Target prediction of candidate miRNAs from Oryza sativa for silencing the RYMV genome

In order to maintain a consistent supply of rice globally, control of pathogens affecting crop production is a matter of due concern. Rice yellow mottle virus(RYMV) is known to cause a variety of symptoms which can result in reduced yield. Four ORFs can be identified in the genome of RYMV encoding for P1 (ORF1), Polyprotein (processed to produce VPg, protease, helicase, RdRp4) (ORF2), putative RdRp (ORF3) and capsid/coat protein (ORF4). This research was aimed at identifying genome encoded miRNAs of O. sativa that are targeted to the genome of Rice Yellow Mottle Virus (RYMV). A consensus of four miRNA target prediction algorithms (RNA22, miRanda, TargetFinder and psRNATarget) was computed, followed by calculation of free energies of miRNA-mRNA duplex formation. A phylogenetic tree was constructed to portray the evolutionary relationships between RYMV strains isolated to date. From the consensus of algorithms used, a total of seven O. sativa miRNAs were predicted and conservation of target site was finally evaluated. Predicted miRNAs can be further evaluated by experiments involving the testing of the success of in vitro gene silencing of RYMV genome; this can pave the way for development of RYMV resistant rice varieties in the future.     

Computational prediction of miRNA/mRNA duplexomes at the whole human genome scale reveals functional subnetworks of interacting genes with embedded miRNA annealing motifs

Perfect annealing between microRNAs (miRNAs) and messenger RNAs (mRNAs) was computationally searched at a broad scale in the human genome to determine whether theoretical pairing is restrictively represented in functional subnetworks or is randomly distributed. Massive RNA interference (RNAi) pairing motifs in genes constitute a remarkable subnetwork that displays highly genetically and biochemically interconnected genes. These analyses show unexpected repertoires of genes defined by their congruence in comatching with miRNAs at numerous sites and by their interconnection based on protein/protein interactions or proteins regulating the activity of others. This offers insights into the putatively coregulated homeostasis of large networks of genes by RNAi, whereas other networks seem to be independent of this regulatory mode. Genes accordingly defined by theoretical RNAi pairing cluster mainly in subnetworks related to cellular, metabolic and developmental processes and their regulation. Indeed, genes harboring numerous potential sites of hybridization with miRNAs are highly enriched with GO terms depicting the abovementioned processes and are grouped in a subnetwork of genes that are significantly more highly connected than they would be according to a random distribution. The significant number of interacting genes that present numerous potential comatches with miRNAs suggests that they may be under the control of the integrative and concerted action of multiple miRNAs.     

In silico analysis of carbohydrate-binding pockets in the lectin genes from various species of Canavalia

Legumes are endowed with an opulent class of proteins called lectins that can detect tenuous variations in carbohydrate structures and bind them reversibly with high affinity and specificity. The genus Canavalia, in the family of Leguminosae, is considered to be an affluent source of lectin. An effort has been made to analyse the sequences encoded by the lectin gene and its carbohydrate binding pockets from three species of Canavalia, including C. virosa, C. rosea, and C. pubescens. Crude seed extract showed highest haemagglutination titer against buffalo RBCs and has high affinity to mannose and trehalose. Amplification of the lectin gene by gene-specific primers showed the presence of an 870 bp amplicon. Physicochemical characterization using various bioinformatic tools showed that the isoelectric point was below 7, suggesting that lectin molecules were acidic. A high aliphatic index and high instability index were observed, which indicated that lectin molecules were stable towards a wide range of temperatures. The occurrence of N-glycosylation sites at two sites was also identified in all three species. Prediction of secondary structure showed that approximately 59.05 %, 56.76 % and 54.88 % of the elements were random coils in the case of C. virosa, C. pubescens and C. rosea, respectively. Comparative modelling of the proteins and docking of hypothetical models with sugar moieties that inhibited the agglutination activity suggested that asparagine, serine, alanine, valine, tyrosine and threonine were the major residues involved in hydrogen bonding and other stacking interactions. This can further provide insights on its prospective antibiosis property.     

In silico models for the prediction of dose-dependent human hepatotoxicity

The liver is extremely vulnerable to the effects of xenobiotics due to its critical role in metabolism. Drug-induced hepatotoxicity may involve any number of different liver injuries, some of which lead to organ failure and, ultimately, patient death. Understandably, liver toxicity is one of the most important dose-limiting considerations in the drug development cycle, yet there remains a serious shortage of methods to predict hepatotoxicity from chemical structure. We discuss our latest findings in this area and present a new, fully general in silico model which is able to predict the occurrence of dose-dependent human hepatotoxicity with greater than 80% accuracy. Utilizing an ensemble recursive partitioning approach, the model classifies compounds as toxic or non-toxic and provides a confidence level to indicate which predictions are most likely to be correct. Only 2D structural information is required and predictions can be made quite rapidly, so this approach is entirely appropriate for data mining applications and for profiling large synthetic and/or virtual libraries.     

