Novel chalcogenides of thymidine and uridine: synthesis, properties and applications

A facile and efficient methodology has been developed for the synthesis of dithymidine and di-uridine derived disulfides using benzyltriethylammonium tetrathiomolybdate as a sulfur transfer reagent. However, a similar reaction of thymidine derivative with tetraethylammonium tetraselenotungstate as a selenium transfer reagent resulted in the formation of an unexpected cyclic diselenide. The disulfide derivatives of nucleosides have been used as precursors in a tandem disulfide cleavage-Michael addition/ring opening reactions to construct aminoacid and carbocyclic derivatives of nucleosides.     

Serum Metabolomic Profiling in Acute Alcoholic Hepatitis Identifies Multiple Dysregulated Pathways

Background and Objectives

While animal studies have implicated derangements of global energy homeostasis in the pathogenesis of acute alcoholic hepatitis (AAH), the relevance of these findings to the development of human AAH remains unclear. Using global, unbiased serum metabolomics analysis, we sought to characterize alterations in metabolic pathways associated with severe AAH and identify potential biomarkers for disease prognosis.

Methods

This prospective, case-control study design included 25 patients with severe AAH and 25 ambulatory patients with alcoholic cirrhosis. Serum samples were collected within 24 hours of the index clinical encounter. Global, unbiased metabolomics profiling was performed. Patients were followed for 180 days after enrollment to determine survival.

Results

Levels of 234 biochemicals were altered in subjects with severe AAH. Random-forest analysis, principal component analysis, and integrated hierarchical clustering methods demonstrated that metabolomics profiles separated the two cohorts with 100% accuracy. Severe AAH was associated with enhanced triglyceride lipolysis, impaired mitochondrial fatty acid beta oxidation, and upregulated omega oxidation. Low levels of multiple lysolipids and related metabolites suggested decreased plasma membrane remodeling in severe AAH. While most measured bile acids were increased in severe AAH, low deoxycholate and glycodeoxycholate levels indicated intestinal dysbiosis. Several changes in substrate utilization for energy homeostasis were identified in severe AAH, including increased glucose consumption by the pentose phosphate pathway, altered tricarboxylic acid (TCA) cycle activity, and enhanced peptide catabolism. Finally, altered levels of small molecules related to glutathione metabolism and antioxidant vitamin depletion were observed in patients with severe AAH. Univariable logistic regression revealed 15 metabolites associated with 180-day survival in severe AAH.

Conclusion

Severe AAH is characterized by a distinct metabolic phenotype spanning multiple pathways. Metabolomics profiling revealed a panel of biomarkers for disease prognosis, and future studies are planned to validate these findings in larger cohorts of patients with severe AAH.     

An inherited variant in the gene coding for vitamin D‐binding protein and survival from cutaneous melanoma: a BioGenoMEL study

An association between low serum vitamin D levels and poorer melanoma survival has been reported. We have studied inheritance of a polymorphism of the GC gene, rs2282679, coding for the vitamin D‐binding protein, which is associated with lower serum levels of vitamin D, in a meta‐analysis of 3137 melanoma patients. The aim was to investigate evidence for a causal relationship between vitamin D and outcome (Mendelian randomization). The variant was not associated with reduced overall survival (OS) in the UK cohort, per‐allele hazard ratio (HR) for death 1.23 (95% confidence interval (CI) 0.93, 1.64). In the smaller cohorts, HR in OS analysis was 1.07 (95% CI 0.88, 1.3) and for all cohorts combined, HR for OS was 1.09 (95% CI 0.93, 1.29). There was evidence of increased melanoma‐specific deaths in the seven cohorts for which these data were available. The lack of unequivocal findings despite the large sample size illustrates the difficulties of implementing Mendelian randomization.     

2'-deoxy-2'-α-fluoro-2'-β-C-methyl 3',5'-cyclic phosphate nucleotide prodrug analogs as inhibitors of HCV NS5B polymerase: discovery of PSI-352938

A series of novel 2'-deoxy-2'-α-fluoro-2'-β-C-methyl 3',5'-cyclic phosphate nucleotide prodrug analogs were synthesized and evaluated for their in vitro anti-HCV activity and safety. These prodrugs demonstrated a 10-100-fold greater potency than the parent nucleoside in a cell-based replicon assay due to higher cellular triphosphate levels. Our structure-activity relationship (SAR) studies provided compounds that gave high levels of active triphosphate in rat liver when administered orally to rats. These studies ultimately led to the selection of the clinical development candidate 24a (PSI-352938).     

