Designing of fragment based inhibitors with improved activity against E. coli AmpC ß-lactamase compared to the conventional antibiotics.

One of the most common primary resistance mechanism of multi-drug resistant (MDR) Gram negative pathogenic bacteria to combat ß-lactam antibiotics, such as penicillins, cephalosporins and carbapenems is the generation of ß- lactamases. The uropathogenic E. coli is mostly getting multi-drug resistance due to the synthesis of AmpC ß-lactamases and therefore new antibiotics and inhibitors are needed to treat the evolving infections. The current study was designed for targetting AmpC ß-lactamase of E. coli using molecular docking based virtual screening, linking fragments for designing novel compounds and binding mode analysis using molecular dynamic simulation with target protein. The FCH group all-purpose fragment library consisting of 9388 fragments has been screened against AmpC ß-lactamase protein of E. coli and the antibiotics and anti-infectives used in treatment of Urinary tract Infections (UTIs) were also screened with AmpC ß-lactamase protein. Among the 9388 fragments, 339 fragment candidates were selected and linked with cefepime antibiotic having maximum binding affinity for AmpC target protein. Computational analysis of interactions as well as molecular dynamics (MD) simulations were also conducted for identifying the most promising ligand-pocket complexes from docking investigations to comprehend their thermodynamic properties and verify the docking outcomes as well. Overall, the linked complexes (LCs) showed good binding interactions with AmpC ß-lactamase. Interestingly, our fragment-based LCs remained relatively stable in comparison with cefepime antibiotic. Moreover, S12 fragment linked complex remained the most stable during 50 ns with remarkable number of interactions indicating it as promising candidate in novel lead discovery against MDR E. coli infections.

Modelling and analysis of the complement system signalling pathways: roles of C3, C5a and pro-inflammatory cytokines in SARS-CoV-2 infection.

The complement system is an essential part of innate immunity. It is activated by invading pathogens causing inflammation, opsonization, and lysis via complement anaphylatoxins, complement opsonin's and membrane attack complex (MAC), respectively. However, in SARS-CoV-2 infection overactivation of complement system is causing cytokine storm leading to multiple organs damage. In this study, the René Thomas kinetic logic approach was used for the development of biological regulatory network (BRN) to model SARS-CoV-2 mediated complement system signalling pathways. Betweenness centrality analysis in cytoscape was adopted for the selection of the most biologically plausible states in state graph. Among the model results, in strongly connected components (SCCs) pro-inflammatory cytokines (PICyts) oscillatory behaviour between recurrent generation and downregulation was found as the main feature of SARS-CoV-2 infection. Diversion of trajectories from the SCCs leading toward hyper-inflammatory response was found in agreement with in vivo studies that overactive innate immunity response caused PICyts storm during SARS-CoV-2 infection. The complex of negative regulators FI, CR1 and DAF in the inhibition of complement peptide (C5a) and PICyts was found desirable to increase immune responses. In modelling role of MAC and PICyts in lowering of SARS-CoV-2 titre was found coherent with experimental studies. Intervention in upregulation of C5a and PICyts by C3 was found helpful in back-and-forth variation of signalling pattern linked with the levels of PICyts. Moreover, intervention in upregulation of PICyts by C5a was found productive in downregulation of all activating factors in the normal SCCs. However, the computational model predictions require experimental studies to be validated by exploring the activation role of C3 and C5a which could change levels of PICyts at various phases of SARS-CoV-2 infection.

Investigating effect of mutation on structure and function of G6PD enzyme: a comparative molecular dynamics simulation study.