In silico modeling and molecular docking insights of kaempferitrin for colon cancer-related molecular targets

Colorectal cancer is one of the most common cancers worldwide, and it is also one of the major causes of mortality from cancer. Chemotherapy drugs are generally limited due to various complications, as well as the development of resistance and recurrence. The in silico docking investigation involved exploration of protein or nucleotide, 3D structural modeling, molecular docking, and binding energy calculation. Protein-protein interactions are significant to many biological processes, and their disruption is a leading cause of disease. The use of small molecules to modulate them is gaining popularity, but protein interfaces usually lack specific cavities for processing small molecules. MMP-2, PARP, iNOS, Chk1, proteins were used in the molecular docking analysis of kaempferitrin and 5-flurouracil. The compound kaempferitrin had the highest binding energy scores with most of the target proteins, according to molecular docking results. The findings suggest it could be used to develop new drugs for cancer therapy.     

The regulation of microRNA in each of cancer stage from two different ethnicities as potential biomarker for breast cancer

miRNA has recently emerged as a potential biomarker for breast cancer. Even though many studies have identified ethnic variation affecting miRNA regulation, the effect of cancer stage within specific ethnicities on miRNA epigenetic remains unclear. The present study is designed to investigate miRNA regulation from two distinct ethnicities in specific cancer stages (non-Hispanic white and non-Hispanic black) using the TCGA dataset. Differentially expressed miRNAs were calculated by using the edgeR package. miRNAs with the highest or lowest log fold Change from each cancer stage were selected as a potential biomarker. miRNA-gene interaction was analyzed by using spearman correlation analysis, CLUEGO, and DIANA-mirpath. The association of biomarker candidates with diagnostic and prognostic performance was assessed using ROC and Kaplan-Meier survival analysis. miRNA-gene interaction analysis revealed the involvement of selected miRNAs in cancer progression. From eleven selected aberrant miRNAs, four of the miRNAs (hsa-mir-495, hsa-mir-592, hsa-mir-6501, and hsa-mir-937) are significantly detrimental to breast cancer diagnosis and prognosis. Hence, our result provides valuable information to explore miRNA’s role in each cancer stage between non-Hispanic white and non-Hispanic black.     

In silico 3-D structure prediction and molecular docking studies of inosine monophosphate dehydrogenase from Plasmodium falciparum

Growing resistance in malarial parasites, particularly in Plasmodium falciparum needs a serious search for the discovery of novel drug targets. Inosine monophosphate dehydrogenase (IMPDH) is an important target for antimalarial drug discovery process in P. falciparum for the treatment of malaria. In the absence of x-ray crystal structure of this enzyme, homology modeling proved to be a reasonable alternate to study substrate binding mechanisms of this enzyme. In this study, a 3-D homology model for P. falciparum IMPDH was constructed taking human IMPDH (PDB code 1NF7) as template. Furthermore, an in-silico combinatorial library of ribavirin (RVP) derivatives (1347 molecules) was designed and virtually screened for ligands having selectively greater binding affinity with Plasmodium falciparum IMPDH relative to human IMPDH II. A total of five Ribavirin derivatives were identified as having greater binding affinity (−126 to −108 Kcal/mol and −9.4 to −8.6 Kcal/mol) with Plasmodium falciparum IMPDH. These five inhibitors should be used as selective and potent for Plasmodium falciparum IMPDH. Such type of study will provide information to synthetic medicinal chemist to enhance the potential of compounds (RVP derivatives) as chemotherapeutic agents to fight against the increasing burden of malarial infections.     

Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M

A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04-0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested.     

In silico analysis of proteins and microRNAs related to human African trypanosomiasis in tsetse fly

