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 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.

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.

Exploring Pakistani Physicians' Knowledge and Practices Regarding High Alert Medications: Findings and Implications.

Introduction: While many low-middle income countries (LMICs), including Pakistan, try and ensure patient safety within available resources, there are considerable concerns with medication use. Unsafe and inappropriate medication use, especially high alert medications (HAMs), is one of the important factors compromising patient safety and quality of care. Besides economic loss, HAMs contribute to greater morbidity, hospitalization, and mortality. Physicians as key members of the provision of healthcare are expected to be well aware of the administration and regulations surrounding HAMs. However, the current status is unknown in Pakistan. Consequently, the objectives of this study were to evaluate the knowledge of Pakistani physicians about the administration, regulation, and practices related to HAMs. This builds on our recently published study with nurses. Methods: An online cross-sectional study design was used, and data were gathered from the physicians throughout Pakistan using previously used self-administered questionnaires during a period of 5 months (January 1 to May 30, 2021). All data were entered and analyzed using SPSS 22 for Windows. Results: Physicians (847) who provided consent were enrolled in the study. Most physicians (62.2%) were male, aged between 25 and 30 years (75.2%) and had 2- to 5-year work experience (50.9%). About 27% were working in the emergency departments. The median (IQR) knowledge score for HAMs administration and regulation was 5 (3) and 5 (2), respectively. About 46.4% of respondents were found to have moderate knowledge about HAMs; increasing age, work experience, and higher qualifications were significantly associated (p < 0.05) with better HAMs knowledge. Around 58% had good practices relating to HAMs during their routine work. Median practice scores increased significantly (p < 0.05) with age, work experience, and postgraduate qualification. Conclusion: Most Pakistani physicians possess moderate knowledge about HAMs administration and regulations. However, their practices relating to the HAMs administration and regulations are typically sub-optimal. Consequently, HAMs awareness needs to be improved by including course content in the current curriculum, provision of hospital-based continuous training programs about patient safety and care, and establishment of multi-disciplinary health care teams, including board-certified pharmacists and specialized nurses, for the effective execution of medication use process in Pakistani hospitals in the future.

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.