Refinement of Draft Genome Assemblies of Pigeonpea (Cajanus cajan).

Genome assembly of short reads from large plant genomes remains a challenge in computational biology despite major developments in next generation sequencing. Of late several draft assemblies have been reported in sequenced plant genomes. The reported draft genome assemblies of Cajanus cajan have different levels of genome completeness, a large number of repeats, gaps, and segmental duplications. Draft assemblies with portions of genome missing are shorter than the referenced original genome. These assemblies come with low map accuracy affecting further functional annotation and the prediction of gene components as desired by crop researchers. Genome coverage, i.e., the number of sequenced raw reads mapped onto a certain location of the genome is an important quality indicator of completeness and assembly quality in draft assemblies. The present work aimed to improve the coverage in reported de novo sequenced draft genomes (GCA_000340665.1 and GCA_000230855.2) of pigeonpea, a legume widely cultivated in India. The two recently sequenced assemblies, A1 and A2 comprised 72% and 75% of the estimated coverage of the genome, respectively. We employed an assembly reconciliation approach to compare the draft assemblies and merge them, filling the gaps by employing an algorithm size sorting mate-pair library to generate a high quality and near complete assembly with enhanced contiguity. The majority of gaps present within scaffolds were filled with right-sized mate-pair reads. The improved assembly reduced the number of gaps than those reported in draft assemblies resulting in an improved genome coverage of 82.4%. Map accuracy of the improved assembly was evaluated using various quality metrics and for the presence of specific trait-related functional genes. Employed pair-end and mate-pair local libraries helped us to reduce gaps, repeats, and other sequence errors resulting in lengthier scaffolds compared to the two draft assemblies. We reported the prediction of putative host resistance genes against Fusarium wilt disease by their performance and evaluated them both in wet laboratory and field phenotypic conditions.

Development and rigorous validation of antimalarial predictive models using machine learning approaches.

The large collection of known and experimentally verified compounds from the ChEMBL database was used to build different classification models for predicting the antimalarial activity against Plasmodium falciparum. Four different machine learning methods, namely the support vector machine (SVM), random forest (RF), k-nearest neighbour (kNN) and XGBoost have been used for the development of models using the diverse antimalarial dataset from ChEMBL. A well-established feature selection framework was used to select the best subset from a larger pool of descriptors. Performance of the models was rigorously evaluated by evaluation of the applicability domain, Y-scrambling and AUC-ROC curve. Additionally, the predictive power of the models was also assessed using probability calibration and predictiveness curves. SVM and XGBoost showed the best performances, yielding an accuracy of ~85% on the independent test set. In term of probability prediction, SVM and XGBoost were well calibrated. Total gain (TG) from the predictiveness curve was more related to SVM (TG = 0.67) and XGBoost (TG = 0.75). These models also predict the high-affinity compounds from PubChem antimalarial bioassay (as external validation) with a high probability score. Our findings suggest that the selected models are robust and can be potentially useful for facilitating the discovery of antimalarial agents.

In silico study directed towards identification of novel high-affinity inhibitors targeting an oncogenic protein: BRD4-BD1.

Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) family of proteins. It epigentically regulates the transcription of growth-promoting genes and has become an attractive target for the development of anticancer and anti-inflammatory agents. In the current study, we performed an in silico screening of a small-molecule chemical library against the acetyl-lysine binding site of the first bromodomain (BD1) in BRD4 protein. Potential inhibitors identified through virtual screening were further studied through molecular dynamics simulations, water entrapment analysis and Molecular Mechanics (MM)/Poisson-Boltzmann surface area (PBSA) binding free energy calculations. Many of the identified compounds exhibit better G-score (-11.64 kcal∙mol-1 to -10.31 kcal∙mol-1) and predicted binding affinity (-9.66 kcal∙mol-1 to -6.63 kcal∙mol-1) values towards BRD4-BD1 than that of the reference compound (+)-JQ1. Molecular dynamics simulation studies show that in free-form BRD4 the reported conserved water molecules are not retained at their specific positoins due to flexibiliy in the ZA-loop. In BRD4-ligand complexes the number and positions of conserved water molecules depends on the bound ligand. Identified potential inhibitors bind stably at the acetyl-lysine binding pocket of BRD4 and form direct and water-mediated hydrogen bonds with higher occupancy which may contribute to ligand specificity towards BRD4-BD1. Further, through MM/PBSA we calculated the binding free energies of selected compounds, which shows that they have comparable energies to that of (+)-JQ1, while NSC744713 shows better binding free energy.

Allosteric inhibition of topoisomerase I by pinostrobin: Molecular docking, spectroscopic and topoisomerase I activity studies.

