Molecular Dynamic Analysis of Carbapenem-Resistant Klebsiella pneumonia's Porin Proteins with Beta Lactam Antibiotics and Zinc Oxide Nanoparticles.

To prevent the rapidly increasing prevalence of bacterial resistance, it is crucial to discover new antibacterial agents. The emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae has been associated with a higher mortality rate in gulf union countries and worldwide. Compared to physical and chemical approaches, green zinc oxide nanoparticle (ZnO-NP) synthesis is thought to be significantly safer and more ecofriendly. The present study used molecular dynamics (MD) to examine how ZnO-NPs interact with porin protein (GLO21), a target of ß-lactam antibiotics, and then tested this interaction in vitro by determining the zone of inhibition (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC), as well as the alteration of KPC's cell surface. The nanoparticles produced were characterized by UV-Vis spectroscopy, zetasizer, Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). In silico investigation was conducted using a variety of computational techniques, including Autodock Vina for protein and ligand docking and Desmond for MD simulation. The candidate ligands that interact with the GLO21 protein were biosynthesized ZnO-NPs, meropenem, imipenem, and cefepime. Analysis of MD revealed that the ZnO-NPs had the highest log P value (-9.1 kcal/mol), which indicates higher permeability through the bacterial surface, followed by cefepime (-7.9 kcal/mol), meropenem (-7.5 kcal/mol), and imipenem (-6.4 kcal/mol). All tested compounds and ZnO-NPs possess similar binding sites of porin proteins. An MD simulation study showed a stable system for ZnO-NPs and cefepime, as confirmed by RMSD and RMSF values during 100 ns trajectories. The test compounds were further inspected for their intersection with porin in terms of hydrophobic, hydrogen, and ionic levels. In addition, the stability of these bonds were measured by observing the protein-ligand contact within 100 ns trajectories. ZnO-NPs showed promising results for fighting KPC, represented in MIC (0.2 mg/mL), MBC (0.5 mg/mL), and ZI (24 mm diameter). To draw the conclusion that ZnO-NP is a potent antibacterial agent and in order to identify potent antibacterial drugs that do not harm human cells, further in vivo studies are required.

MD Simulation Studies for Selective Phytochemicals as Potential Inhibitors against Major Biological Targets of Diabetic Nephropathy.

Diabetes is emerging as an epidemic and is becoming a public health concern worldwide. Diabetic nephropathy is one of the serious complications of diabetes, and about 40% of individuals with diabetes develop diabetic nephropathy. The consistent feature of diabetes and its associated nephropathy is hyperglycemia, and in some cases, hyperamylinemia. Currently, the treatment includes the use of medication for blood pressure control, sugar control, and cholesterol control, and in the later stage requires dialysis and kidney transplantation, making the management of this complication very difficult. Bioactive compounds, herbal medicines, and extracts are extensively used in the treatment and prevention of several diseases, and some are reported to be efficacious in diabetes too. Therefore, in this study, we tried to identify the therapeutic potential of phytochemicals used in in silico docking and molecular dynamic simulation studies using a library of 5284 phytochemicals against the two potential targets of type 2 diabetes-associated nephropathy. We identified two phytochemicals (i.e., gentisic acid and michelalbine) that target human amylin peptide and dipeptidyl peptidase-4, respectively, with good binding affinity. These phytochemicals can be further evaluated using in vitro and in vivo studies for their anti-hyperglycemia and anti-hyperamylinemia effects.

Molecular cloning, expression, purification, and functional characterization of SP-22 gene from Bombyx mori.

Serine protease (SPs) is one of the immune enzyme's molecules that play a main role in the variation of a physiological process by controlling protease actions in vertebrates. For example, signaling cells, protector and improvement, which are included in melanization, are utilized to cascade with the meddling pathogens and defense the harmed tissue in insects. In this study, we explore the biochemical process of (SP-22) from Bombyx mori. Reverse-transcription polymerase chain reaction (RT-PCR) discloses that BmSP-22 is expressed in all tissues including the fat body. The formative expression profile of BmSP-22 reveal that BmSP-22 messenger RNA is expressed constitutively in larvae. Injection of recombinant BmSP-22 into B. mori larvae reduces significantly the transcript levels of antimicrobial peptides in the fat body. Our results suggest that BmSP-22 plays an important role in the innate immunity of B. mori and possibly in other insects.

