Genome-wide association studies of seedling quantitative trait loci against salt tolerance in wheat.

Salinity is one of the significant factors in decreasing wheat yield and quality. To counter this, it is necessary to develop salt-tolerant wheat varieties through conventional and advanced molecular techniques. The current study identified quantitative trait loci in response to salt stress among worldwide landraces and improved varieties of wheat at the seedling stage. A total of 125 landraces and wheat varieties were subjected to salt treatment (50, 100, and 150 mM) with control. Morphological seedling traits, i.e., shoot length, root length, and fresh and dry shoot and root weights for salinity tolerance were observed to assess salt tolerance and genetic analysis using SNP data through DArT-seq. The results showed that, at the seedling stage, 150 mM NaCl treatment decreased shoot length, root length, and fresh and dry weights of the shoot and root. The root length and dry root weight were the most affected traits at the seedling stage. Effective 4417 SNPs encompassing all the chromosomes of the wheat genome with marker density, i.e., 37%, fall in genome B, genome D (32%), and genome A (31%). Five loci were found on four chromosomes 6B, 6D, 7A, and 7D, showing strong associations with the root length, fresh shoot weight, fresh root weight, and dry root weight at the p < 0.03 significance level. The positive correlation was found among all morphological traits under study.

In-silico Studies Calculated a New Chitin Oligomer Binding Site Inside Vicilin: A Potent Antifungal and Insecticidal Agent.

Vicilins are major seed storage proteins and show differential binding affinities toward sugar moieties of fungal cell wall and insect gut epithelium. Hence, purpose of study is the thorough in-silico characterization of interactions between vicilin and chitin oligomer followed by fungal and insecticidal bioassays. This work covers the molecular simulation studies explaining the interactions between Pisum sativum vicilin (PsV) and chitin oligomer followed by protein bioassay against different pathogens. LC-MS/MS of purified PsV (∼50 kDa) generated residual data along high pea vicilin homology (UniProtKB ID; P13918). Predicted model (PsV) indicated the characteristic homotrimer joined through head-to-tail association and each monomer is containing a bicupin domain. PsV site map analysis showed a new site (Site 4) into which molecular docking confirmed the strong binding of chitin oligomer (GlcNAc)4. Molecular dynamics simulation data (50 ns) indicated that chitin-binding site was comprised of 8 residues (DKEDRNEN). However, aspartate and glutamate significantly contributed in the stability of ligand binding. Computational findings were further verified via significant growth inhibition of Aspergillus flavus, A. niger, and Fusarium oxysporum against PsV. Additionally, the substantial adult population of Brevicoryne brassicae was reduced and different life stages of Tribolium castaneum also showed significant mortality.

Comprehensive Structural and Functional Characterization of a Seed γ-thionin as a Potent Bioactive Molecule Against Fungal Pathogens and Insect Pests.