Cancer, the second major cause of mortality trailing the cardiovascular diseases, is a multifactorial heterogeneous disease and growing public health problem worldwide. Owing to severe adverse effects of currently available therapies, there is a growing interest in natural compounds present in our daily diet. Among various natural products, flavonoids-polyphenolic compounds have attracted much attention and have been well-documented for their biological activities. Some flavonoids may inhibit cancer cell proliferation by modulating the action of different enzymes and signal transduction pathways. In the present study, we have evaluated the effect of pinostrobin, a natural flavonoid, on the catalytic activity of topoisomerase I, an essential enzyme for normal DNA replication. Catalytic inhibition of topoisomerase I activity would impair DNA replication of rapidly dividing cancer cells and hence inhibits tumor progression. Pinostrobin interaction with the topoisomerase I and DNA assessed in silico indicated it to form a ternary complex with both. In silico data also suggested pinostrobin to be an effective allosteric inhibitor for topoisomerase I. Further, in vitro investigations such as ethidium bromide displacement assay and spectroscopic studies supported in silico results on the binding of pinostrobin at the interface of topoisomerase I and DNA. Pinostrobin effectively inhibited topoisomerase I activity in vitro further confirming our in silico and in vitro findings. Since topoisomerase I is essential for DNA replication, inhibition of its activity by pinostrobin highlights the therapeutic potential of pinostrobin as an anti-proliferative agent.

Transcription regulation of nuclear receptor PXR: Role of SUMO-1 modification and NDSM in receptor function.

Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the nuclear receptor superfamily of ligand-modulated transcription factors. PXR plays an important role in metabolism and elimination of diverse noxious endobiotics and xenobiotics. Like in case of some nuclear receptors its function may also be differentially altered, positively or negatively, by various post-translational modifications. In this context, regulation of PXR function by SUMOylation is the subject of present investigation. Here, we report that human PXR is modified by SUMO-1 resulting in its enhanced transcriptional activity. RT-PCR analysis showed that PXR SUMOylation in presence of rifampicin also enhances the endogenous expression levels of key PXR-regulated genes like CYP3A4, CYP2C9, MDR1 and UGT1A1. In addition, mammalian two-hybrid assay exhibited enhanced interaction between PXR and co-activator SRC-1. EMSA results revealed that SUMOylation has no influence on the DNA binding ability of PXR. In silico analysis suggested that PXR protein contains four putative SUMOylation sites, centered at K108, K129, K160 and K170. In addition to this, we identified the presence of NDSM (Negative charge amino acid Dependent SUMOylation Motif) in PXR. Substitution of all its four putative lysine residues along with NDSM abolished the effect of SUMO-1-mediated transactivation function of PXR. Furthermore, we show that interaction between PXR and E2-conjugation enzyme UBCh9, an important step for implementation of SUMOylation event, was reduced in case of NDSM mutant PXRD115A. Overall, our results suggest that SUMOylation at specific sites on PXR protein are involved in enhancement of transcription function of this receptor.

In silico screening for novel inhibitors of DNA polymerase III alpha subunit of Mycobacterium tuberculosis (MtbDnaE2, H37Rv).

Tuberculosis, a pandemic disease is caused by Mycobacterium tuberculosis (Mtb). DNA polymerase III encoded by DnaE2 of Mtb is specifically required for its survival in vivo, and hence can be considered to be a potential drug target. Amino acid sequence analysis of the MtbDnaE2 and its human counterpart does not show any significant similarity. Therefore, a 3D model of the MtbDnaE2 was generated using Modeller 9v10 with the template structure of E. Coli DNA polymerase III alpha subunit (2HNH_A). The generated models were validated using a number of programmes such as RAMPAGE/PROCHECK, VERIFY_3D, and ProSA. MtbDnaE2 has few conserved residues and four conserved domains similar to that present in DNA polymerase III of E. coli. In silico screening was performed with bioactive anti-tuberculosis compounds and 6-AU (a known inhibitor of DNA polymerase III of Bacillus subtilis) and its analogues against the modeled MtbDnaE2 structure. Docking was performed using GOLD v5.2 software which resulted in the identification of top ten compounds with high GOLD fitness scores and binding affinity (X-Score). To further evaluate the efficacy of these compounds, in silico ADMET analysis was performed using MedChem Designer v3. Given their high binding affinity to the targeted MtbDnaE2, which is essential for DNA replication in the Mtb and good ADMET properties, these compounds are promising candidates for further evaluation and development as anti-tubercular agents.

A case of leprosy with multiple cranial neuropathy mimicking Melkerson Rosenthal syndrome.

Involvement of cranial nerves is not uncommon in leprosy with trigeminal and facial nerves being commonly affected. Other cranial nerves can also be involved especially in longstanding cases of leprosy towards the lepromatous pole. Herein, we report a case of leprosy with multiple cranial neuropathy mimicking Melkerson Rosenthal syndrome.

Bilateral lagophthalmos in leprosy: is it a rare phenomenon?