Molecular dynamic analyses of the interaction of SARS-CoV-1 or 2 variants with various angiotensin-converting enzyme-2 species.

The transmembrane glycoprotein angiotensin-converting enzyme 2 (ACE2) is a key component of the renin-angiotensin system (RAS). It was shown to be the receptor of severe acute respiratory syndrome coronavirus 2 in the COVID-19 outbreak (SARS-COV-2). Furthermore, ACE2 aids in the transport of amino acids across the membrane. ACE2 is lost from the membrane, resulting in soluble ACE2 (sACE2). We aim to examine the structural conformation alterations between SARS-CoV-1 or 2 variants at various periods with ACE2 from various sources, particularly in the area where it interacts with the viral protein and the receptor. It is important to study the molecular dynamics of ACE2/SARS-COV RBD when the structure is available on the database. Here we analyzed the crystal structure of ACE2 from Human, Dog, Mus, Cat, and Bat ACE2 in complex with RBD from SARS-COV-1 and SARS-COV-2. The result shows, there is a variation in the type of residues, number of contact atoms and hydrogen bonds in ACE2 and RBD during the interaction interfaces. By using molecular dynamics simulation, we can measure RMSD, RMSF, SASA, Rg and the difference in the percentage of α helix and ß strand. As bat ACE2 & SARS-CoV-2 RBD found to have a high amount of ß strand compared to another structure complex, while hACE2 & SARS-CoV-1 RBD has fewer amounts of ß strand. Our study provides a deep view of the structure which is available and a summary of many works around ACE2/SARS-CoV RBD interaction.Communicated by Ramaswamy H. Sarma.

Study on Molecular Anti-tumor Mechanism of 2-Thiohydantoin Derivative based on Molecular Docking and Bioinformatic Analyses.

OBJECTIVE: Several methods for synthesizing 2-thiohydantoin derivatives have been devised and exploited, and they have found widespread application as antioxidants, antimicrobials, antivirals, and anticancer agents. As a result, we tried to understand the underlying processes of the 2- thiohydantoin derivative's anti-LIHC activity. METHODS: We predicted the anticancer mechanism of N-(4-oxo-5-(2-oxo-2-(p-tolylamino)ethyl)-3- phenyl-2-thioxoimidazolidin-1-yl)benzamide as a derivative of 2-thiohydantoin by utilizing molecular docking and molecular dynamic simulation. Furthermore, based on the results of molecular dynamic modelling, we employed bioinformatics to anticipate the immunotherapy of this molecule in the tumor microenvironment (TME) of Liver Hepatocellular Carcinoma (LIHC) patients. Next, we examined how this derivative affected proliferation, cell cycle progression, reactive oxygen species production, and apoptosis in HepG2 cancer cells. RESULTS: Substantially, our investigation revealed that the IC50 value was 2.448 µM and that it arrested the cell cycle of HepG2 in the S phase. Furthermore, molecular docking and dynamics studies revealed a worthy interaction of this compound with AKT1 and CDK2 proteins. Considerably, AKT1 and CDK2 have negative affinity energies of -10.4 kcal/mol and -9.6 kcal/mol, respectively. Several bioinformatic tools were used in this investigation to provide insight into the future clinical application of this derivative as a novel candidate to target immune cells such as macrophages, neutrophils, eosinophils, and CD8+ T cells. CONCLUSION: The relevance of this 2-thiohydantoin derivative was demonstrated by our experimental tests, docking studies, and bioinformatics analysis, and it may be investigated as a lead molecule for anticancer medicines, notably as AKT1 and CKD2 inhibitors.

Novel slow-binding reversible acetylcholinesterase inhibitors based on uracil moieties for possible treatment of myasthenia gravis and protection from organophosphate poisoning.

A series of new compounds in which uracil and 3,6-dimethyluracil moieties are bridged with different spacers were prepared and evaluated in vitro for the acetyl- and butyrylcholinesterase (AChE and BChE) inhibitory activities. These bisuracils are shown to be very effective inhibitors of AChE, inhibiting the enzyme at nano- and lower molar concentrations with extremely high selectivity for AChE vs. BChE. Kinetic analysis showed that the lead compound 2h acts as a slow-binding inhibitor of AChE and possess a long drug-target residence time (τ = 1/koff = 18.6 ± 7.5 min). Moreover, compound 2h ameliorated muscle weakness in myasthenia gravis rat model with a lower effective dose and longer lasting effect than pyridostigmine bromide. Besides, it was shown that compound 2h has an effect of increasing efficiency of antidotal therapy as a pretreatment for poisoning by organophosphates.