BACKGROUND: Fungi and insect pests ruin stored crop grain, which results in millions of dollars of damage, presenting an ongoing challenge for farmers in addition to diminishing the safety of stored food. A wide-range defensive system against pathogens is needed to reduce or even eliminate the dependence of the crop yield upon the use of pesticides. Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) that are a component of the host defense system. They are known to interact with cell membranes to exhibit antifungal and insecticidal activity. They exhibit a broad range of activities against fungi and insects and are effective at low concentrations. Thionins act on membranes, greatly reducing the development of pathogen resistance. OBJECTIVE: The aim of this study is to investigate a bioactive molecule that acts against fungal pathogens and stored grain insect pests. METHODS: γ-thionin protein was extracted from Brassica oleracea L. var. capitata f. alba (white cabbage) seed powder in phosphate buffer (100 mM, pH 7.0) and was identified by MALDI-TOF/TOF. The crude extract was subjected to 70% ammonium sulfate saturation followed by gel filtration chromatography. The disc diffusion assay along with a microtiter bioassay was used to determine the antifungal activity of the protein against phytopathogenic fungi. The insecticidal efficacy was evaluated by feeding insect pests with food contaminated with the purified protein. Additionally, an in silico molecular structure prediction study of the protein was performed using Auto Dock Vina for molecular docking of the protein with either fungal membrane moieties or α-amylase from Tenebrio molitor L. MD simulations of protein-ligand complexes were conducted using Schrodinger's Desmond module. RESULTS: γ-Thionin (BoT) was purified from white cabbage seeds and showed 100% homology with thionin (Brassica oleracea L. var. viridis) and 80% homology with defensin-like protein 1 (Raphanus sativus L.), respectively. BoT significantly inhibited the mycelial growth of Aspergillus niger van Tieghem and Aspergillus flavus Link at a concentration of 2 µM. Similarly, 0.12 µM BoT treatment resulted in significant mortality of Tribolium castaneum Herbst and Sitophilus oryzae L. Molecular docking and MD simulation of BoT confirmed the strong binding affinity with fungal membrane moieties (phosphatidylinositol 4,5-bisphosphate and phosphatidic acid), which causes disruption of the cell membrane and leakage of the cellular contents, leading to cell death. BoT blocked the active site of α-amylase, and as a result of the inactivation of this gut enzyme, the digestive systems of insects were disturbed, resulting in their deaths. CONCLUSION: This study revealed that γ-thionin is a good antifungal and insecticidal agent that could be used as an alternate to fungicides and insecticides.

In Silico Analysis and Functional Characterization of Antimicrobial and Insecticidal Vicilin from Moth Bean (Vigna aconitifolia (Jacq.) Marechal) Seeds.

Vicilin has nutraceutical potential and different noteworthy medicative health-promoting biotic diversions, and it is remarkable against pathogenic microorganisms and insects. In this study, Vigna aconitifolia vicilin (VacV) has been identified and characterized from the seed of Vigna aconitifolia (Jacq.) Marechal (Moth beans). LC-MS/MS analysis of VacV provided seven random fragmented sequences comprising 238 residues, showing significant homology with already reported Vigna radiata vicilin (VraV). VacV was purified using ammonium sulfate precipitation (60%) followed by size exclusion chromatography on Hi-Load 16/60 Superdex 200 pg column and anion-exchange chromatography (Hi trap Q FF column). Purified VacV showed a major ~50 kDa band and multiple lower bands on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under both reduced and non-reduced conditions. After all, a three-dimensional molecular structure of VacV was predicted, which showed ß-sheeted molecular conformation similar to crystallographic structure of VraV. All Vicilins from V. aconitifolia and other plants were divided into six sub-groups by phylogenetic analysis, and VacV shared a high degree of similarity with vicilins of Vigna radiata, Pisum sativum, Lupinus albus, Cicer arietinum and Glycine max. Additionally, VacV (20 µg) has significant growth inhibition against different pathogenic bacteria along strong antifungal activity (50 µg). Likewise, VacV (3.0 mg) produced significant growth reduction in Rice Weevil Sitophilus oryzae larvae after 9 days compared with control. Furthermore, by using MMT assay, the cytotoxicity effect of VacV on the growth of HepG2 liver cancerous cells was tested. VacV showed cytotoxicity against the HepG-2 line and the acquired value was 180 µg after 48 h. Finally, we performed molecular docking against caspase-3 protein (PDB ID: 3DEI) for VacV bioactive receptor interface residues. Hence, our results reveal that VacV, has nutraceutical potential and moth beans can be used as a rich resource of functional foods.

Comparative Study to Characterise the Pharmaceutical Potential of Synthesised Snake Venom Bradykinin-Potentiating Peptides In Vivo.

