Short and Straightforward Synthesis of Triphosphates of Artificial Nucleobase Pairs Displaying Unconventional Pairing Scheme

Concise, facile and efficient synthesis of 5′-O-triphosphates of 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2a]-1,3,5-triazin-4(8H)-one (dP) is reported using a one-pot synthetic procedure.     

Synthesis of Zidovudine Derivatives with Anti-HIV-1 and Antibacterial Activities

Twelve novel zidovudine derivatives were prepared by modifying 5 ′-hydroxyl group of sugar moiety (1–8) and 5-methyl group of thymidine nucleus (9–12) and characterized spectrally. The compounds were evaluated for anti-HIV-1, antitubercular and antibacterial activities. Compound (3-azido-tetrahydro-5- (3,4-dihydro-5-methyl-2,4-dioxopyrimidin- 1 (2H)-yl) furan-2-yl)methyl 7- (4- (2-phenylacetoyloxy) -3,5- dimethylpiperazin-1-yl) -5- (2-phenylacetoyloxyamino) -1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate (5) was found to be the most potent anti-HIV-1 agent with EC₅₀ of 0.0012 μM against HIV-1_IIIB and CC₅₀ of 34.05 μM against MT-4 with selectivity index of 28,375. Compound 5 inhibited Mycobacterium tuberculosis with MIC of 1.72 μM and inhibited four pathogenic bacteria with MIC of less than 1 μM.     

Design,Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K_i and IC₅₀ values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC₅₀ values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).     

Behavioural Lateralization in Budgerigars Varies with the Task and the Individual

Handedness/footedness and side biases are a well-known phenomenon in many animals, including humans. However, these so-called biases have mostly been studied at the population level - individual biases have received less attention, especially with regard to consistency over different tasks. Here we investigate behavioral lateralization in 12 male Budgerigars, Melopsittacus undulatus, a social parrot inhabiting the Australian bushlands. We performed 5 types of experiments to investigate lateralization, in tasks that involved climbing onto a perch, or landing on perches arranged in various configurations. The birds displayed highly significant, individually varying biases. The bias displayed by any particular individual varied with the task, in strength as well as polarity. Analysis of the data revealed that the preferred foot used for climbing did not coincide with the foot that was used while landing. Thus, landing choices are probably not determined by foot bias. Furthermore, these individual preferences were overridden completely when a bird had to perform a task simultaneously with another bird.     

The Histone Variant His2Av is Required for Adult Stem Cell Maintenance in the Drosophila Testis

Many tissues are sustained by adult stem cells, which replace lost cells by differentiation and maintain their own population through self-renewal. The mechanisms through which adult stem cells maintain their identity are thus important for tissue homeostasis and repair throughout life. Here, we show that a histone variant, His2Av, is required cell autonomously for maintenance of germline and cyst stem cells in the Drosophila testis. The ATP-dependent chromatin-remodeling factor Domino is also required in this tissue for adult stem cell maintenance possibly by regulating the incorporation of His2Av into chromatin. Interestingly, although expression of His2Av was ubiquitous, its function was dispensable for germline and cyst cell differentiation, suggesting a specific role for this non-canonical histone in maintaining the stem cell state in these lineages.     

Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive ³H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive ³H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive ³H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.     

Improved Detection of Remote Homologues Using Cascade PSI-BLAST: Influence of Neighbouring Protein Families on Sequence Coverage

Background

Development of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST.

Methodology/Principal Findings

We have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of ∼100% and Mathew’s correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families.

Conclusions/Significance

Our study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for covering the search space effectively in minimal computational time. This study also provides an understanding of the ‘bridging’ role of related families.     

Adherence to Antiretroviral Therapy and Its Effect on Survival of HIV-Infected Individuals in Jharkhand,India

Introduction

Research in India has extensively examined the factors associated with non-adherence to antiretroviral therapy (ART) with limited focus on examining the relationship between adherence to ART regimen and survival status of HIV infected patients. This study examines the effect of optimal adherence to ART on survival status of HIV infected patients attending ART centers in Jharkhand, India.

Materials and Methods

Data from a cohort of 239 HIV infected individuals who were initiated ART in 2007 were compiled from medical records retrospectively for 36 months. Socio-demographic characteristics, CD4 T cell count, presence of opportunistic infections at the time of ART initiation and ART regimen intake and survival status was collected periodically. Optimal adherence was assessed using pill count methods; patients who took <95% of the specified regimens were identified as non-adherent. Cox-proportional hazard model was used to determine the relative hazards of mortality.

Results

More than three-fourths of the patients were male, on an average 34 year old and median CD4 T cell count was 118 cells/cmm at the time of ART registration. About 57% of the patients registered for ART were found to be adherent to ART. A total of 104 patients died in 358.5 patient-years of observation resulting in a mortality rate of 29 per 100 patient-years (95% confidence interval (CI): 23.9–35.2) and median survival time of 6.5 months (CI: 2.7–10.9). The mortality rate was higher among patients who were non-adherent to ART (64.5, CI: 50.5–82.4) than who were adherent (15.4, CI: 11.3–21.0). The risk of mortality was fourfold higher among individuals who were non-adherent to ART than who were adherent (Adjusted hazard ratio: 3.9, CI: 2.6–6.0).

Conclusion

Adherence to ART is associated with a higher chance of survival of HIV infected patients, ascertaining the need for interventions to improve the ART adherence and early initiation of ART.     

Oxidative Stress Induced Mitochondrial Protein Kinase A Mediates Cytochrome C Oxidase Dysfunction

Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.     

More Than Meets the Eye: Associations of Vaginal Bacteria with Gram Stain Morphotypes Using Molecular Phylogenetic Analysis

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the “Bacteroides morphotype,” whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.