Lagophthalmos is one of the well known complications of leprosy due to involvement of the facial nerve. Herein, we report three cases of bilateral lagophthalmos due to leprosy which presented to us within a span of just three months. In all these cases, lagophthalmos was not the presenting complaint and it was detected by the treating doctor during examination. This report is being presented to highlight the importance of cranial nerve examination in all cases of leprosy as at times early changes of lagophthalmos may go unnoticed by the patient.

Interaction of nalidixic acid and ciprofloxacin with wild type and mutated quinolone-resistance-determining region of DNA gyrase A.

The quinolones exert their anti-bacterial activity by binding to DNA gyrase A (GyrA), an essential enzyme in maintenance of DNA topology within bacterial cell. The mutations conferring resistance to quinolones arise within the quinolone-resistance-determining region (QRDR) of GyrA. Therefore, quinolones interaction with wild and mutated GyrA can provide the molecular explanation for resistance. Resistant strains of Salmonella enterica of our hospital have shown mutations in the QRDR of GyrA of serine 83 (to phenylalanine or tyrosine) or aspartic acid 87 (to glycine or tyrosine). In order to understand the association between observed resistance and structural alterations of GyrA with respect to quinolone binding, we have studied the interaction of mutated QRDR of GyrA with nalidixic acid and ciprofloxacin by molecular modeling using GLIDE v4. Analysis of interaction parameters like G-score has revealed reduced interaction between nalidixic acid/ciprofloxacin with QRDR of GyrA in all four mutated cases of resistant strains. The mutation of Ser83 to Phe or Tyr shows least binding for nalidixic acid, while Asp87 to Gly or Tyr exhibits minimal binding for ciprofloxacin. The study also highlights the important role of arginines at 21, 91 and His at 45, which form strong hydrogen bonds (at < 3 A) with quinolones. The hydrophilic OH group of Serine 83, which is in close proximity to the quinolone binding site is replaced by aromatic moieties of Tyr or Phe in mutated GyrA. This replacement leads to steric hindrance for quinolone binding. Therefore, quinolone resistance developed by Salmonella appears to be due to the decreased selectivity and affinity of nalidixic acid/ciprofloxacin to QRDR of GyrA.

Molecular modeling on pyruvate phosphate dikinase of Entamoeba histolytica and in silico virtual screening for novel inhibitors.

Pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica. Inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, we modeled the three dimensional structure of the PPDK protein. We used templates with 51% identity and 67% similarity to employ homology-modeling approach. Stereo chemical quality of protein structure was validated by protein structure validation program PROCHECK and VERIFY3D. Experimental proof available in literature along with the in silico studies indicated Lys21, Arg91, Asp323, Glu325 and Gln337 to be the probable active sites in the target protein. Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica. Further, the phylogeny study was carried out to assess the possibility of using the proposed ligands as inhibitors in related pathogens.

Fluorescence studies of spectrin and its subunits.

To better understand the solution structure of spectrin, the environment of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its alpha and beta subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the beta subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated beta chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2-p-toluidinyl napthalene-6-sulfonic acid (TNS). It therefore appears that low ionic strength, alpha-beta association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solution does not involve exposure of significant numbers of hydrophobic sites.

Lipid unsaturation influences melittin-induced leakage of vesicles.

Investigation of vesicles composed of different phosphatidylcholines revealed that the extent of leakage of internal contents induced by the lytic agent melittin can range from practically none to essentially complete, depending upon the fatty acyl chain composition of the phospholipid. The extent of leakage increases with the number of double bonds in the series dioleoylphosphatidylcholine < dilinoleoylphosphatidylcholine < dilinolenoylphosphatidylcholine. It depends on the length of the saturated chain with 1-myristoyl-2-arachidonoylphosphatidylcholine vesicles being more sensitive to melittin induced leakage than 1-palmi-toyl-2-arachidonoylphosphatidylcholine, 1-stearoyl-2-arachidonoylphosphatidylcholine or 1-palmitoyl-2- docosahexaenoylphosphatidylcholine vesicles. The extent of leakage induced by melittin from vesicles composed of 1-palmitoyl-2-oleoylphosphatidylcholine, dioleoylphosphatidylcholine, 1-palmitoyl-2-arachidonoylphosphatidylcholine and 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine increases with the free volume parameter of these lipids for 1,6-diphenylhexatriene (Straume, M. and Litman, B.J. (1987) Biochemistry 26, 5113-5120). Among the lipid examined here, diphytanoylphosphatidylcholine vesicles were least susceptible to melittin induced leakage. The results indicate that lipid fatty acyl structure may be important in lipid-protein interactions of the kind simulated by melittin.

Experimental method to correct fluorescence intensities for the inner filter effect.

