Prebiotic Oligosaccharides: Special Focus on Fructooligosaccharides,Its Biosynthesis and Bioactivity

MicroRNAs (miRNAs) are important nonprotein-coding genes involved in almost all biological processes during biotic and abiotic stresses in plants. To investigate the miRNA-mediated plant response to drought stress, two drought-tolerant (C-306 and NI-5439) and two drought-sensitive (HUW-468 and WL-711) wheat genotypes were exposed to 25 % PEG 6000 for 1, 12 and 24 h. Temporal expression patterns of 12 drought-responsive miRNAs and their corresponding nine targets were monitored by quantitative real-time PCR (qRT-PCR). The results showed differential expression of miRNAs and their targets with varying degree of upregulation and downregulation in drought-sensitive genotypes. Likewise, in drought-tolerant wheat genotypes, maximum accumulation of miR393a and miR397a was observed at 1 h of stress. In addition, nearly perfect negative correlation was observed in four miRNA and target pairs (miR164-NAC, miR168a-AGO, miR398-SOD and miR159a-MYB) across all the temporal period studied which could be a major player during drought response in wheat. We, for the first time, validated the presence of miR529a and miR1029 in wheat. These findings gives a clue for temporal and variety-specific differential regulation of miRNAs and their targets in wheat in response to osmotic shock and could help in defining the potential roles of miRNAs in plant adaptation to osmotic stress in future.     

Exploratory Study on the RNA‐Binding Structural Motifs by Library Screening Targeting pre‐miRNA‐29 a

The metabolic stream of microRNA (miRNA) production, the so‐called maturation process of miRNAs, became one of important metabolic paths for drug‐targeting to modulate the expression of genes related to a number of diseases. We carried out discovery studies on small molecules binding to the precursor of miR‐29a (pre‐miR‐29a) from a chemical library containing 41 119 compounds (AQ library) by the fluorescent indicator displacement (FID) assay using the xanthone derivative X2SdiMe as a fluorescent indicator. The FID assay provided 1075 compounds, which showed an increase of fluorescence. These compounds were subsequently submitted to a binding analysis in a surface plasmon resonance (SPR) assay on a pre‐miR‐29a immobilized surface. 21 hit compounds were identified with a good reproducibility in the binding. These compounds have not been reported to bind to RNA until now and can be classified into two groups on the basis of the kinetics in the binding. To gain more information on the motif structures that could be necessary for the binding to pre‐miR‐29a, 19 substructures were selected from the hit compounds. The substructure library (SS library) which consisted of 362 compounds was prepared from the AQ library. An SPR assay of the SS library on pre‐miR‐29a‐immobilized surface suggested that five substructures could potentially be important structural motifs to bind to pre‐miR‐29a. These studies demonstrate that the combination of FID‐based screening of chemical library and subsequent SPR assay would be one way for obtaining practical solutions for the discovery of molecules which bind to the target pre‐miRNAs.     

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.     

Curcumin loaded PEG400‐OA nanoparticles: A suitable system to increase apoptosis,decrease migration,and deregulate miR‐125b/miR182 in MDA‐MB‐231 human breast cancer cells

Curcumin is an anti‐cancerous agent, but its low‐solubility limits its clinical use. The relationship between deregulation of miRNAs and their targets suggested that miRNAs can be interest targets of curcumin in treatment of different cancers. In this study, to overcome essential defects of the clinical usage of this golden drug, curcumin‐encapsulated polymersome nanoparticles (CPNs) have been developed, and the cytotoxicity effects were studied on MDA‐MB‐231 breast cancer cells. The expression level of miR‐182/125b and the expression pattern of some potential targets in apoptotic pathway, predicted by in silico approaches, were analyzed by RT‐qPCR in CPNs‐treated and untreated cells. Moreover, the amount of CASP9 and CASP8 proteins were determined by Western blotting. The effect of CPNs on cell migration were studied by scratch test and the level of EGFR, E‐cadherin, and beta‐catenin proteins were monitored in CPNs‐treated and untreated cells by western blotting. RT‐qPCR analysis identified the downregulation of miR‐125b and miR‐182 in CPNs‐treated cells and the upregulation of some predicted apoptotic target genes such as P53, CASP9 and BAX after 24 hours. Western blotting confirmed the effects of curcumin on the increase of cleaved CASP9 protein. Based on data from the current experiment, the migration of MDA‐MB‐231 cells was decreased after CPNs treatment. According to the results, CPNs, as suitable and compatible nanocarriers, can deliver curcumin into cancerous cells more effectively and can increase the therapeutic effects of curcumin on MDA‐MB‐231 cells partly by suppression of miR‐125b and miR‐182 as well as induction of apoptosis and inhibition of metastatic progression.     