Human African trypanosomiasis (HAT), also known as sleeping sickness, causes millions of deaths worldwide. HAT is primarily transmitted by the vector tsetse fly (Glossina morsitans). Early diagnosis remains a key objective for treating this disease. MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that play key roles in vector-borne diseases. To date, the roles of proteins and miRNAs in HAT disease have not been thoroughly elucidated. In this study, we have re-annotated the function of protein-coding genes and identified several miRNAs based on a series of bioinformatics tools. A batch of 81.1 % of tsetse fly proteins could be determined homology in mosquito genome, suggesting their probable similar mechanisms in vector-borne diseases. A set of 11 novel salivary proteins and 14 midgut proteins were observed in the tsetse fly, which could be applied to the development of vaccine candidates for the control of HAT disease. In addition, 35 novel miRNAs were identified, among which 10 miRNAs were found to be unique in tsetse fly. Pathway analysis of these 10 miRNAs indicated that targets of miR-15a-5p were significantly enriched in the HAT-related neurotrophin signaling pathway. Besides, topological analysis of the miRNA-gene network indicated that miR-619-5p and miR-2490-3p targeted several genes that respond to trypanosome infection, including thioester-containing protein Tep1 and heat shock protein Hsp60a. In conclusion, our work helps to elucidate the function of miRNAs in tsetse fly and establishes a foundation for further investigations into the molecular regulatory mechanisms of HAT disease.     

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli

BackgroundThe recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing.ObjectiveThe aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli.MethodsThirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package.Results and ConclusionComputational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.     

The auxiliary determination of the binding site of berberine binding to human serum albumin by surface-enhanced Raman scattering

We report on the joint application of fluorescence, ultraviolet-visible (UV-Vis) and Raman spectroscopy to the study of berberine with human serum albumin (HSA). We propose the surface-enhanced Raman scattering (SERS) technique to improve the understanding of the quenching interaction caused by berberine which could be applied in recognition process of fluorescent drugs with large biomolecules. The fluorescence and UV-Vis spectroscopic results show that the fluorescence intensity of HSA is significantly decreased in the presence of berberine, and the quenching mechanism is static. The SERS technique demonstrates clear advantages over direct measurements in physiological conditions. By means of this method, we are able to deduce important information concerning the binding property of berberine when interacting with HSA. We show the nitrogen atom is free but the dioxolane is involved in the spontaneously electrostatic inducement and subsequently hydrophobic binding.     

In silico design,synthesis and activity of potential drug-like chrysin scaffold-derived selective EGFR inhibitors as anticancer agents

Background & objectiveEpidermal growth factor receptor (EGFR) signaling pathway is one of the promising and well-established targets for anticancer therapy. The objective of the present study was to identify new EGFR inhibitors using ligand and structure-based drug designing methods, followed by a synthesis of selected inhibitors and evaluation of their activity.MethodsA series of C-7-hydroxyproton substituted chrysin derivatives were virtually drawn to generate a small compound library that was screened using 3D QSAR model created from forty-two known EGFR tyrosine kinase inhibitors. Next, the obtained hits with fitness score ≥ 1.0 were subjected to molecular docking analysis. Based on the predicted activity and XP glide score, three EGFR inhibitors were synthesized and characterized using ¹H-NMR, ¹³C-NMR and MS. Finally, comparative in vitro investigation of the biological activity of synthesized inhibitors was performed with that of the parent molecule, chrysin.ResultsThe data depicted a 3.2–fold enhanced cytotoxicity of chrysin derivative, CHM-04 against breast cancer cells as compared with chrysin as well as its binding with EGFR protein. Furthermore, the biological activity of CHM-04 was comparable to the standard EGFR inhibitor, AG1478 in increasing apoptosis and decreasing the migratory potential of triple-negative breast cancer cells as well as significantly lowering the mammosphere forming ability of breast cancer stem cells.ConclusionThe present study suggests CHM-04, an EGFR inhibitor possessing drug-like properties as a plausible therapeutic candidate against breast cancer.     

In silico validation of anti-viral drugs obtained from marine sources as a potential target against SARS-CoV-2 Mpro

COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has threatened the whole world affecting almost 243 million people globally. Originating from China, it has now spread worldwide with USA and India being the two most affected countries which emphasizes the immense potential of the coronaviruses to cause severity in the human population. This study validates the efficacy of some marine antiviral agents to target the viral main protease (Mpro) of SARS-CoV-2 by in silico studies. A total of 14 marine-derived antiviral agents were screened from several databases including PubChem and DrugBank and docked against the crystallised 3D structure of SARS-CoV-2 Mpro. MD simulation of the top two ligands was carried out for 100 ns to validate the protein-ligand stability. Later, their physicochemical, pharmacokinetics, and drug-likeness properties were evaluated and toxicity prediction was performed using eMOLTOX webtool. We found that all the 14 compounds are acting as a good target for Mpro. Among them, avarol and AcDa-1 procured the best docking results with the estimated docking score of −8.05 and −7.74 ​kcal/mol respectively. MD simulation revealed good conformational stability. The docked conformation was visualised and subsequent ligand-amino acid interactions were analysed. Avarol revealed good pharmacokinetic properties with oral bioavailability. The overall finding suggested that these marine compounds may have the potential to be used for the treatment of COVID-19 to tackle this pandemic.