring cadmium stress resistance in Arabidopsis thaliana. In silico analysis of PCR2 gene and promoter revealed the role, in cadmium stress resistance of A. thaliana. Real-time expression analysis employing qRT-PCR ratified the upregulation of AtPCR2 transcript under cadmium stress up to 6 folds. Total leaf (50%), biomass (23%), chlorophyll content (chlorophyll-a and b 40%, and 36 %) silique number (50%), and other growth parameters significantly improved on bacterial treatment of the 2mM Cd-stressed plants. CONCLUSION: Moreover, generated 35s-promoter driven AtPCR2 over-expressing transgenic lines that exhibited resistance to cadmium and other heavy metal stress. Taken together, a crucial interplay of P. fluorscens mediated enhanced expression of AtPCR2 significantly induced cadmium stress resistance in Arabidopsis plants.

Functional characterization of novel mutations in the conserved region of EPSPS for herbicide resistance in pigeonpea: structure-based coherent design.

Prospectively, agroecosystems for the growth of crops provide the potential fertile, productive, and tropical environment which attracts infestation by weedy plant species that compete with the primary crop plants. Infestation by weed is a major biotic stress factor faced by pigeonpea that hampers the productivity of the crop. In the modern era with the development of chemicals the problem of weed infestation is dealt with armours called herbicides. The most widely utilized, post-emergent, broad-spectrum herbicide has an essential active ingredient called glyphosate. Glyphosate mechanistically inhibits a chloroplastic enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by competitively interacting with the PEP binding site which hinders the shikimate pathway and the production of essential aromatic amino acids (Phe, Tyr, Trp) and other secondary metabolites in plants. Moreover, herbicide spray for weed management is lethal to both the primary crop and the weeds. Therefore, it is critical to develop herbicide-resistant crops for field purposes to reduce the associated yield and economic losses. In this study, the in-silico analysis drove the selection and validation of the point mutations in the conserved region of the EPSPS gene, which confers efficient herbicide resistance to mutated-CcEPSPS enzyme along with the retention of the normal enzyme function. An optimized in-silico validation of the target mutation before the development of the genome-edited resistant plant lines is a prerequisite for testing their efficacy as a proof of concept. We validated the combination of GATIPS mutation for its no-cost effect at the enzyme level via molecular dynamic (MD) simulation.Communicated by Ramaswamy H. Sarma.

HIGHLIGHTSWeed infestation is a major biotic stress factor and a consistent problem in agriculture.Development of glyphosate-resistant mutation is crucial to minimize the yield loss in agriculturally or nutritionally important crops for field application.Present in-silico approach is a proof-of-concept for validation of the selected glyphosate-resistant mutations.The current study has validated the combination of GATIPS mutation for its glyphosate-resistant phenotype and no negative cost effect at the enzyme simulation level.

CRISPR/Cas9-mediated homology donor repair base editing confers glyphosate resistance to rice (Oryza sativa L.).

Globally, CRISPR-Cas9-based genome editing has ushered in a novel era of crop advancements. Weeds pose serious a threat to rice crop productivity. Among the numerous herbicides, glyphosate [N-(phosphonomethyl)-glycine] has been employed as a post-emergent, broad-spectrum herbicide that represses the shikimate pathway via inhibition of EPSPS (5'-enolpyruvylshikimate-3-phosphate synthase) enzyme in chloroplasts. Here, we describe the development of glyphosate-resistant rice lines by site-specific amino acid substitutions (G172A, T173I, and P177S: GATIPS-mOsEPSPS) and modification of phosphoenolpyruvate-binding site in the native OsEPSPS gene employing fragment knockout and knock-in of homology donor repair (HDR) template harboring desired mutations through CRISPR-Cas9-based genome editing. The indigenously designed two-sgRNA OsEPSPS-NICTK-1_pCRISPR-Cas9 construct harboring rice codon-optimized SpCas9 along with OsEPSPS-HDR template was transformed into rice. Stable homozygous T2 edited rice lines revealed significantly high degree of glyphosate-resistance both in vitro (4 mM/L) and field conditions (6 ml/L; Roundup Ready) in contrast to wild type (WT). Edited T2 rice lines (ER1-6) with enhanced glyphosate resistance revealed lower levels of endogenous shikimate (14.5-fold) in contrast to treated WT but quite similar to WT. ER1-6 lines exhibited increased aromatic amino acid contents (Phe, two-fold; Trp, 2.5-fold; and Tyr, two-fold) than WT. Interestingly, glyphosate-resistant Cas9-free EL1-6 rice lines displayed a significant increment in grain yield (20%-22%) in comparison to WT. Together, results highlighted that the efficacy of GATIPS mutations in OsEPSPS has tremendously contributed in glyphosate resistance (foliar spray of 6 ml/L), enhanced aromatic amino acids, and improved grain yields in rice. These results ensure a novel strategy for weed management without yield penalties, with a higher probability of commercial release.