Sequence-based analysis of 5′UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5′UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5′UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p’s binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5′UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5′UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.     

In silico studies on phytochemicals to combat the emerging COVID-19 infection

The current COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, remains a serious health hazard globally. The SARS-CoV-2 Mpro and spike proteins, as well as the human ACE2 receptor, have previously been reported as good targets for the development of new drug leads to combat COVID-19. Various ligands, including synthetic and plant-derived small molecules, can interact with the aforementioned proteins. In this study, we investigated the interaction of eight phytochemicals, from selected medicinal plants (Aegle marmelos, Azadirachta indica, and Ocimum sanctum) commonly used in Indian traditional medicine, with SARS-CoV-2 Mpro (PDBID: 6LU7), SARS-CoV-2S spike protein (PDB ID: 6M0J) and the human ACE2 receptor (PDB ID: 6M18). All compounds were subjected to density functional theory (DFT) and frontier molecular orbitals (FMO) analysis to determine their geometry, and key electronic and energetic properties. Upon examining the interactions of the phytochemicals with the human ACE2 receptor and the SARS-CoV-2 Mpro, spike protein targets, two compounds (C-5 and C-8) were identified as the best binding ligands. These were further examined in MD simulation studies to determine the stability of the ligand–protein interactions. QSAR, pharmacokinetic and drug-likeness properties studies revealed that C-5 may be the best candidate to serve as a template for the design and development of new drugs to combat COVID-19.     

In silico prediction of human genes as potential targets for rice miRNAs

BackgroundExogenous microRNAs (miRNAs) enter the human body through food, and their effects on metabolic processes can be considerable. It is important to determine which miRNAs from plants affect the expression of human genes and the extent of their influence.MethodThe binding sites of 738Oryza sativa miRNAs (osa-miRNAs) that interact with 17 508 mRNAs of human genes were determined using the MirTarget program.ResultThe characteristics of the binding of 46 single osa-miRNAs to 86 mRNAs of human genes with a value of free energy (ΔG) interaction equal 94%–100% from maximum ΔG were established. The findings showed that osa-miR2102-5p, osa-miR5075-3p, osa-miR2097-5p, osa-miR2919 targeted the largest number of genes at 38, 36, 23, 19 sites, respectively. mRNAs of 86 human genes were identified as targets for 93 osa-miRNAs of all family osa-miRNAs with ΔG values equal 94%–98% from maximum ΔG. Each miRNA of the osa-miR156-5p, osa-miR164-5p, osa-miR168-5p, osa-miR395-3p, osa-miR396-3p, osa-miR396-5p, osa-miR444-3p, osa-miR529-3p, osa-miR1846-3p, osa-miR2907-3p families had binding sites in mRNAs of several human target genes. The binding sites of osa-miRNAs in mRNAs of the target genes for each family of osa-miRNAs were conserved when compared to flanking nucleotide sequences.ConclusionTarget mRNA human genes of osa-miRNAs are also candidate genes of cancer, cardiovascular and neurodegenerative diseases.     