BACKGROUND: Bradykinin-potentiating peptides (BPPs) are snake venom peptides inhibiting the angiotensin-converting enzyme (ACE). ACE plays an important role in the regulation of blood pressure. BPPs lead to the development of ACE inhibitors for the treatment of hypertension. OBJECTIVE: The objective of the present work was to carry out a comprehensive comparative study of four synthesised snake venom BPPs in vivo. METHODS: Four synthesised snake venom BPPs were administered to rats via the intraperitoneal route for 15 days at a fixed dose. Lisinopril was used as a comparative standard. Thirty male albino rats were divided into six groups: A, B, C, D, E (lisinopril), and F (control). Group F was maintained as the control group and given only saline. After 15 days, blood samples and tissues were removed for the study of selective biochemical parameters and histomorphometric analysis. Statistical evaluation of all results was also performed. RESULTS: The results indicated that peptide I, with the sequence ZSAPGNEAIPP, was highly toxic and adversely affected all the biochemical and histological parameters studied in this work. Peptide II (ZNWPHPQIPP) and peptide IV (ZQWAQGRAPHPP) showed lower toxicity. None of the BPPs raised the serum creatinine level and exhibited nephroprotective effects. Although lisinopril raised the creatinine level, it showed a protective role towards the pancreas and lungs in parallel. CONCLUSION: The present work shows that although there is a high sequence similarity between the four BPPs, their in vivo activity varies. The sequences of peptide II and peptide IV can be used to improve the design of current ACE inhibitors used for hypertension treatment.

Genome-wide association mapping of bread wheat genotypes using yield and grain morphology-related traits under different environments.

Depleting water resources and increasing global temperature due to climate change are major challenges to agriculture and food security worldwide. Deciphering the underlying mechanisms of traits contributing to grain development and yield is essential for the development of climate-resilient cultivars. Therefore, this study assessed 105 bread wheat genotypes grown under control, drought, and heat-stress conditions for two crop seasons and performed a genome-wide association study (GWAS) using a 90k SNP array. The genotypes showed significant trait differences under all environmental conditions. Highly significant variation was observed, with moderate (50.09%) to high (76.19%) heritability in the studied germplasms. The studied traits were all also significantly positively correlated. A total of 541 significant associations (p ≤ 10-3) between marker and trait (MTAs) were observed after crossing the FDR <0.05 threshold for all traits. Among these, 195, 179, and 167 significant MTAs were detected under control, drought, and heat-stress conditions, respectively. Under the control and drought conditions, pleiotropic loci BS00010616_51 and BS00010868_51 were observed on chromosomes 7B and 1B situated at 186.24 cM and 35.47 cM, respectively. Pleiotropic loci BS00010868_51, Kukri_c11154_1723, and Ex_c10068_1509 were identified on chromosomes 1B, 5B, and 2A, respectively, under control and heat stress conditions. A stable and consistent locus (Excalibur_c20796_395) on chromosome 7A, located at 372.34 cM, was also linked to grain morphology and yield-related attributes in control, drought, and heat-stress conditions. The results of the current study confirmed several previously reported MTAs for the traits under consideration and identified new MTAs under harsh climatic conditions. These SNPs will aid in the discovery of novel genes in wheat. SNPs showing significant associations may be used in marker-assisted selection and allow the development of drought- and heat-tolerant genotypes with high yields to address global food security concerns.

Antibiograms of Gut Flora of Poultry Farms Workers Reveal Higher Resistance Levels as Compared to Non-Workers.

Introduction: Antibiotics are being used in humans and animals for treatment and control of bacterial infections. Excessive use of antibiotics in the production of poultry is a popular practice, but it poses serious health issues by transferring resistance from farm to humans via food or direct exposure. Study Objective: The objective of this study was to carry out a comparison of the resistance and sensitivity profile of isolated isolates from sewage of toilets that were in use of workers inside the farm and from sewage of household toilets. Methodology: In this study, a total of 320 sewage samples were collected. The antibiotic susceptibility profile was checked by Kirby-Bauer disc diffusion method, and the statistical analysis was carried out by MS excel. Chi-square test was performed to determine whether the antibiograms from two sample types were statistically different from each other or not. Results: From 320 sewage samples, a total of 296 bacterial isolates were isolated among which the leading bacterium was E. coli. The proportion of resistance, ESBL production and MDR was significantly higher in bacteria isolated from sewage of toilets under use of poultry farm workers as compared to the sewage from domestic use toilets. Conclusion: Resistance significantly increased in the bacteria isolated from toilets under use of poultry farm workers as compared to the ones isolated from control sewage samples.

Eruca sativa seed napin structural insights and thorough functional characterization.

