Demystifying emerging bulk RNA-Seq applications: the application and utility of bioinformatic methodology.

Significant innovations in next-generation sequencing techniques and bioinformatics tools have impacted our appreciation and understanding of RNA. Practical RNA sequencing (RNA-Seq) applications have evolved in conjunction with sequence technology and bioinformatic tools advances. In most projects, bulk RNA-Seq data is used to measure gene expression patterns, isoform expression, alternative splicing and single-nucleotide polymorphisms. However, RNA-Seq holds far more hidden biological information including details of copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens. Recent novel and advanced bioinformatic algorithms developed the capacity to retrieve this information from bulk RNA-Seq data, thus broadening its scope. The focus of this review is to comprehend the emerging bulk RNA-Seq-based analyses, emphasizing less familiar and underused applications. In doing so, we highlight the power of bulk RNA-Seq in providing biological insights.

Splicing of long non-coding RNAs primarily depends on polypyrimidine tract and 5' splice-site sequences due to weak interactions with SR proteins.

Many nascent long non-coding RNAs (lncRNAs) undergo the same maturation steps as pre-mRNAs of protein-coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences using the ncRNA-a2 as a model. Genome-wide analyses of intergenic lncRNAs (lincRNAs) revealed that lincRNA splicing efficiency positively correlates with 5'ss strength while no such correlation was identified for PCGs. In addition, efficiently spliced lincRNAs have higher thymidine content in the polypyrimidine tract (PPT) compared to efficiently spliced PCGs. Using model lincRNAs, we provide experimental evidence that strengthening the 5'ss and increasing the T content in PPT significantly enhances lincRNA splicing. We further showed that lincRNA exons contain less putative binding sites for SR proteins. To map binding of SR proteins to lincRNAs, we performed iCLIP with SRSF2, SRSF5 and SRSF6 and analyzed eCLIP data for SRSF1, SRSF7 and SRSF9. All examined SR proteins bind lincRNA exons to a much lower extent than expression-matched PCGs. We propose that lincRNAs lack the cooperative interaction network that enhances splicing, which renders their splicing outcome more dependent on the optimality of splice sites.

In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-ß-hydroxylase and docking studies.

Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-ß-hydroxylase (TßH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, the inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) was tested on cloned Drosophila tyramine-ß-hydroxylase (DmTßH) expressed in Bombyx mori strain. Radiolabelled 3H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTßH, whose ID50 values range from 0.02 to 2511nM where DmTßH was inhibited in a dose-dependent manner at pH 7.6 and 25°C during a 30min of incubation. To understand the catalytic role of the TßH, a three dimensional structure of the TßH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TßH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TßH to control insect's population.

A transcriptomic insight into the infective juvenile stage of the insect parasitic nematode, Heterorhabditis indica.

BACKGROUND: Nematodes are the most numerous animals in the soil. Insect parasitic nematodes of the genus Heterorhabditis are capable of selectively seeking, infecting and killing their insect-hosts in the soil. The infective juvenile (IJ) stage of the Heterorhabditis nematodes is analogous to Caenorhabditis elegans dauer juvenile stage, which remains in 'arrested development' till it finds and infects a new insect-host in the soil. H. indica is the most prevalent species of Heterorhabditis in India. To understand the genes and molecular processes that govern the biology of the IJ stage, and to create a resource to facilitate functional genomics and genetic exploration, we sequenced the transcriptome of H. indica IJs. RESULTS: The de-novo sequence assembly using Velvet-Oases pipeline resulted in 13,593 unique transcripts at N50 of 1,371 bp, of which 53 % were annotated by blastx. H. indica transcripts showed higher orthology with parasitic nematodes as compared to free living nematodes. In-silico expression analysis showed 30 % of transcripts expressing with ≥100 FPKM value. All the four canonical dauer formation pathways like cGMP-PKG, insulin, dafachronic acid and TGF-ß were active in the IJ stage. Several other signaling pathways were highly represented in the transcriptome. Twenty-four orthologs of C. elegans RNAi pathway effector genes were discovered in H. indica, including nrde-3 that is reported for the first time in any of the parasitic nematodes. An ortholog of C. elegans tol-1 was also identified. Further, 272 kinases belonging to 137 groups, and several previously unidentified members of important gene classes were identified. CONCLUSIONS: We generated high-quality transcriptome sequence data from H. indica IJs for the first time. The transcripts showed high similarity with the parasitic nematodes, M. hapla, and A. suum as opposed to C. elegans, a species to which H. indica is more closely related. The high representation of transcripts from several signaling pathways in the IJs indicates that despite being a developmentally arrested stage; IJs are a hotbed of signaling and are actively interacting with their environment.

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite.

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.

De novo transcriptome sequencing and analysis of the cereal cyst nematode, Heterodera avenae.

The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptome analysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases (CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction.

Structural and functional analysis of cathepsin S of Heterodera spp: a promising candidate for its control.