Cloning and comparative modeling identifies a highly stress tolerant Cu/Zn cytosolic super oxide dismutase 2 from a drought tolerant maize inbred line.

Plants have a complex system of stress response that deals with different types of stresses. Maize (Zea mays L.), one of the most important crops grown throughout the world, across a range of agro-ecological environments, employs complex mechanisms of gene regulation in response to drought stress. HKI 335 is a tropical maize inbred line showing remarkable adaptation to drought stress. Abiotic stresses, like drought, trigger the production of reactive oxygen species (ROS) due to the incomplete reduction or excitation of molecular oxygen, eventually leading to cell damage. Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme that acts as the first line of defense against ROS. We cloned the Sod2 gene from HKI 335 inbred line and analyzed its protein through detailed in silico characterization. Our comparative modeling revealed that at the level of tertiary structure, the HKI 335 SOD2 protein is highly similar to Potentilla atrosanguinea SOD2, which had been previously identified as highly thermostable SOD that can tolerate autoclaving as well as sub-zero temperatures. We performed phylogenetic analysis, estimated physicochemical properties, post-translational modifications, protein-protein interactions, and domain composition of this SOD2. The phylogenetic analysis showed that orthologous sequences of SOD from different species were clustered into two clusters. Secondary structure prediction indicates that SOD2 is a soluble protein and no transmembrane domains have been found. Most of the beta sheets have RSA value greater than 2. The Ramachandran plot from PDBsum revealed that most of the residues fall in the highly favored region. It was estimated that the value of the instability index was less than 40, the value of the aliphatic index was extremely high and the GRAVY value lies between -2 and +2. We could identify only one phosphorylation site, located at position 20 with a score of 0.692. Overall, the unique stress-tolerant properties of the HKI 335 SOD2, may be one of the reasons contributing to the high drought tolerance trait exhibited by HKI 335 maize inbred line. Further research may reveal more insights into the drought adaptation mechanism in maize and the eventual deployment of the trait in maize hybrids.

De novo genome assembly of rice bean (Vigna umbellata) - A nominated nutritionally rich future crop reveals novel insights into flowering potential, habit, and palatability centric - traits for efficient domestication.

Rice bean is a less-known underutilized legume crop with a high nutritional value among members of the Vigna family. As an initiative to compose rice bean (Vigna umbellata) genomic resource, the size of 414 mega-base pairs with an estimated identification of 31,276 high confidence index genes via 15,521 scaffolds generated from Illumina and PacBio platform 30X coverage data has achieved 96.08% functional coverage data from Illumina and PacBio platform. Rice bean genome assembly was found to be exquisitely close to Vigna angularis (experimental control/outgroup), Vigna radiata, and Vigna unguiculata, however, Vigna angularis being the closest. The assembled genome was further aligned with 31 leguminous plants (13 complete genomes and 18 partial genomes), by collinearity block mapping. Further, we predicted similar discriminant results by complete coding sequence (CDS) alignment. In contrast, 17 medically influential genomes from the National Institute of General Medical Sciences-National Institutes of Health NIGMS-NIH, when compared to rice bean assembly for LCB clusters, led to the identification of more than 18,000 genes from the entire selected medicinal genomes. Empirical construction of all genome comparisons revealed symplesiomorphic character in turn uncovering the lineage of genetic and functional features of rice beans. Significantly, we found deserving late-flowering genes, palatably indexed uncommon genes that regulate various metabolite pathways, related to abiotic and biotic stress pathways and those that are specific to photoperiod and disease resistance and so on. Therefore, the findings from this report address the genomic value of rice bean to be escalated via breeding by allied and applied approaches.

Expression of OsDREB2A transcription factor confers enhanced dehydration and salt stress tolerance in rice (Oryza sativa L.).

Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants improved stress tolerance indicating the DRE/DREB regulon is conserved across plants. We developed 30 transgenic T(0) rice plants overexpressing OsDREB2A which were devoid of any growth penalty or phenotypic abnormalities during stressed or non-stressed conditions. Integration of T-DNA in the rice genome and stress inducible overexpression of OsDREB2A had occurred in these transgenic lines. Functional analyses of T(1)-3 and T(1)-10 lines revealed significant tolerance to osmotic, salt and dehydration stresses during simulated stress conditions with enhanced growth performance as compared to wild type. OsDREB2A, thus, confers stress tolerance in homologous rice system that failed in the heterologous Arabidopsis system earlier.

