A Nano-MgO and Ionic Liquid-Catalyzed ‘Green’ Synthesis Protocol for the Development of Adamantyl-Imidazolo-Thiadiazoles as Anti-Tuberculosis Agents Targeting Sterol 14α-Demethylase (CYP51)

In this work, we describe the ‘green’ synthesis of novel 6-(adamantan-1-yl)-2-substituted-imidazo[2,1-b][1,3,4]thiadiazoles (AITs) by ring formation reactions using 1-(adamantan-1-yl)-2-bromoethanone and 5-alkyl/aryl-2-amino1,3,4-thiadiazoles on a nano material base in ionic liquid media. Given the established activity of imidazothiadiazoles against M. tuberculosis, we next examined the anti-TB activity of AITs against the H₃₇Rv strain using Alamar blue assay. Among the tested compounds 6-(adamantan-1-yl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (3f) showed potent inhibitory activity towards M. tuberculosis with an MIC value of 8.5 μM. The inhibitory effect of this molecule against M. tuberculosis was comparable to the standard drugs such as Pyrazinamide, Streptomycin, and Ciprofloxacin drugs. Mechanistically, an in silico analysis predicted sterol 14α-demethylase (CYP51) as the likely target and experimental activity of 3f in this system corroborated the in silico target prediction. In summary, we herein report the synthesis and biological evaluation of novel AITs against M. tuberculosis that likely target CYP51 to induce their antimycobacterial activity.     

Losses at chromosome 4q are associated with poor survival in operable ductal pancreatic adenocarcinoma

Here we tested the prognostic impact of genomic alterations in operable localized pancreatic ductal adenocarcinoma (PDAC). Fifty-two formalin-fixed and paraffin-embedded primary PDAC were laser micro-dissected and were investigated by comparative genomic hybridization after whole genome amplification using an adapter-linker PCR. Chromosomal gains and losses were correlated to clinico-pathological parameters and clinical follow-up data. The most frequent aberration was loss on chromosome 17p (65%) while the most frequent gains were detected at 2q (41%) and 8q (41%), respectively. The concomitant occurrence of losses at 9p and 17p was found to be statistically significant. Higher rates of chromosomal losses were associated with a more advanced primary tumor stage and losses at 9p and 18q were significantly associated with presence of lymphatic metastasis (chi-square: p = 0.03, p = 0.05, respectively). Deletions on chromosome 4 were of prognostic significance for overall survival and tumor recurrence (Cox-multivariate analysis: p = 0.026 and p = 0.021, respectively). In conclusion our data suggest the common alterations at chromosome 8q, 9p, 17p and 18q as well as the prognostic relevant deletions on chromosome 4q as relevant for PDAC progression. Our comprehensive data from 52 PDAC should provide a basis for future studies with a higher resolution to discover the relevant genes located within the chromosomal aberrations identified.     

EPHA2 polymorphisms and age-related cataract in India

Objective

We investigated whether previously reported single nucleotide polymorphisms (SNPs) of EPHA2 in European studies are associated with cataract in India.

Methods

We carried out a population-based genetic association study. We enumerated randomly sampled villages in two areas of north and south India to identify people aged 40 and over. Participants attended a clinical examination including lens photography and provided a blood sample for genotyping. Lens images were graded by the Lens Opacification Classification System (LOCS III). Cataract was defined as a LOCS III grade of nuclear ≥4, cortical ≥3, posterior sub-capsular (PSC) ≥2, or dense opacities or aphakia/pseudophakia in either eye. We genotyped SNPs rs3754334, rs7543472 and rs11260867 on genomic DNA extracted from peripheral blood leukocytes using TaqMan assays in an ABI 7900 real-time PCR. We used logistic regression with robust standard errors to examine the association between cataract and the EPHA2 SNPs, adjusting for age, sex and location.

Results

7418 participants had data on at least one of the SNPs investigated. Genotype frequencies of controls were in Hardy-Weinberg Equilibrium (p>0.05). There was no association of rs3754334 with cataract or type of cataract. Minor allele homozygous genotypes of rs7543472 and rs11260867 compared to the major homozygote genotype were associated with cortical cataract, Odds ratio (OR) = 1.8, 95% Confidence Interval (CI) (1.1, 3.1) p = 0.03 and 2.9 (1.2, 7.1) p = 0.01 respectively, and with PSC cataract, OR = 1.5 (1.1, 2.2) p = 0.02 and 1.8 (0.9, 3.6) p = 0.07 respectively. There was no consistent association of SNPs with nuclear cataract or a combined variable of any type of cataract including operated cataract.

Conclusions

Our results in the Indian population agree with previous studies of the association of EPHA2 variants with cortical cataracts. We report new findings for the association with PSC which is particularly prevalent in Indians.     

Decreased serologic response in vaccinated military recruits during 2011 correspond to genetic drift in concurrent circulating pandemic A/H1N1 viruses

Background

Population-based febrile respiratory illness surveillance conducted by the Department of Defense contributes to an estimate of vaccine effectiveness. Between January and March 2011, 64 cases of 2009 A/H1N1 (pH1N1), including one fatality, were confirmed in immunized recruits at Fort Jackson, South Carolina, suggesting insufficient efficacy for the pH1N1 component of the live attenuated influenza vaccine (LAIV).