In silico carborane docking to proteins and potential drug targets

The presence of boron atoms has made carboranes, C(2)B(10)H(12), attractive candidates for boron neutron capture therapy. Because of their chemistry and possible conjugation with proteins, they can also be used to enhance interactions between pharmaceuticals and their targets and to increase the in vivo stability and bioavailability of compounds that are normally metabolized rapidly. Carboranes are isosteric to a rotating phenyl group, which they can substitute successfully in biologically active systems. A reverse ligand-protein docking approach was used in this work to identify binding proteins for carboranes. The screening was carried out on the drug target database PDTD that contains 1207 entries covering 841 known potential drug targets with structures taken from the Protein Data Bank. First, for validation, the protocol was applied to three crystal structures of proteins in which carborane derivatives are present. Then, the model was applied to systems for which the protein structure is available, but the binding site of carborane has not been reported. These systems were used for further validation of the protocol, while simultaneously providing new insight into the interactions between cage and protein. Finally, the screening was carried out on the database to reveal potential carborane binding targets of interest for biological and pharmacological activity. Carboranes are predicted to bind well to protease and metalloprotease enzymes. Other carborane pharmaceutical targets are also discussed, together with possible protein carriers.     

In-silico investigations of selective miRNA-gene targets and their validation studies in obstructive sleep apnea (OSA) patient cohorts

BackgroundObstructive sleep apnoea (OSA) is a prevalent form of sleep disordered breathing which results in sleep fragmentation and deprivation. Obesity and cardiovascular disorders are the major risk factors associated with OSA. Molecular analysis of the factors associated with OSA could demarcate the clinical analysis pattern in a population.ObjectiveThis study pertains to in-silico analyses of miRNA and their gene targets with validation for their potential role in OSA as putative biomarker candidates.MethodsmiRDB, TargetScan and miRanda databases were used to identify targets of miR-27 and let-7 that have documented role in OSA and co-related obesity and cardiovascular disorders. Quantitative PCR was used to analyze expression pattern of miR-27 and let-7 in obese and non-obese OSA patient cohorts with respective controls. In-silico analysis was done using PatchDoc to obtain atomic contact energy (ACE) scores that indicated the docked gene targets to the predicted miRNA structures. The docked structures were analysed using Maestro Suite 11 for the hydrogen and aromatic interactions.ResultsDownregulation of miR-27 and let-7 in OSA compared to controls was observed. In-silico data analysis was performed for gene targets (TGFBR1, TGFBR2, SMAD2, SMAD4, CRY2 and CNR1) of the selected miRNAs (miR-27 and let-7). Among all, CNR1 and CRY2 were found to be better targets for miR-27 and let-7 respectively as per ACE scores, ROC scores and expression fold change in OSA.ConclusionOur study gives insights to the expression profiling of miR-27 and let-7 and explore a set of potential target genes (CNR1 and CRY2) of these two miRNAs for a promising clinical relevance in OSA.     

Identification of novel vaccine candidates against carbapenem resistant Klebsiella pneumoniae: A systematic reverse proteomic approach

Klebsiella pneumoniae is declared as antibiotic resistant by WHO, with the critical urgency of developing novel antimicrobial therapeutics as drug resistance is the second most dangerous threat after terrorism. Besides many attempts still, there is no effective vaccine available against K. pneumoniae. By utilizing all the available proteomic data we prioritized the novel proteins ideal for vaccine development using bioinformatics tools and techniques. Among the huge data, eight proteins passed all the barriers and were considered ideal candidates for vaccine development. These include: copper silver efflux system outer membrane protein (CusC), outer membrane porin protein (OmpN), Fe⁺⁺ enterobactin transporter substrate binding protein (fepB), zinc transporter substrate binding protein (ZnuA), ribonuclease HI, tellurite resistant methyltransferase (the B), and two uncharacterized hypothetical proteins (WP_002918223 and WP_002892366). These proteins were also subjected to epitope analysis and were found best for developing subunit vaccine against K. pneumoniae. The study shows that the potential vaccine targets are sufficiently efficient being virulent, of outer membranous origin and can be proposed for the DNA third-generation vaccines development that would help to cope up infections caused by multidrug-resistant K. pneumoniae.     