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC-MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

Intensive Poultry Farming Practices Influence Antibiotic Resistance Profiles in Pseudomonas aeruginosa Inhabiting Nearby Soils.

BACKGROUND: The drastic increase in use of antibiotics as a mandatory part of production in poultry and livestock has led to the development of bacterial resistance against antibiotics. The spread of resistant bacteria from poultry to humans increases the risk of treatment failure by antibiotics because of resistance genes transfer. STUDY OBJECTIVE: The objective of the study was to estimate and compare the P. aeruginosa resistance profile collected from areas around the poultry farm premises and areas at least 500 meters away from the nearest poultry farm. We studied the effect of antibiotic usage in farms on the bacterial profile present in the upper layer of soil. METHODOLOGY: A total of 1,200 moist soil samples were collected from areas within a 25 meters range of poultry farms and areas that had no poultry farms in its 500 meters vicinity. P. aeruginosa was cultured and isolated. The antibiotic susceptibility profile was carried out by Kirby-Bauer disc diffusion method and results were analyzed according to CLSI guidelines. Statistical analysis was carried out to check the significance of results. RESULTS: A total of 300 P. aeruginosa isolates were isolated, among which 140 isolates were isolated from areas around the poultry farm premises and had higher prevalence of antibiotic resistance. A total of 160 isolates were isolated from areas outside the poultry farm range. Resistance was not as high as in the isolates from around the farm. The ESBL production was higher in the isolates that were in close contact with the poultry farm as compared to the isolates away from the farm. CONCLUSION: Use of antibiotics in the poultry farm for production significantly increases the resistance in bacterial strains present in the upper layer of soil around the poultry farm within at least a 25 meter range.

Investigations on Acinetophage, QAB 3.4, Targeting Extensively Drug-Resistant Acinetobacter baumannii Isolates.

PURPOSE: Drug resistance against antimicrobials is on the rise at alarmingly high rates. Acinetobacter baumannii is one of the six ESKAPE pathogens which are a significant "one health" issue. Clinical isolates of A. baumannii exhibit MDR phenotype mostly and infrequently the XDR and PDR phenotype. As a result, these infections have one of the highest mortality rates in hospitals. Alternative therapies are urgently needed. METHODS: Various phages were enriched against XDR clinical strain of A. baumannii. A potent phage, QAB 3.4, was further tested against 100 clinical strains. Because of its broad lytic activity, it was further tested for stability, resistance development and as an infection control agent. RESULTS: Phage QAB 3.4 showed broad lytic activity against 100 MDR and XDR clinical isolates representing a wide diversity of infection sites. Assays conducted to document the phage's stability, and ability of clinical isolates to develop resistance against it, showed promising outcomes for its potential use in clinical applications. Phage QAB 3.4 was able to eradicate A. baumannii from pre-inoculated solid surfaces. It provides a proof of concept that phages can be used as environmentally friendly infection control agents. CONCLUSION: We propose the phage QAB 3.4 is a promising candidate for further pre-clinical and clinical studies to test its biosafety and efficacy.

Bioherbicidal Activity and Metabolic Profiling of Potent Allelopathic Plant Fractions Against Major Weeds of Wheat-Way Forward to Lower the Risk of Synthetic Herbicides.

