Nematicidal activity of paucimannose-type glycoconjugates from acacia honey.

Natural honey contains glycoconjugates as minor components. We characterized acacia honey glycoconjugates with molecular masses in the range of 2-5 kDa. The glycoconjugates were separated by RP-HPLC into three peaks (termed RP-2-5 k-I, RP-2-5 k-II, and RP-2-5 k-III) which demonstrated paralyzing effects on the model nematode C. elegans (ED50 of 50 ng glycoconjugates/µL). To examine molecular mechanisms underlying the nematicidal effects of honey glycoconjugates, expressional analyses of genes that are essential for the growth, development, reproduction, and movement of C. elegans were carried out. Quantitative PCR-based assays showed that these molecules moderately regulate the expression of genes involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1 and act-2). MALDI-ToF-MS/MS analysis of RP-HPLC peaks revealed the presence of paucimannose-like N-glycans which are known to play important roles in invertebrates e.g., worms and flies. These findings provided novel information regarding the structure and nematicidal function of honey glycoconjugates.

Understanding the impact of structural modifications at the NNAT gene's post-translational acetylation site: in silico approach for predicting its drug-interaction role in anorexia nervosa.

PURPOSE: Anorexia nervosa (AN) is a neuropsychological public health concern with a socially disabling routine and affects a person's healthy relationship with food. The role of the NNAT (Neuronatin) gene in AN is well established. The impact of mutation at the protein's post-translational modification (PTM) site has been exclusively associated with the worsening of the protein's biochemical dynamics. METHODS: To understand the relationship between genotype and phenotype, it is essential to investigate the appropriate molecular stability of protein required for proper biological functioning. In this regard, we investigated the PTM-acetylation site of the NNAT gene in terms of 19 other specific amino acid probabilities in place of wild type (WT) through various in silico algorithms. Based on the highest pathogenic impact computed through the consensus classifier tool, we generated 3 residue-specific (K59D, P, W) structurally modified 3D models of NNAT. These models were further tested through the AutoDock Vina tool to compute the molecular drug binding affinities and inhibition constant (Ki) of structural variants and WT 3D models. RESULTS: With trained in silico machine learning algorithms and consensus classifier; the three structural modifications (K59D, P, W), which were also the most deleterious substitution at the acetylation site of the NNAT gene, showed the highest structural destabilization and decreased molecular flexibility. The validation and quality assessment of the 3D model of these structural modifications and WT were performed. They were further docked with drugs used to manage AN, it was found that the ΔGbind (kcal/mol) values and the inhibition constants (Ki) were relatively lower in structurally modified models as compared to WT. CONCLUSION: We concluded that any future structural variation(s) at the PTM-acetylation site of the NNAT gene due to possible mutational consequences, will serve as a basis to explore its relationship with the propensity of developing AN. LEVEL OF EVIDENCE: No level of evidence-open access bioinformatics research.

Characterization of the genome and serine protease of a novel Bacillus subtilis isolate.

Current study was undertaken to carry out the genome-wide analysis of a multipotent isolate from desert soil which was previously identified as Bacillus tequilensis based on 16S rDNA analysis. This study also aims to characterize the serine protease and its biocatalytic potentials implying a combination of empirical and in-silico approaches. Next generation sequencing and short read de novo assembly generated the 4,235,084 bp draft genome of Bacillus sp. ZMS-2. Genome sequence analysis by digital DNA:DNA hybridization (dDDH) and average nucleotide identity classified the isolate as Bacillus subtilis ZMS-2 (Bioproject ID: PRJNA691551). Genome annotation revealed 10 antibiotic resistance genes, 8 antibiotic/antifungal gene clusters and 25 genes encoding proteases including subtilisin E, an extracellular alkaline protease. This extracellular protease (ZMS-2 protease) was produced using a statistically optimized medium, purified partially and characterized as alkaline serine protease. The partially purified ZMS-2 protease (780 U/mL) showed a 21 mm zone of casein hydrolysis and dehaired goat skin by pulling out hair with roots. These catalytic potentials of ZMS-2 protease were further confirmed using scanning electron microscopy of casein beads and dehaired skin. The study concludes B. subtilis ZMS-2 as a potent producer of a protease with promising potentials of commercial importance.

In silico identification of the rare-coding pathogenic mutations and structural modeling of human NNAT gene associated with anorexia nervosa.