Several natural mutants of the human G6PD enzyme exist and have been reported. Because the enzymatic activities of many mutants are different from that of the wildtype, the genetic polymorphism of G6PD plays an important role in the synthesis of nucleic acids via ribulose-5-phosphate and formation of reduced NADP in response to oxidative stress. G6PD mutations leading to its deficiency result in the neonatal jaundice and acute hemolytic anemia in human. Herein, we demonstrate the molecular dynamics simulations of the wildtype G6PD and its three mutants to monitor the effect of mutations on dynamics and stability of the protein. These mutants are Chatham (A335T), Nashville (R393H), Alhambra (V394L), among which R393H and V394L lie closer to binding site of structural NADP+. MD analysis including RMSD, RMSF and protein secondary structure revealed that decrease in the stability of mutants is key factor for loss of their activity. The results demonstrated that mutations in the G6PD sequence resulted in altered structural stability and hence functional changes in enzymes. Also, the binding site, of structural NADP+, which is far away from the catalytic site plays an important role in protein stability and folding. Mutation at this site causes changes in structural stability and hence functional deviations in enzyme structure reflecting the importance of structural NADP+ binding site. The calculation of binding free energy by post processing end state method of Molecular Mechanics Poisson Boltzmann SurfaceArea (MM-PBSA) has inferred that ligand binding in wildtype is favorable as compared to mutants which represent destabilised protein structure due to mutation that in turn may hinder the normal physiological function. Exploring individual components of free energy revealed that the van der Waals energy component representing non-polar/hydrophobic energy contribution act as a dominant factor in case of ligand binding. Our study also provides an insight in identifying the key inhibitory site in G6PD and its mutants which can be exploited to use them as a target for developing new inhibitors in rational drug design.

Genome-wide analysis, identification, evolution and genomic organization of dehydration responsive element-binding (DREB) gene family in Solanum tuberosum.

BACKGROUND: The dehydration responsive element-binding (DREB) gene family plays a crucial role as transcription regulators and enhances plant tolerance to abiotic stresses. Although the DREB gene family has been identified and characterized in many plants, knowledge about it in Solanum tuberosum (Potato) is limited. RESULTS: In the present study, StDREB gene family was comprehensively analyzed using bioinformatics approaches. We identified 66 StDREB genes through genome wide screening of the Potato genome based on the AP2 domain architecture and amino acid conservation analysis (Valine at position 14th). Phylogenetic analysis divided them into six distinct subgroups (A1-A6). The categorization of StDREB genes into six subgroups was further supported by gene structure and conserved motif analysis. Potato DREB genes were found to be distributed unevenly across 12 chromosomes. Gene duplication proved that StDREB genes experienced tandem and segmental duplication events which led to the expansion of the gene family. The Ka/Ks ratios of the orthologous pairs also demonstrated the StDREB genes were under strong purification selection in the course of evolution. Interspecies synteny analysis revealed 45 and 36 StDREB genes were orthologous to Arabidopsis and Solanum lycopersicum, respectively. Moreover, subcellular localization indicated that StDREB genes were predominantly located within the nucleus and the StDREB family's major function was DNA binding according to gene ontology (GO) annotation. CONCLUSIONS: This study provides a comprehensive and systematic understanding of precise molecular mechanism and functional characterization of StDREB genes in abiotic stress responses and will lead to improvement in Solanum tuberosum.

Therapeutic Potential of Novel Mastoparan-Chitosan Nanoconstructs Against Clinical MDR Acinetobacter baumannii: In silico, in vitro and in vivo Studies.

PURPOSE: Acinetobacter baumannii antibiotic resistant infections in high-risk patients are a great challenge for researchers and clinicians worldwide. In an effort to achieve potent bactericidal outcomes, a novel chitosan-mastoparan nanoconstruct (Mast-Cs NC) was designed and assessed for its therapeutic potential through in silico, in vitro and in vivo experimentation against clinical multidrug-resistant (MDR) A. baumannii. METHODS: Optimized 3D structures of mastoparan and chitosan were coupled computationally through an ionic cross-linker to generate a circular ring of chitosan encasing mastoparan. The complex was assessed for interactions and stability through molecular dynamic simulation (MDS). Binding pocket analysis was used to assess the protease-peptide interface. Mast-Cs NC were prepared by the ionic gelation method. Mast-Cs NC were evaluated in vitro and in vivo for their therapeutic efficacy against drug-resistant clinical A. baumannii. RESULTS: MDS for 100 ns showed stable bonds between chitosan and mastoparan; the first at chitosan oxygen atom-46 and mastoparan isoleucine carbon atom with a distance of 2.77 Å, and the second between oxygen atom-23 and mastoparan lysine nitrogen atom with a distance of 2.80 Å, and binding energies of -3.6 and -7.4 kcal/mol, respectively. Mast-Cs complexes approximately 156 nm in size, with +54.9 mV zeta potential and 22.63% loading capacity, offered >90% encapsulation efficiency and were found to be geometrically incompatible with binding pockets of various proteases. The MIC90 of Mast-Cs NC was significantly lower than that of chitosan (4 vs 512 µg/mL, respectively, p<0.05), with noticeable bacterial damage upon morphological analysis. In a BALB/c mouse sepsis model, a significant reduction in bacterial colony count in the Mast-Cs treated group was observed compared with chitosan and mastoparan alone (p<0.005). Mast-Cs maintained good biocompatibility and cytocompatibility. CONCLUSION: Novel mastoparan-loaded chitosan nanoconstructs signify a successful strategy for achieving a synergistic bactericidal effect and higher therapeutic efficacy against MDR clinical A. baumannii isolates. The Mast-Cs nano-drug delivery system could work as an alternative promising treatment option against MDR A. baumannii.