Methodology/Principal Findings

To test serologic protection, serum samples were collected at least 30 days post-vaccination from recruits at Fort Jackson (LAIV), Parris Island (LAIV and trivalent inactivated vaccine [TIV]) at Cape May, New Jersey (TIV) and responses measured against pre-vaccination sera. A subset of 78 LAIV and 64 TIV sera pairs from recruits who reported neither influenza vaccination in the prior year nor fever during training were tested by microneutralization (MN) and hemagglutination inhibition (HI) assays. MN results demonstrated that seroconversion in paired sera was greater in those who received TIV versus LAIV (74% and 37%). Additionally, the fold change associated with TIV vaccination was significantly different between circulating (2011) versus the vaccine strain (2009) of pH1N1 viruses (ANOVA p value = 0.0006). HI analyses revealed similar trends. Surface plasmon resonance (SPR) analysis revealed that the quantity, IgG/IgM ratios, and affinity of anti-HA antibodies were significantly greater in TIV vaccinees. Finally, sequence analysis of the HA1 gene in concurrent circulating 2011 pH1N1 isolates from Fort Jackson exhibited modest amino acid divergence from the vaccine strain.

Conclusions/Significance

Among military recruits in 2011, serum antibody response differed by vaccine type (LAIV vs. TIV) and pH1N1 virus year (2009 vs. 2011). We hypothesize that antigen drift in circulating pH1N1 viruses contributed to reduce vaccine effectiveness at Fort Jackson. Our findings have wider implications regarding vaccine protection from circulating pH1N1 viruses in 2011–2012.     

Heterologous expression and characterization of Indian Sahiwal cattle (Bos indicus) alpha inhibin

Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major action as negative feedback control of the production of FSH by the anterior pituitary gland. The physiological role of inhibin has led to the development of inhibin immunogens for fertility enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion would increase FSH concentrations and thus offers a potential for increasing the number of ovulatory follicles in the ovary. The present work was carried out to produce recombinant bovine (Indian Sahiwal Cattle; Bos indicus) alpha inhibin (bINH-α) in E. coli by optimizing its expression and purification in biologically active form and to study its immunological characterization. A bacterial protein expression vector system based on the phage T(5) promoter was used. The bINH-α encoding gene was successfully cloned and expressed in E. coli and the purified recombinant bINH-α was characterized. Recombinant bINH-α (25?μg?mL(-1)) immunized guinea pigs had a significant increase in litter size compared to the control group. These results indicate a role for recombinant bINH-α as a fecundity vaccine to enhance the ovulation rate and litter size in animals.     

Bacterial HA1 vaccine against pandemic H5N1 influenza virus: evidence of oligomerization, hemagglutination, and cross-protective immunity in ferrets

The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. We have developed bacterial systems for expression and purification of properly folded functional hemagglutinin as a rapid response to emerging pandemic strains. A recombinant H5N1 (A/Vietnam/1203/2004) hemagglutinin globular domain (HA1) was produced in Escherichia coli under controlled redox refolding conditions. Importantly, the properly folded HA1(1-320), i.e., HA1 lacking amino acids 321 to 330, contained ≥75% functional oligomers without addition of foreign oligomerization sequence. Site-directed mutagenesis mapped the oligomerization signal to the HA1 N-terminal Ile-Cys-Ile residues at positions 3 to 5. The purified HA1 oligomers (but not monomers) bound fetuin and agglutinated red blood cells. Upon immunization of rabbits, the oligomeric HA1(1-320) elicited potent neutralizing antibodies against homologous and heterologous H5N1 viruses more rapidly than HA1(28-320) containing only monomers. Ferrets vaccinated with oligomeric HA1 (but not monomeric HA1 with the N terminus deleted) at 15 and 3 μg/dose were fully protected from lethality and weight loss after challenge with homologous H5N1 (A/Vietnam/1203/2004, clade 1) virus, as well as heterologous clade 2.2 H5N1 (A/WooperSwan/Mongolia/244/2005) virus. Protection was associated with a significant reduction in viral loads in the nasal washes of homologous and heterologous virus challenged ferrets. This is the first study that describes the presence of an N-terminal oligomerization sequence in the globular domain of influenza virus hemagglutinin. Our findings suggest that functional oligomeric rHA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.     

Different regions of HIV-1 subtype C env are associated with placental localization and in utero mother-to-child transmission

HIV infections are initiated by a limited number of variants that diverge into a diverse quasispecies swarm. During in utero mother-to-child transmission (IU MTCT), transmitted viral variants must pass through multiple unique environments, and our previously published data suggest a nonstochastic model of transmission. As an alternative to a stochastic model of viral transmission, we hypothesize that viral selection in the placental environment influences the character of the viral quasispecies when HIV-1 is transmitted in utero. To test this hypothesis, we used single-template amplification to isolate HIV-1 envelope gene (env) sequences from both peripheral plasma and the placentas of eight nontransmitting (NT) and nine IU-transmitting participants. Statistically significant compartmentalization between peripheral and placental HIV-1 env was detected in one of the eight NT cases and six of the nine IU MTCT cases. In addition, viral sequences isolated from IU MTCT placental tissue showed variation in env V1 loop lengths compared to matched maternal sequences, while NT placental env sequences did not. Finally, comparison of env sequences from NT and IU MTCT participants indicated statistically significant differences in Kyte-Doolittle hydropathy in the signal peptide, C2, V3, and C3 regions. Our working hypothesis is that the hydropathy differences in Env associated with IU MTCT alter viral cellular tropism or affinity, allowing HIV-1 to efficiently infect placentally localized cells.