PURPOSE: Increased susceptibility towards anorexia nervosa (AN) was reported with reduced levels of neuronatin (NNAT) gene. We sought to investigate the most pathogenic rare-coding missense mutations, non-synonymous single-nucleotide polymorphisms (nsSNPs) of NNAT and their potential damaging impact on protein function through transcript level sequence and structure based in silico approaches. METHODS: Gene sequence, single nucleotide polymorphisms (SNPs) of NNAT was retrieved from public databases and the putative post-translational modification (PTM) sites were analyzed. Distinctive in silico algorithms were recruited for transcript level SNPs analyses and to characterized high-risk rare-coding nsSNPs along with their impact on protein stability function. Ab initio 3D-modeling of wild-type, alternate model prediction for most deleterious nsSNP, validation and recognition of druggable binding pockets were also performed. AN 3D therapeutic compounds that followed rule of drug-likeness were docked with most pathogenic variant of NNAT to estimate the drugs' binding free energies. RESULTS: Conclusively, 10 transcript (201-205)-based nsSNPs from 3 rare-coding missense variants, i.e., rs539681368, rs542858994, rs560845323 out of 840 exonic SNPs were identified. Transcript-based functional impact analyses predicted rs539681368 (C30Y) from NNAT-204 as the high-risk rare-coding pathogenic nsSNP, deviating protein functions. The 3D-modeling analysis of AN drugs' binding energies indicated lowest binding free energy (ΔG) and significant inhibition constant (Ki) with mutant models C30Y. CONCLUSIONS: Mutant model (C30Y) exhibiting significant drug binding affinity and the commonest interaction observed at the acetylation site K59. Thus, based on these findings, we concluded that the identified nsSNP may serve as potential targets for various studies, diagnosis and therapeutic interventions. LEVEL OF EVIDENCE: No level of evidence-open access bioinformatics research.

3-substituted coumarin derivatives over-activate the HslV protease: A potential drug target for antibacterial activity.

The bacterial HslVU complex consists of two different proteins, i.e., the HslV protease and the HslU ATPase. The functional HslVU enzyme complex forms only when the HslU c-terminal helix is inserted into the cavity located between two adjacent HslV monomers in order to allosterically activate the HslV protease. Based on its essential role in maintaining microbial proteostasis as well its absence from human beings, it is considered a promising therapeutic target for designing antibacterial agents. The goal of the present study was to find out potential drug candidates that could over-activate the HslV protease and produce aberrant proteolysis in pathogenic bacteria. Derivatives of 3-substituted coumarin have been identified as potential HslV protease activators based on their highest docking scores, ideal interaction patterns, and significant in-vitro HslV activation potential. Their ED50 values were in the sub-micromolar range, i.e., 0.4-0.48µM. The conformational stability of the contacts between the HslV dimer and the active compounds was further confirmed by molecular dynamics studies. Correspondingly, the ADMET characteristics of these lead molecules considerably demonstrated their significant non-toxic drug-like abilities. This research not only identified small non-peptidic HslV protease activators but also improved the understanding of the mode of action of 3-substituted coumarin derivatives as antibacterials.

Analysis of Inflammatory Gene Expression Profile of Peripheral Blood Leukocytes in Type 2 Diabetes.

The pathophysiology of type 2 diabetes (T2DM) is associated with perturbation of innate immune response. Several studies indicated alteration of pro-inflammatory and anti-inflammatory cytokines, chemokines and other mediators of innate immune response in T2DM. This study was designed to perform quantitative PCR-based expression profiling of genes involved in inflammation (i.e. CASP1, CASP5, CCL5, CXC11, CCR5, NF-Κb, IL-4, PPARG and PGC1α) in peripheral blood leukocytes of T2DM patients. The T2DM patients are often prescribed with metformin and insulin while metformin has also been reported to possess anti-inflammatory activity. To address the question whether metformin exerts any effect on inflammatory mediators in bloodstream, human subjects in this study were divided into four groups on the basis of medication they were taking during last 6 month. These groups included NT-T2DM (T2DM patients not taking medication, n = 34), Met-T2DM (T2DM patients taking metformin, n = 33), INS-T2DM (T2DM patients taking insulin, n = 15) and NGT (normoglycemic subjects, n = 34) groups. Differential expression of gene transcripts at a cutoff of fourfold was considered significant. In the NT-T2DM group, transcripts of inflammation-related genes (i.e. CASP1, CASP5, CCL5, CCR5 and NF-kB) were up-regulated while transcripts of PPARG and PGC1α genes were down-regulated compared to NGT group. On the other hand, down-regulation of CASP1, CASP5, CCL5, CCR5 and NF-kB transcripts was evident in Met-T2DM and INS-T2DM groups when compared to the NT-T2DM group. The Met-T2DM group and INS-T2DM group showed a significant difference in the transcript level of CASP1 and CCL5 which are more down-regulated in the Met-T2DM group compared to INS-T2DM group. These findings indicated that (a) in T2DM, expression of inflammation-related genes is up-regulated and (b) anti-inflammatory activity of metformin appears to be independent of its anti-hyperglycemic activity.