Genotype-Independent Regeneration and Transformation Protocol for Rice Cultivars.

Developing an efficient and reproducible plant transformation protocol relies on callus induction and plant regeneration, which is prerequisite for genetic enhancement of crops, especially rice. The present study has been carried out in order to establish a genotype-independent regeneration and biolistic transformation protocol for rice varieties. Putative transgenic rice lines were confirmed by PCR analysis, DNA sequencing, and Southern analysis. The transformation protocol reported here is relatively simple and consistent and can be exploited in future biotechnological investigations particularly for gene transformation studies.

Phylogenetic and modelling analysis of purple acid phosphatase 18 (SiPAP18) from Setaria italica.

Purple acid phosphatases belong to metallo-phosphatase family. Intracellular phosphatases are crucial for phosphorus (P) distribution in the cell and are highly induced in phosphorus-deprived conditions in the soil. Disparate PAP isoforms exist within discrete subcellular compartments in Setaria italica and their expression in P deprived conditions fosters phosphorus amelioration. We isolated the SiPAP18 gene and developed the homology SiPAP18 protein model based on the crystal structure of the Kidney bean PvPAP (PDB ID: 2QFP) as template (sequence similarity 42.7%) using Modeller 9.12 with adequate validation. Structure model analysis shows the significance of five conserved signatures with seven metal-paired amino acid residues during P-deprivation induced phosphorus amelioration.

Structure-based virtual screening, pharmacokinetic prediction, molecular dynamics studies for the identification of novel EGFR inhibitors in breast cancer.

Breast cancer is one of the most prevalent malignancy cancer types especially affecting women globally. EGFR is a proto onco gene as well as the first identified tyrosine kinase receptor. It plays a dynamic role in many biological tasks such as apoptosis, cell cycle progression, differentiation, development and transcription. Somatic mutation in the EGFR kinase domain derails the normal kinase activity and over expression leads to the progression of cancer especially breast cancer. EGFR is one of the well-known therapeutic targets for breast cancer. In this scenario, we attempt to identify novel potent inhibitors of EGFR. Initially, we performed structure-based virtual screening and identified four potential compounds effective against EGFR. Further, the compounds were subjected to ADME prediction as part of evaluation of the druggability and all the four compounds found to fall under satisfactory range with predicted pharmacokinetic properties. Eventually, the conformational stability of protein-ligand complex was analyzed at different time scale by using Gromacs software. Molecular dynamics simulation run of 20 ns is carried out and results were analyzed using root mean square deviation (RMSD), root mean square fluctuation (RMSF) to signify the stability of protein-igand complex. The stability of the protein-ligand complex is more stable throughout entire simulation. From the results obtained from in silico studies, we propose that these compounds are exceptionally useful for further lead optimization and drug development.Communicated by Ramaswamy H. Sarma.

Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses.

Molecular chaperones (Hsps) have been shown to facilitate protein folding or assembly under various developmental and adverse environmental conditions. The aim of this study was to unravel a possible role of heat-shock proteins in conferring abiotic stress tolerance to plants. We isolated a cDNA encoding a cytoplasmic Hsp70 (PgHsc70) from Pennisetum glaucum by screening heat-stress cDNA library. PgHsc70 cDNA encoding 649 amino acids represents all conserved signature motifs characteristic of Hsp70s. The predicted molecular model of PgHsc70 protein suggests that the N-terminus ATP-binding region is evolutionarily conserved, in comparison to C-terminus peptide-binding domains. A single intron in ATPase domain coding region of PgHsc70 exhibited a high degree of conservation with respect to its position and phasing among other plant Hsp70 genes. Recombinant PgHsc70 protein purified from E. coli possessed in vitro chaperone activity and protected PgHsc70 expressing bacteria from damage caused by heat and salinity stress. Nucleotide sequence analysis of 5' flanking promoter region of PgHsc70 gene revealed a potential heat-shock element (HSE) and other putative stress-responsive transcription factor binding sites. Positive correlation existed between differentially up-regulated PgHsc70 transcript levels and the duration and intensity of different environmental stresses. Molecular and biochemical analyses revealed that PgHsc70 gene was a member of the Hsp70 family and suggested that its origin was from duplication of a common ancestral gene. Transcript induction data, presence of several putative stress-responsive transcription factor-binding sites in the promoter region of PgHsc70 and the presence of a protective in vitro chaperone activity of this protein against damage caused by heat and salinity, when expressed in E. coli, suggest its probable role in conferring abiotic stress tolerance to this plant.