The productivity of major field crops is highly compromised due to weed infestation. Inefficient weed management practices and undue and excessive use of chemical herbicides have drastically contaminated the environment and human health, in addition to resistance development in weed species. Therefore, utilization of allelopathic plants to explore phytochemicals as potent organic alternatives to such chemical herbicides has become indispensable. The current study evaluates the comparative bio-herbicidal potential of methanolic extracts of castor (Ricinus communis), artemisia (Artemisia santolinifolia), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) to suppress growth of major weeds, i.e., wild mustard (Sinapis arvensis), Italian ryegrass (Lolium multiflorum), and carrot grass (Parthenium hysterophorus). The results demonstrated a concentration-dependent effect on weeds' growth. Overall, in vitro seed germination was reduced from 60 to 100% in response to 5% (w/v) extract concentration. Significant reduction in radicle length, hypocotyl length, and fresh biomass of the weeds was also observed. A strong inhibitory effect was seen in in vivo pot experiments, revealing that application of 10-20% methanolic extracts induced permanent wilting and substantial reduction in the chlorophyll content of weeds along with 20-80% increase in oxidative stress. Artemisia showed the most significant allelopathic effect, on account of highest phenolic and flavonoid contents, followed by castor, wheat, and sorghum, against S. arvensis, L. multiflorum, and P. hysterophorus, respectively. Phytochemical analysis, through high-performance liquid chromatography (HPLC), also exhibited a correlation between extract's phytotoxicity and their antioxidant potential due to their major constituents (rutin, quercetin, catechin, gallic acid, vanillic acid, syringic acid, ferulic acid, p-hydroxy benzoic acid, p-coumaric acid, and sinapic acid), among the total of 13 identified in methanolic fractions. Comprehensive profiling of allelochemicals with liquid chromatography-mass spectrometry (LC-MS) determined 120, 113, 90, and 50 derivates of phenolic acids, flavonoids, and alkaloids, reported for the first time through this study, demonstrating significant allelopathic potential of the targeted plant fractions, which can be explored further to develop a sustainable bio-herbicidal formulation.

The response of culturally important plants to experimental warming and clipping in Pakistan Himalayas.

The relative effects of climate warming with grazing on medicinally important plants are not fully understood in Hindukush-Himalaya (HKH) region. Therefore, we combined the indigenous knowledge about culturally important therapeutic plants and climate change with experimental warming (open-top chambers) and manual clipping (simulated grazing effect) and compared the relative difference on aboveground biomass and percent cover of plant species at five alpine meadow sites on an elevation gradient (4696 m-3346 m) from 2016-2018. Experimental warming increased biomass and percent cover throughout the experiment. However, the interactive treatment effect (warming x clipping) was significant on biomass but not on percent cover. These responses were taxa specific. Warming induced an increase of 1 ± 0.6% in Bistorta officinalis percent cover while for Poa alpina it was 18.7 ± 4.9%. Contrastingly, clipping had a marginally significant effect in reducing the biomass and cover of all plant species. Clipping treatment reduced vegetation cover & biomass by 2.3% and 6.26%, respectively, but that was not significant due to the high variability among taxa response at different sites. It was found that clipping decreased the effects of warming in interactive plots. Thus, warming may increase the availability of therapeutic plants for indigenous people while overgrazing would have deteriorating effects locally. The findings of this research illustrate that vegetation sensitivity to warming and overgrazing is likely to affect man-environment relationships, and traditional knowledge on a regional scale.

Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles.

Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.

Model prediction of a Kunitz-type trypsin inhibitor protein from seeds of Acacia nilotica L. with strong antimicrobial and insecticidal activity.

A Kunitz-type trypsin inhibitor protein has been purified and characterized from seeds of Acacia nilotica L. LC-MS/MS analysis of Acacia nilotica trypsin inhibitor (AnTI) provided the N-terminal fragment of 11 amino acids which yielded 100% identity with already reported Kunitz-type trypsin inhibitor protein of Acacia confusa (AcTI) in UniProtKB database search. SDS-PAGE showed a single band of ~21 kDa under nonreduced condition and appearance of a daughter band (17 kDa) in the presence of ß-mercaptoethanol indicating the presence of interchain disulfide linkage typical for Kunitz-type trypsin inhibitors. AnTI was purified from seed extract by using a combination of anion exchange and gel filtration chromatography. Since AnTI showed maximum homology with AcTI, a molecular structure of AcTI was predicted which showed highly ß-sheeted molecular conformation similar to crystallographic structure of Enterolobium contortisiliquum trypsin inhibitor (EcTI). AnTI (20 µg) produces significant population inhibition against different human pathogenic bacteria along strong antifungal activity (50 µg). Entomotoxin potential of AnTI was evaluated against two stored grain insect pests Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera) and Sitophilus oryzae (Linnaeus) (Curculionidae: Coleoptera). Statistically significant mortality of T. castaneum adults was observed at 1.5 mg after 15 days in comparison to control. Additionally, number of total eggs, larvae, pupae, adults, and their male/female ratio were also severely reduced in comparison to control. Similarly, two generation progeny of S. oryzae was studied after mixing AnTI with rice kernels. Mean percent mortality of adult population was significantly higher after 9 days of exposure in comparison to control group. AnTI significantly reduced the F1 generation while little mortality was observed for F2 generation. Exploration of such potent molecules is the prerequisite of our time regarding the anticipation of postantibiotic era and the development of insect resistance against chemical pesticides.