Silencing lung cancer genes using miRNAs identified by 7mer-seed matching

Lung cancer (LC) is the main cause of cancer-associated deaths in both men and women globally with a very high mortality rate. The microRNAs (miRNAs) are a class of noncoding RNAs consisting of 18–25 nucleotides. They inhibit translation of protein through binding to complementary target mRNAs. The non-coding miRNAs are recognized as potent biomarkers for detection, development and treatment of malignancy. In this study, we screened a set of 12 genes over expressed in small cell lung cancer, non small cell lung cancer and the genes involved in both categories and their binding sites for human miRNAs as no work was reported yet. Screening of human miRNAs revealed that a few genes showed numerous miRNA binding sites. Free energy values of mRNA sequences revealed that they might acquire compact folded structure causing complexity for miRNAs to interact. GC content in the target site was relatively higher than that of their flanks. It was observed through analysis of cosine similarity metric and compAI parameters that the genes related to lung cancer were encoded with non optimal codons and thus might be translationally less efficient for producing polypeptides. Gene ontology analysis was carried out to understand the diverse functions of these 12 genes.     

Drug search for leishmaniasis: a virtual screening approach by grid computing

The trypanosomatid protozoa Leishmania is endemic in ~100 countries, with infections causing ~2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational “snapshots” were chosen from each MD trajectory to simulate the protein’s flexibility. A Relaxed Complex Scheme methodology was used to screen ~2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays.     

Computational analysis revealed miRNAs produced by Chikungunya virus target genes associated with antiviral immune responses and cell cycle regulation

Chikungunya virus (CHIKV) that causes chikungunya fever, is an alphavirus that belongs to the Togaviridae family containing a single-stranded RNA genome. Mosquitoes of the Aedes species act as the vectors for this virus and can be found in the blood, which can be passed from an infected person to a mosquito through mosquito bites. CHIKV has drawn much attention recently because of its potential of causing an epidemic. As the detailed mechanism of its pathogenesis inside the host system is still lacking, in this in silico research we have hypothesized that CHIKV might create miRNAs, which would target the genes associated with host cellular regulatory pathways, thereby providing the virus with prolonged refuge. Using bioinformatics approaches we found several putative miRNAs produced by CHIKV. Then we predicted the genes of the host targeted by these miRNAs. Functional enrichment analysis of these targeted genes shows the involvement of several biological pathways regulating antiviral immune stimulation, cellular proliferation, and cell cycle, thereby provide themselves with prolonged refuge and facilitate their pathogenesis, which in turn may lead to disease conditions. Finally, we analyzed a publicly available microarray dataset (GSE49985) to determine the altered expression levels of the targeted genes and found genes associated with pathways such as cell differentiation, phagocytosis, T-cell activation, response to cytokine, autophagy, Toll-like receptor signaling, RIG-I like receptor signaling and apoptosis. Our finding presents novel miRNAs and their targeted genes, which upon experimental validation could facilitate in developing new therapeutics to combat CHIKV infection and minimize CHIKV mediated diseases.     

An in-silico approach to study the possible interactions of miRNA between human and SARS-CoV2

BackgroundThe progressive SARS-CoV2 outbreaks worldwide have evoked global investigation. Despite the numerousin-silico approaches, the virus-host relationship remains a serious concern. MicroRNAs are the small non-coding RNAs that help in regulating gene profiling. The current study utilized miRNA prediction tools along with the PANTHER classification system to demonstrate association and sequence similarities shared between miRNAs of SARS-CoV2 and human host.MethodAn in-silico approach was carried out using Vmir analyzer to predict miRNAs from SARS-CoV2 viral genomes. Predicted miRNAs from SARS-CoV2 viral genomes were used for effective hybridization sequence identification along the nucleotide similarities with human miRNAs from miRbase database. Further, it was proceeded to analyze the gene ontology using miRDB with PANTHER classification.ResultBased on the prediction and analysis, we have identified 22 potential miRNAs from five genomes of SARS-CoV2 linked with 12 human miRNAs. Analysis of human miRNAs hsa-mir-1267, hsa-mir-1-3p, hsa-mir-5683 were found shared between all the five viral SARS-CoV2 miRNAs. Further, PANTHER classification analyzed the gene-ontology being carried by these associations showed that 44 genes were involved in biological functions that includes genes specific for signaling pathway, immune complex generation, enzyme binding with effective role in the virus-host relationship.ConclusionOur analysis concludes that the genes identified in this study can be effective in analyzing the virus-host interaction. It also provides a new direction to understand viral pathogenesis with a probable new way to link, that can be used to understand and relate the miRNAs of the virus to the host conditions.     

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.