Synthesis and anti-HCV activity of 3′,4′-oxetane nucleosides

Hepatitis C virus afflicts approximately 180 million people worldwide and currently there are no direct acting antiviral agents available to treat this disease. Our first generation nucleoside HCV inhibitor, RG7128 has already established proof-of-concept in the clinic and is currently in phase IIb clinical trials. As part of our continuing efforts to discover novel anti-HCV agents, 3′,4′-oxetane cytidine and adenosine nucleosides were prepared as inhibitors of HCV RNA replication. These nucleosides were shown not to be inhibitors of HCV as determined in a whole cell subgenomic replicon assay. However, 2′-mono/diflouro analogs, 4, 5, and 6 were readily phosphorylated to their monophosphate metabolites by deoxycytidine kinase and their triphosphate derivatives were shown to be inhibitors of HCV NS5B polymerase in vitro. Lack of anti-HCV activity in the replicon assay may be due to the inability of the monophosphates to be converted to their corresponding diphosphates.     

Effect of exogenous insulin administration on ovarian function, embryo/fetal development during pregnancy in goats

The effect of insulin was investigated on ovarian follicle population, ovulation rate, hormonal profiles and embryo/fetal development during pregnancy using transrectal ultrasonography in goats. Twelve goats synchronized in estrus were selected for the experiment. They were divided into two groups, viz. (untreated control, n=6) and (insulin treated, n=6). In treated group long acting bovine insulin was administered @ 0.2IU/kg body weight subcutaneously for three consecutive days, i.e. days 7-9 of estrous cycle. Thereafter, weekly single injection of insulin was continued for rest of the experiment. However, in control group only normal saline was injected as placebo. Breeding was allowed by natural service in both the groups. The does were subjected to B-mode transrectal ultrasound scanning of ovary and uterus weekly up to 120 and 98 days of gestation, respectively. Blood samples were collected weekly up to 135 days of gestation for the estimation of estradiol 17beta and progesterone (P4). The result revealed no difference in mean number of total follicles between the control and insulin treated groups. The diameter of medium follicle did not differ where as diameter of large follicle was comparatively higher in treated than control goats. The average number of corpus luteum (CL) was higher in insulin treated group as compared to control (1.66 vs. 1.16). However, the number as well as mean diameter of CL did not differ significantly between treated and control group. Serum concentrations of estradiol 17beta and progesterone were significantly (P<0.01) higher in treated than control goats. Embryonic vesicle was detected by day 21 in both the groups, however, its diameter did not differ significantly (0.73 and 0.72cm) between the groups. The twinning percentage was higher (50 vs. 16%) in insulin treated than the control goats. Placentome diameter was also higher (P>0.05) in treated animals. The results demonstrated beneficial effect of exogenous administration of insulin on ovarian function and twinning percentage in goats.     

Energetics of the loop-to-helix transition leading to the coiled-coil structure of influenza virus hemagglutinin HA2 subunits

Fusion of influenza virus with the endosomal membrane of the host cell is mediated by the homotrimer-organized glycoprotein hemagglutinin (HA). Its fusion activity is triggered by a low pH-mediated conformational change affecting the structure of the HA1 and HA2 subunits. The HA2 subunits undergo a loop-to-helix transition leading to a coiled-coil structure, a highly conserved motif for many fusion mediating viral proteins. However, experimental studies showed that the HA2 coiled-coil structure is stable at neutral and low pH, implying that there is no direct relationship between low pH and the HA2 loop-to-helix transition. To interpret this observation, we used a computational approach based on the dielectric continuum solvent model to explore the influence of water and pH on the free energy change of the transition. The computations showed that the electrostatic interaction between HA2 fragments and water is the major driving force of the HA2 loop-to-helix transition leading to the coiled-coil structure, as long as the HA1 globular domain covering the HA2 subunits in the nonfusion competent conformation is reorganized and thereby allows water molecules to interact with the whole loop segments of the HA2 subunits. Moreover, we show that the energy released by the loop-to-helix transition may account for those energies required for driving the subsequent steps of membrane fusion. Such a water-driven process may resemble a general mechanism for the formation of the highly conserved coiled-coil motif of enveloped viruses.     

Purification,characterization and molecular cloning of a monocot mannose-binding lectin from <Emphasis Type="Italic">Remusatia vivipara</Emphasis> with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80°C and under wide range of pH (2.0–9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 μg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.     

Retardation of dark inducedin vitro alterations in photosystem 2 organisation of cowpea leaf discs by combination of Ca2+ and benzyladenine

Cowpea (Vigna unguiculata L.) leaf discs incubated in the dark in solutions with CaCl₂ and benzyladenine (BA) maintained higher concentration of chlorophylls and proteins and higher activity of photosystem 2 (PS2) than controls. The effect of CaCl₂ and BA in combination was additive.