Design,Synthesis and Molecular Docking Studies of Novel Indole–Isoxazole–Triazole Conjugates as Potent Antibacterial Agents

Russian Journal of Bioorganic Chemistry - In search of better antibacterial agents, a series of novel 5-((aryl)methyl)-3-(1H-indol-2-yl)isoxazole (IIIa–e) and...     

Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo–devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion–deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi_non/syn was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.     

Effects of methyl parathion (o,o-dimethyl o-4-nitrophenyl phosphorothioate) on rat sperm morphology and sperm count, but not fertility, are associated with decreased ascorbic acid level in the testis

Methyl parathion (MP; o,o-dimethyl o-4-nitrophenyl phosphorothioate) is an organophosphorous pesticide used world wide to spray agricultural crops. The present study was aimed to investigate the genotoxic and cytotoxic effects on male germ cells and their possible relation with testicular ascorbic acid levels. Adult male Wistar rats (n=5/group) received MP at 0, 0.5, or 1 mg/kg (experiments 1 and 2) for 12 days and 0, 0.75 or 1.5 mg/kg (experiment 3) for 25 days (i.p.) everyday at intervals of 24 h. The epididymal sperm count, sperm abnormalities and testicular ascorbic acid levels (by 2,4-dinitrophenyl hydrazine method) were estimated on days 130, 77 and 17 following the last exposure in experiments 1, 2, and 3, respectively. Virgin untreated female rats were mated with treated males from experiments 2 and 3 for a week effective from day 35 to 41 after the first treatment, and fertility indices were measured after the birth of pups. Sperm count was decreased in experiments 2 and 3 (P<0.01), and in all three experiments, the abnormal sperms increased (P<0.001). Concomitantly, the ascorbic acid levels decreased in the testis (P<0.05-0.001; one-way ANOVA and Bonferroni's post hoc test). The body weights of offspring of treated males did not show significant changes from those of the controls, although there were some decreases observed. MP reduced the lactation index in experiment 2 (P<0.001; Chi-square test). The number of pups/parent along with fertility indices showed some numerical decrease but without any statistical significance. The present findings suggest that MP is a weak genotoxic and cytotoxic agent in the rat exposed to human exposure dose-levels, and that these effects, except the fertility are well correlated with decreased ascorbic acid level in the testis. Furthermore, MP-induced changes in the germ cells do not have any significant effects on F1 generation.     

Dysbiosis in the Gut Bacterial Microbiome of Patients with Uveitis,an Inflammatory Disease of the Eye

Uveitis (UVT), an inflammatory disease of the eye significantly contributes to vision impairment and blindness. Uveitis is associated with systemic infectious and autoimmune diseases, but in most cases, the aetiology remains unidentified. Dysbiosis in the gut microbiome has been implicated in autoimmune diseases, inflammatory diseases, cancers and mental disorders. In a mice model of autoimmune UVT, it was observed that manipulating the gut microbiome reduces the inflammation and disease severity. Further, alterations in the bacterial gut microbiome and their metabolites were reported in UVT patients from a Chinese cohort. Hence, it is worth comparing the bacterial gut microbiome of UVT patients with that of healthy controls (HC) to ascertain whether dysbiosis of the gut microbiome has implications in UVT. Our analyses showed reduced diversity of several anti-inflammatory organisms including Faecalibacterium, Bacteroides, Lachnospira, Ruminococcus and members of Lachnospiraceae and Ruminococcaceae families, and enrichment of Prevotella (proinflammatory) and Streptococcus (pathogenic) OTUs in UVT microbiomes compared to HC. In addition, decrease in probiotic and antibacterial organisms was observed in UVT compared to HC microbiomes. Heatmap and PCoA plots also indicated significant variations in the microbiomes of UVT versus HC. This is the first study demonstrating dysbiosis in the gut bacterial communities of UVT patients in an Indian cohort and suggests a role of the gut microbiome in the pathophysiology of UVT.     

Alterations in gut bacterial and fungal microbiomes are associated with bacterial Keratitis,an inflammatory disease of the human eye

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n?=?21) and bacterial Keratitis patients (BK, n?=?19). An increase in abundance of several anti-inflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut–eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.     

Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells

Background

Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines.

Methodology/Principal Findings

In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5–50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3.

Conclusions/Significance

Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.     

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.     

Comparative Endothelial Cell Response on Topographically Patterned Titanium and Silicon Substrates with Micrometer to Sub-Micrometer Feature Sizes

In this work, we evaluate the in vitro response of endothelial cells (EC) to variation in precisely-defined, micrometer to sub-micrometer scale topography on two different substrate materials, titanium (Ti) and silicon (Si). Both substrates possess identically-patterned surfaces composed of microfabricated, groove-based gratings with groove widths ranging from 0.5 to 50 µm, grating pitch twice the groove width, and groove depth of 1.3 µm. These specific materials are chosen due to their relevance for implantable microdevice applications, while grating-based patterns are chosen for the potential they afford for inducing elongated and aligned cellular morphologies reminiscent of the native endothelium. Using EA926 cells, a human EC variant, we show significant improvement in cellular adhesion, proliferation, morphology, and function with decreasing feature size on patterned Ti substrates. Moreover, we show similar trending on patterned Si substrates, albeit to a lesser extent than on comparably patterned Ti substrates. Collectively, these results suggest promise for sub-micrometer topographic patterning in general, and sub-micrometer patterning of Ti specifically, as a means for enhancing endothelialization and neovascularisation for novel implantable microdevice applications.