Cysteine proteinases are required for a wide range of physiological processes in all living organisms. In parasitic nematodes, they are particularly crucial for the digestion of host tissues and evasion of host immune responses. Therefore, in general, these are identified as primary targets for the control of parasitic nematodes. Herein, cathepsin S-like cysteine proteinase of Heterodera avenae (Hacp-s) has been cloned and analysed for the first time. The predicted protein is 298 amino acids long and showed significant similarity with cathepsin S of Heterodera glycines (Hgcp-s). The sequence of cathepsin S contains a signal peptide of 30 amino acids which suggests its role in extracellular functions. Multiple sequence alignment revealed the presence of ERFNIN motif and conserved catalytic residues. Three dimensional structure (3D) of Hgcp-s was modelled using homology modelling. In order to illustrate the plausible mode of interaction of cathepsin S (Hgcp-s), docking analysis was performed with E-64 cysteine proteinase inhibitor. Docking studies revealed the hydrogen bonding of E-64 with Gln153, His299 and Gly203 as well as close interaction with catalytic residues Cys159 and Asn320 Expression analysis of Hacp-s using qRT-PCR showed high expression of cathepsin S in pre parasitic J2s and female stages suggesting its significant role in both pre-parasitic and parasitic stages of the nematode life cycle.

In vitro and in silico antimalarial activity of 2-(2-hydrazinyl)thiazole derivatives.

A series of 2-(2-hydrazinyl)thiazole derivatives with a wide range of substitutions at 2-, 4- and 5-positions were synthesized, characterized and evaluated their inhibitory potentials against plasmodium falciparum, NF54, by in vitro blood stage assay. The compounds, ethyl-4-methyl-2-[(E)-2-[1-(pyridin-2-yl)ethylidene]hydrazin-1-yl]-1,3-thiazole-5-carboxylate, 4d, and 1-{4-methyl-2-[(E)-2-[1-(pyridin-2-yl)ethylidene]hydrazin-1-yl]-1,3-thiazol-5-yl}ethan-1-one, 5d showed significant antimalarial activity with IC50 values of 0.725 µM and 0.648 µM respectively. To understand the mechanism, the binding interactions between 2-(2-hydrazinyl)thiazole derivatives and trans-2-enoyl acyl carrier protein reductase of P. falciparum were studied through docking studies. The half maximal inhibitory concentration (IC50) through docking studies for the compounds, 4d and 5d were found to be 22.88 µM and 631.84 µM respectively.

Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L).

BACKGROUND: Analysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR. RESULTS: We have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant. CONCLUSION: 18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

Search of potential inhibitor against New Delhi metallo-beta-lactamase 1 from a series of antibacterial natural compounds.

BACKGROUND: New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Gram-negative bacteria are today's major worldwide health concern. The enzyme NDM-1 provides bacterial resistance by its hydrolytic activity against the ß-lactam ring of antibiotics. Inhibition of NDM-1 may prevent the hydrolysis of ß-lactam ring of the antibiotics, and therefore, plays an important role against antibacterial resistance. MATERIALS AND METHODS: Here we made an attempt to design suitable inhibitors against NDM-1 from different natural antibacterial compounds using molecular docking approach. RESULTS: We observed that natural compounds such as Nimbolide and Isomargololone are showing an appreciable IC50 value as well as significant binding energy value for NDM-1. We further observed these compounds showing better affinity to NDM-1 on comparison with 14 ß-lactam antibiotics. CONCLUSION: Finally, our study provides a platform for the development of a potent inhibitor of NDM-1, which may be considered as a potential drug candidate against bacterial resistance.

Identification of neuropeptides, flp-1 and flp-12 targeting neuromuscular system of rice root knot nematode (RRKN) Meloidogyne graminicola.

Root-knot nematodes (RKNs), Meloidogyne spp, are found in all temperate and tropical areas, and are among the most damaging plant pathogens worldwide. M. graminincola is an economically important root parasite on upland, lowland and deepwater rice. FMRFamide-like peptides (FLPs) play significant role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time, we have cloned and characterized two neuropeptide genes (flp-1 and flp-12) from the cDNA of preparasitic second stage juveniles of M. graminicola. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting function as an extra-cellular protein. We have found highly conserved motif LFRGR in flp-1. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.

Discovering a potent small molecule inhibitor for gankyrin using de novo drug design approach.

Gankyrin is an oncoprotein composed of six ankyrin repeats, over-expressed in the Hepatocellular Carcinoma (HCC), and directly involved in the cell cycle regulation. Therefore, it is a potential drug target to restrict the growth of cancer cell and activation of apoptosis. We have successfully designed a potent ligand to inhibit the activity of gankyrin. Using docking approach we designed a potential ligand, which is exactly fitting in the cavity of gankyrin and forming many close interactions to protein atoms including its active site residues. This molecule shows minimum energy and good binding affinity for gankyrin.