Graphene quantum dots as cysteine protease nanocarriers against stored grain insect pests.

Storing grains remain vulnerable to insect pest attack. The present study developed a biopesticide using biomolecules and their encapsulation in nanoparticles. A 25 kDa cysteine protease extracted from seeds of Albizia procera (ApCP) was encapsulated in graphene quantum dots (GQDs). The insecticidal activity of ApCP, with or without GQDs, against two stored grain insect pests, Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius) was explored. Insects were exposed to three concentrations 7.0, 3.5 and 1.7 mg of ApCP per a gram of wheat flour and grains. The insecticidal activity of ApCP encapsulated with GQDs was improved compared to that of ApCP without GQDs for both insect pests. The number of eggs and larvae of T. castaneum was reduced by 49% and 86%, respectively. Larval mortality was increased to 72%, and adult eclosion of T. castaneum was reduced by 98% at a 7.0 mg/g concentration of ApCP with GQDs compared to that of ApCP without GQDs. Exposure to 7.0 mg/g ApCP with GQDs, the number of R. dominica eggs and larvae was reduced by 72% and 92% respectively, larval mortality was increased by 90%, and eclosion was reduced by 97%. The extraction, purification, characterization, quantification and encapsulation of ApCP with GQDs were also studied. Cysteine protease nanocarriers have the potential to control stored grain insect pests.

Genome-wide identification, classification, expression profiling and DNA methylation (5mC) analysis of stress-responsive ZFP transcription factors in rice (Oryza sativa L.).

Cytosine DNA methylation (5mC) is an epigenetic mark that regulates gene expression in plant responses to environmental stresses. Zinc-finger protein (ZFP) is the largest family of DNA-binding transcription factors that also plays an essential role in eukaryote. In plant we have already identified and characterized different useful ZFP-genes. While, the main objective of this research was to observe and identify more targeted stress responsive genes of ZFPs epigenetically throughout genome in rice for the first time. A comprehensive correlation analysis was performed through methylated DNA immunoprecipitation (MeDIP)-chip hybridization in rice under salt and osmotic stresses. High salinity and drought are two major abiotic hazards that are destroying the crop world-wide. As a result, Through-out genome 14 unique stress responsive transcription factors of ZFP-genes with varying level of methylation and expression under two conditions (control vs. stress) were isolated. All the identified genes were confirmed from different databases for their specific structure, cis-regulatory elements, phylogenetic analysis, and synteny analysis. Moreover, the tissue-specific expression patterns, and expression under abiotic and phytohormones stresses were also investigated. Phylogenetically all the genes were divided into 6 distinct subgroups with Arabidopsis and orthologous proteins were find-out through synteny analysis. Available RNA-seq data in response to various phytohormones provided hormone inducible gene expression profile. Through Reverse Transcriptase qPCR (RT-qPCR) analysis tissue-specific expression in shoot and root over various time points against salt and osmotic stresses exhibited the diverse expression patterns of identified genes. Overall, the present study providing a foundation for in-depth characterization of identified genes and to further understand the epigenetic role of DNA methylation for genes expression and environmental stresses regulation in higher plant.

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus.

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl ß-d-N,N',N''-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (ß/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 µg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 µg/well Similarly, insect toxicity bioassays of GgChi (50 µg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase.

Snake Venom Peptides: Tools of Biodiscovery.