MicroRNAs and their target mRNAs as potential biomarkers among smokers and non-smokers with lung adenocarcinoma.

Lung adenocarcinoma is one of the major causes of mortality. Current methods of diagnosis can be improved through identification of disease specific biomarkers. MicroRNAs are small non-coding regulators of gene expression, which can be potential biomarkers in various diseases. Thus, the main objective of this study was to gain mechanistic insights into genetic abnormalities occurring in lung adenocarcinoma by implementing an integrative analysis of miRNAs and mRNAs expression profiles in the case of both smokers and non-smokers. Differential expression was analysed by comparing publicly available lung adenocarcinoma samples with controls. Furthermore, weighted gene co-expression network analysis is performed which revealed mRNAs and miRNAs significantly correlated with lung adenocarcinoma. Moreover, an integrative analysis resulted in identification of several miRNA-mRNA pairs which were significantly dysregulated in non-smokers with lung adenocarcinoma. Also two pairs (miR-133b/Protein Kinase C Zeta (PRKCZ) and miR-557/STEAP3) were found specifically dysregulated in smokers. Pathway analysis further revealed their role in important signalling pathways including cell cycle. This analysis has not only increased the authors' understanding about lung adenocarcinoma but also proposed potential biomarkers. However, further wet laboratory studies are required for the validation of these potential biomarkers which can be used to diagnose lung adenocarcinoma.

Formal modeling and analysis of the hexosamine biosynthetic pathway: role of O-linked N-acetylglucosamine transferase in oncogenesis and cancer progression.

The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP) drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation) and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT), an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer.

On the modelling and analysis of the regulatory network of dengue virus pathogenesis and clearance.

Dengue virus can ignite both protective and pathogenic responses in human. The pathogenesis is related with modified functioning of our immune system during infection. Pattern recognition receptors like Toll like receptor 3 is vital for the induction of innate immunity in case of Dengue infection. Toll like receptor 3 induces TRIF mediated activation of Type 1 interferons and Fc receptor mediated induction of cytokines. Interferons have been related with clearance of Dengue virus but it has adopted modified regulatory mechanisms to counter this effect. SOCS protein is also induced due to the interferon and cytokine mediated signalling which can subsequently play its part in the regulation of interferon and cytokine production. Our hypothesis in this study relates the pathogenesis of Dengue virus with the SOCS mediated inhibition of our innate immunity. We used the qualitative formalism of René Thomas to model the biological regulatory network of Toll like receptor 3 mediated signalling pathway in an association with pathogenesis of dengue. Logical parameters for the qualitative modelling were inferred using a model checking approach implemented in SMBioNet. A linear hybrid model, parametric linear hybrid automaton, was constructed to incorporate the activation and inhibition time delays in the qualitative model. The qualitative model captured all the possible expression dynamics of the proteins in the form of paths, some of which were observed as abstract cycles (representing homoeostasis) and diverging paths towards stable states. The analysis of the qualitative model highlighted the importance of SOCS protein in elevating propagation of dengue virus through inhibition of type 1 interferons. Detailed qualitative analysis of regulatory network endorses our hypothesis that elevated levels of cytokine subsequently induce SOCS expression which in turn results into the continuous down-regulation of Toll like receptor 3 and interferon. This may result into the Dengue pathogenesis during the stage of immunosuppression. Further analysis with HyTech (HYbrid TECHnology) tool provided us with the real-time constraints (delay constraints) of the proteins involved in the cyclic paths of the regulatory network backing the evidence provided by the qualitative analysis. The HyTech results also suggest that the role of SOCS is vital in homoeostasis.