Molecular dynamic study of SARS-CoV-2 with various S protein mutations and their effect on thermodynamic properties.

Studying the structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein is important to understand the infection process. The S protein is necessary in completing the virus life cycle and is responsible for the appearance of new variants and drug and vaccine resistance. Understanding the structure and dynamics of biological macromolecules is essential for understanding how they function. In this work, we investigated the effects of mutations on S protein stability and solubility through molecular dynamic (MD) simulation in a 100 ns (ns) period. We screened four variants in addition to the wild type (WT). Results show that changes on MD simulation parameters of S protein indicate fluctuations and changes in the conformation, especially in the area between 300 and 600 amino acids (aa). This provides us an image of how the virus protein can reshape itself to adapt to any changes that occur in human angiotensin-converting enzyme 2 or drugs that can target the protein region. Our results also show that the Brazil variant has high fluctuations and unstable folding at some stages compared with other variants.

Antibacterial and antibiofilm activity of mannose-modified chitosan/PMLA nanoparticles against multidrug-resistant Helicobacter pylori.

Because of the apparent stasis in antibiotic discoveries and the growth of multidrug resistance, Helicobacter pylori-associated gastric infections are difficult to eradicate. In the search for alternative therapy, the reductive amination of chitosan with mannose, followed by ionic gelation, produced mannose functionalized chitosan nanoparticles. Then, molecular docking and molecular dynamics (MD) simulations were conducted with H. pylori lectin (HPLectin) as a target protein involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. Changes in zeta potential and FTIR spectroscopy revealed that chitosan was functionalized with mannose. Time-kill, polystyrene adherence, and antibiofilm studies were utilized to assess nanoparticles as an alternative antibacterial treatment against a resistant gastric pathogen. Man-CS-Nps were discovered to have effective anti-adherence and biofilm disruption characteristics in suppressing the development of resistant H. pylori. In addition, bioimaging studies with CLSM, TEM, and SEM illustrated that Man-CS-Nps interacted with bacterial cells and induced membrane disruption by creating holes in the outer membranes of the bacterial cells, resulting in the leakage of amino acids. Importantly, molecular docking and 20 ns MD simulations revealed that Man-CS-Nps inhibited the target protein through slow-binding inhibition and hydrogen bond interactions with active site residues. As a consequence of the findings of this study, the Man-CS-Nps is an excellent candidate for developing alternative therapies for the increasing incidences of resistant gastric infections.

Botrytis cinerea alcohol dehydrogenase mediates fungal development, environmental adaptation and pathogenicity.

Botrytis cinerea is an economically critical necrotrophic fungus that infecting many types of plants species. Although the lifestyle adaptations and genetic foundations of several enzymes and metabolites involved in B. cinerea virulence during host plant infection are well studied, the role of B. cinerea alcohol dehydrogenase (ADH) enzymes in these processes is poorly understood. Herein, we identified a significant up-regulation of the transcriptional levels of the BcADH1 gene during the tomato - B. cinerea strain B0510 interaction and at the early stage of infection. Substantially, we used a recent approach for replacement of gene by utilizing homologous recombination to generate knock-out mutants (Δbcadh1) and their effective complementary strains (Δbcadh1/C). A strong difference in the morphology of Δbcadh1 mutants from the wild type (WT) was detected, with respect to the conidiospore, conidial germination, and formation of branches, sporulation and sclerotia. In addition, the Δbcadh1 mutants showed significant differences in their virulence on tomato leaves relative to the WT. Moreover, the Δbcadh1 mutants appeared to have higher sensitivity to oxygen limitation (hypoxia) and reactive oxygen species, and had lost their ability of alcoholic fermentation compared with the WT and complementary strains. These results provide strong evidence for the requirement of the ADH1 gene for fungal development, environmental adaptation and its ability for full pathogenicity.Communicated by Ramaswamy H. Sarma.

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.

Ethnic practices in treating skin diseases: The traditional dermatologist's role.