An empirical procedure for the correction of measured fluorescence intensity for the inner filter effect is described. The procedure is more reliable than those described previously as fluorophores with the fluorescence properties of interest are sequestered from the external aqueous phase. The inner filter effect can therefore be assessed in the absence of chemical reaction or Forster energy transfer between the fluorophore and chromophore. A correction curve for the inner filter effect is obtained by measuring the fluorescence intensity of the sequestered fluorophore after adding a chromophore to increase the absorbance of the solution. The procedure was tested with liposomes containing calcein and two commercial polymer bead preparations containing embedded fluorophores. The former have the advantage of a wider choice of fluorophores and the latter of stability and convenience. The inner filter effect was varied using small aliquots of potassium chromate or pyridinium chloride solution. It was shown that the magnitude of the inner filter effect must be experimentally determined for each instrument and whenever the instrumental configuration is altered if an accurate correction for the inner filter effect is to be obtained. The magnitude of the correction depends on the wavelength range and the pathlength but not on slit-width or sample turbidity for the instrument employed for most of the experiments reported here. This empirical procedure makes possible studies of protein fluorescence quenching by agents previously unsuitable because of high absorbances.

A robust and efficient automated docking algorithm for molecular recognition.

A completely automated method is described for determining the most likely mode of binding of two (macro)molecules from the knowledge of their three-dimensional structures alone. The method is based on well-known graph theoretical techniques and has been used successfully to determine and rationalize the binding of a number of known macromolecular complexes. In this article we present results for a special case of the general molecular recognition problem--given the information concerning the particular atoms involved in the binding for one of the molecules, the algorithm can correctly identify the corresponding (contacting) atoms of the other molecule. The approach used can be easily extended to the general molecular recognition problem and requires the extraction of maximal common subgraphs. In these studies the docking of the macromolecules was achieved without the aid of computer graphics or other visual aids. The algorithm has been used to determine the correct mode of binding of a protein antigen to an antibody in approximately 100 min on a DEC micro VAX 3600.

Defining topological equivalences in macromolecules.

We describe a completely automated and objective method for defining topological equivalents in macromolecules. The method is based on well established techniques for identifying topologically and topographically equivalent atoms in small molecules and has been used in structural alignment of proteins and RNA molecules, and to extract fragments of molecules (protein secondary structures and RNA and DNA double helices) from structural databases consistent with some specified template structure.

Characteristics of spectrin-induced leakage of extruded, phosphatidylserine vesicles.

At neutral pH spectrin induces modest leakage of trapped calcein from reverse-phase or extruded, but not sonicated, vesicles composed of phosphatidylserine, but not phosphatidylcholine. The extent of leakage from extruded vesicles is not or is only slightly affected by magnesium ions at a physiological concentration or calcium ions at a greater than physiological concentration, respectively. In addition to accounting for several previously discrepant observations on the lytic effects of spectrin, these findings indicate that some proteins like spectrin may destabilize vesicles with low curvature more readily than vesicles of high curvature, in contrast to certain amphiphilic peptides. 60% less leakage is induced from phosphatidylserine vesicles by heat-denatured than by native spectrin. In contrast, both trypsin- and subtilisin-treated spectrins, if sufficiently digested, induce several-fold more leakage than undigested spectrin. Since spectrin prepared either by 1 M Tris dissociation of Triton-extracted cytoskeletons or by low ionic strength extraction of ghosts released the same amounts of calcein from vesicles of various compositions, these effects are unlikely to reflect artifacts of spectrin preparation. Furthermore, spectrin is unlikely to promote leakage in vivo, since vesicles composed of phosphatidylserine, cholesterol and/or phosphatidylethanolamine, which constitute the lipid composition of the inner monolayer of the red cell membrane, did not leak on addition of spectrin, whereas vesicles composed of phosphatidylserine and phosphatidylcholine, did leak in the presence of spectrin.

Small-volume extrusion apparatus for preparation of large, unilamellar vesicles.

The design and performance of a filter holder which enables convenient preparation of volumes of up to a milliliter of large, unilamellar vesicles formed by extrusion (LUVETs) from multilamellar vesicles (MLVs) are described. The filter holder provides for back-and-forth passage of the sample between two syringes, a design that minimizes filter blockage, eliminates the need to change filters during LUVET preparation and reduces preparation time to a few minutes. Replicas of slam-frozen LUVETs in the electron microscope are unilamellar and reasonably homogeneous with an average diameter close to the pore size of the filters used to extrude them. Extrusion per se does not destabilize the vesicles, which trapped a fluorescent dye only when they were disrupted on freeze-thawing and during the first extrusion when most of the MLVs were apparently converted to LUVETs.

Association of a pH-sensitive peptide with membrane vesicles: role of amino acid sequence.

The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.

Lecithin:cholesterol acyltransferase activation by synthetic amphipathic peptides.

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.