Nature endowed snakes with a lethal secretion known as venom, which has been fine-tuned over millions of years of evolution. Snakes utilize venom to subdue their prey and to survive in their natural habitat. Venom is known to be a very poisonous mixture, consisting of a variety of molecules, such as carbohydrates, nucleosides, amino acids, lipids, proteins and peptides. Proteins and peptides are the major constituents of the dry weight of snake venoms and are of main interest for scientific investigations as well as for various pharmacological applications. Snake venoms contain enzymatic and non-enzymatic proteins and peptides, which are grouped into different families based on their structure and function. Members of a single family display significant similarities in their primary, secondary and tertiary structures, but in many cases have distinct pharmacological functions and different bioactivities. The functional specificity of peptides belonging to the same family can be attributed to subtle variations in their amino acid sequences. Currently, complementary tools and techniques are utilized to isolate and characterize the peptides, and study their potential applications as molecular probes, and possible templates for drug discovery and design investigations.

Isolation and characterization of Bradykinin potentiating peptides from Agkistrodon bilineatus venom.

BACKGROUND: Snake venom is a source of many pharmacologically important molecules. Agkistrodon bilineatus commonly known as Cantil, is spread over Central America particularly in Mexico and Costa Rica. From the venom of Agkistrodon bilineatus we have isolated and characterised six hypotensive peptides, and two bradykinin inhibitor peptides. The IC-50 value of four synthesized peptides was studied, towards angiotensin converting enzyme, in order to study the structure-function relationship of these peptides. RESULTS: The purification of the peptides was carried out by size exclusion chromatography, followed by reverse phase chromatography. Sequences of all peptides were determined applying MALDI-TOF/TOF mass spectrometry. These hypotensive peptides bear homology to bradykinin potentiating peptides and venom vasodilator peptide. The peptide with m/z 1355.53 (M + H)(+1), and the corresponding sequence ZQWAQGRAPHPP, we identified for the first time. A precursor protein containing a fragment of this peptide was reported at genome level, (Uniprot ID P68515), in Bothrops insularis venom gland. These proline rich hypotensive peptides or bradykinin potentiating peptides are usually present in the venom of Crotalinae, and exhibit specificity in binding to the C domain of somatic angiotensin converting enzyme. Four of these hypotensive peptides, were selected and synthesized to obtain the required quantity to study their IC50 values in complex with the angiotensin converting enzyme. The peptide with the sequence ZLWPRPQIPP displayed the lowest IC50 value of 0.64 µM. The IC50 value of the peptide ZQWAQGRAPHPP was 3.63 µM. CONCLUSION: The canonical snake venom BPPs classically display the IPP motif at the C-terminus. Our data suggest that the replacement of the highly conserved hydrophobic isoleucine by histidine does not affect the inhibitory activity, indicating that isoleucine is not mandatory to inhibit the angiotensin converting enzyme. The evaluation of IC 50 values show that the peptide with basic pI value exhibits a lower IC 50 value.

MOLECULAR MODEL OF CYTOTOXIN-1 FROM NAJA MOSSAMBICA MOSSAMBICA VENOM IN COMPLEX WITH CHYMOTRYPSIN.

Snake venom is a myriad of biologically active proteins and peptides. Three finger toxins are highly conserved in their molecular structure, but interestingly possess diverse biological functions. During the course of evolution the introduction of subtle mutations in loop regions and slight variations in the three dimensional structure, has resulted in their functional versatility. Cytotoxin-1 (UniProt ID: P01467), isolated from Naja mossambica mossambica, showed the potential to inhibit chymotrypsin and the chymotryptic activity of the 20S proteasome. In the present work we describe a molecular model of cytotoxin-1 in complex with chymotrypsin, prepared by the online server ClusPro. Analysis of the molecular model shows that Cytotoxin-1 (P01467) binds to chymotrypsin through its loop I located near the N-terminus. The concave side of loop I of the toxin fits well in the substrate binding pocket of the protease. We propose Phe10 as the dedicated P1 site of the ligand. Being a potent inhibitor of the 20S proteasome, cytotoxin-1 (P01467) can serve as a potential antitumor agent. Already snake venom cytotoxins have been investigated for their ability as an anticancer agent. The molecular model of cytotoxin-1 in complex with chymotrypsin provides important information towards understanding the complex formation.