Mutational landscape of head and neck squamous cell carcinomas in a South Asian population.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type globally and contributes significantly to burden of disease in South Asia. In Pakistan, HNSCC is among the most commonly diagnosed cancer in males and females. The increasing regional burden of HNSCC along with a unique set of risk factors merited a deeper investigation of the disease at the genomic level. Whole exome sequencing of HNSCC samples and matched normal genomic DNA analysis (n=7) was performed. Significant somatic single nucleotide variants (SNVs) were identified and pathway analysis performed to determine frequently affected signaling pathways. We identified significant, novel recurrent mutations in ASNS (asparagine synthetase) that may affect substrate binding, and variants in driver genes including TP53, PIK3CA, FGFR2, ARID2, MLL3, MYC and ALK. Using the IntOGen platform, we identified MAP kinase, cell cycle, actin cytoskeleton regulation, PI3K-Akt signaling and other pathways in cancer as affected in the samples. This data is the first of its kind from the Pakistani population. The results of this study can guide a better mechanistic understanding of HNSCC in the population, ultimately contributing new, rational therapeutic targets for the treatment of the disease.

Nematicidal activity of 'major royal jelly protein'-containing glycoproteins from Acacia honey.

Parasitic nematodes infect more than two billion people worldwide particularly in developing countries. We previously reported nematicidal activity of natural honey using model nematode Caenorhabditis elegans. In this study, characterization of nematicidal effects of natural honey and its glycoproteins has been carried out. Chromatographically separated honey glycoproteins showed potent anti-C. elegans activity (LD50 = 100 ng proteins/µL). Honey glycoproteins with molecular masses of ∼260 kD and ∼160 kD comprised of 'major royal jelly protein-1'-containing complexes. In these complexes, MRJP1 was present in different glycosylation forms. Quantitative PCR based gene expression assays described molecular functions of C. elegans affected by honey and honey glycoproteins. Expression of 14 gene transcripts associated with key cellular and molecular functions including energy metabolism, cytoskeleton, cell division, transcription and translation was analyzed. Acacia honey exerted a concentration-dependent alteration of gene transcripts involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1, act-2, and arp6). Likewise, MRJP1-containing glycoproteins caused down-regulation of arp-6 and idhg-1; and up-regulation of act-1 and mdh-1 gene transcripts. Consistent down-regulation of isocitrate dehydrogenase encoding idhg-1 gene which is among the rate-controlling enzymes of the citric acid cycle was considered as main biochemical factor involved in the nematicidal activity of honey and MRJP-containing glycoproteins. Acacia honey suppressed the expression of gene transcripts encoding actin-2, while honey glycoproteins did not. Hence, honey partly exerted anti-C. elegans activity by decreasing the transcription of actin-2 gene transcripts, demonstrated by a defect in the movement and egg laying. Moreover, arp-6 gene transcripts encoding actin-related protein 6 was significantly and constantly down-regulated by honey and honey proteins.

Carbohydrazones as new class of carbonic anhydrase inhibitors: Synthesis, kinetics, and ligand docking studies.

Discovery and development of carbonic anhydrase inhibitors is crucial for their clinical use as antiepileptic, diurectic and antiglaucoma agents. Keeping this in mind, we have synthesized carbohydrazones 1-27 and evaluated them for their in vitro carbonic anhydrase inhibitory potential. Out of twenty-seven compounds, compounds 1 (IC50=1.33±0.01µM), 2 (IC50=1.85±0.24µM), 3 (IC50=1.37±0.06µM), and 9 (IC50=1.46±0.12µM) have showed carbonic anhydrase inhibition better than the standard drug zonisamide (IC50=1.86±0.03µM). Moreover, compounds 4 (IC50=2.32±0.04µM), 5 (IC50=3.96±0.35µM), 7 (IC50=2.33±0.02µM), and 8 (IC50=2.67±0.01µM) showed good inhibitory activity. Cheminformatic analysis has shown that compounds 1 and 2 possess lead-like properties. In addition, kinetic and molecular docking studies were also performed to investigate the binding interaction between carbohydrazones and carbonic anhydrase enzyme. This study has identified a novel and potent class of carbonic anhydrase inhibitors with the potential to be investigated further.

Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.

We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.