This study documents the role of traditional medicinal and therapeutic plants in treating various skin ailments by indigenous communities of the tribal district of North Waziristan, Pakistan. A total of 130 informants and traditional dermatologists were interviewed. They employed 77 plant species belonging to 49 families to treat various skin ailments. The leading life form reported was herbs (41 species), whereas the dominant family was Lamiaceae (5 species). Leaves were the most commonly used plant part (37 species). The most preferred mode of preparation was paste (30.38%), and the dominant mode of administration was topical (69.23%). Important medicinal plants reported in this study are recommended for further phytochemical screening for bioactive constituents, which may lead to novel drug discoveries.

In Silico Discovery of GPCRs and GnRHRs as Novel Binding Receptors of SARS-CoV-2 Spike Protein Could Explain Neuroendocrine Disorders in COVID-19.

Despite the intense research work since the beginning of the pandemic, the pathogenesis of COVID-19 is not yet clearly understood. The previous mechanism of COVID-19, based on ACE2 tropism and explained through a single receptor, is insufficient to explain the pathogenesis due to the absence of angiotensin-converting enzyme 2 (ACE2) receptors in most of the affected organs. In the current study, we used the PatchDock server to run a molecular docking study of both the gonadotropin-releasing hormone receptor (GnRHR) and G-protein-coupled-receptor (GPCR) with the SARS-CoV-2 spike protein. Molecular Dynamics (MD) simulations were run to analyze the stability of the complexes using the GROMACS package. The docking results showed a high affinity between the spike protein with the GnRHR (-1424.9 kcal/mol) and GPCR (-1451.8 kcal/mol). The results of the MD simulations revealed the significant stability of the spike protein with the GnRHR and GPCR up to 100 ns. The SARS-CoV-2 spike protein had strong binding interactions with the GPCRs and GnRHRs, which are highly expressed in the brain, endocrine organs, and olfactory neurons. This study paves the way towards understanding the complex mechanism of neuroendocrine involvement and peripheral organ involvement, may explain the changing symptoms in patients due to new variants, and may lead to the discovery of new drug targets for COVID-19. In vitro studies involving genetic engineering or gene knockdown of the GPCRs and GnRHRs are needed to further investigate the role of these receptors in COVID-19 pathogenesis.

Comparison of structures among Saccharomyces cerevisiae Grxs proteins.

Glutaredoxins (Grxs) comprise a group of glutathione (GSH)-dependent oxidoreductase enzymes that respond to oxidative stress and sustain redox homeostasis. Saccharomyces cerevisiae Grx has a similar interaction patterns through its residues between the residues and the environment. The glutaredoxin domain covers 100% of the entire mature Grx1 and Grx8, while the glutaredoxin domain covers ~ 52% of the entire mature Grx6 and Grx7, which have approximately 74 additional amino acids in their N-terminal regions, whereas Grx3 and Grx4 have two functional domains: glutaredoxin and thioredoxin. We have presented the prediction of disordered regions within these protein sequences. Multiple sequence alignment combined with a phylogenetic tree enabled us to specify the key residues contributing to the differences between Saccharomyces cerevisiae Grxs and the proportion symmetry.

Structure analysis of yeast glutaredoxin Grx6 protein produced in Escherichia coli.

BACKGROUND: Grx6 is a yeast Golgi/endoplasmic reticulum protein involved in iron-sulfur binding that belongs to monothiol glutaredoxin-protein family. Grx6 has been biochemically characterized previously. Grx6 contains a conserved cysteine residue (Cys-136). Depending on the active-site sequences, Grxs can be classified to classic dithiol Grxs with a CXXC motif known as classes II and monothiol Grxs with a CXXS motif known as classes I, and Grx6 belongs to the class I with a CSYS motif. RESULTS: Our results showed how the loop between the N-terminal and C-terminal can affect the stability. When Grx6 was incubated with FeSO4·7H2O and (NH4)2Fe(SO4)2·6H2O, a disulfide bond was formed between the cysteine 136 and glutathione, and the concentration of dimer and tetramer was increased. The results presented various levels of stability of Grx6 with mutant and deleted amino acids. We also highlighted the difference between the monomer and dimer forms of the Grx6, in addition to comparison of the Fe-S cluster positions among holo forms of poplar Grx-C1, human Grx2 and Saccharomyces cerevisiae Grx6. CONCLUSIONS: In this paper, we used a combination of spectroscopic and proteomic techniques to analyse the sequence and to determine the affected mutations and deletions in the stability of Grx6. Our results have increased the knowledge about the differences between monomer and dimer structures in cellular processes and proteins whose roles and functions depend on YCA1 in yeast.