Genetic differentiation between three ecological variants (‘type’, ‘mysorensis’ and ‘intermediate’) of malaria vector Anopheles stephensi (Diptera: Culicidae)

Abstract Anopheles stephensi is the main vector of urban malaria in South Asia. Three ecological variants (‘type’, ‘mysorensis’and‘intermediate’) of An. stephensi have been reported on the basis of ecology and egg morphology. However, it is unclear if there is any genetic isolation between the three variants. We analyzed the three variants of An. stephensi using eight microsatellite loci and found that large and significant genetic differentiation exists between them (mean F_ST= 0.393 and mean R_ST= 0.422). Pairwise estimates of genetic differentiation between the variants were ‘type’ versus ‘mysorensis’ (mean F_ST= 0.411 and mean R_ST= 0.308), ‘type’ versus ‘intermediate’ (mean F_ST= 0.388 and mean R_ST= 0.518) and ‘intermediate’ versus ‘mysorensis’ (mean F_ST= 0.387 and mean R_ST= 0.398) and all were statistically significant (P < 0.05). The greater sensitivity of R_ST in differentiation indicated that mutations and not genetic drift had generated the differences between three variants of An. stephensi. The present study indicated large genetic differentiation and presence of non-significant low level of gene flow between the three variants (‘type’, ‘mysorensis’and‘intermediate’) of An. stephensi.     

UDP-N-acetylglucosamine enolpyruvyl transferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K _m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.     

Cholic acid modified 2 kDa polyethylenimine as efficient transfection agent

New lipopolymers were synthesized by conjugating cholic acid (ChA) to polyethylenimines (PEI; 2 and 25 kDa) and a polyallylamine (PAA; 15 kDa) via N‐acylation to develop effective gene delivery systems. The extent of ChA substitution linearly varied with the feed ratio during synthesis, indicating good control over grafting ratio. While ChA did not affect binding to plasmid DNA (pDNA) for higher molecular weight (MW) polymers, ChA substitution to 2 kDa PEI significantly affected the pDNA binding. Toxicity of the 2 kDa PEI was unaffected by ChA substitution, but it was improved for the higher MW polymers. Using immortal 293T cells and primary cord blood‐derived mesenchymal stem cells, low MW (2 kDa) PEI was shown to display much better transfection efficiency as a result of ChA substitution, unlike the higher MW polymers. We conclude that ChA could be a suitable substituent for non‐toxic (low MW) PEIs in order to improve their transfection efficiency. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1337–1341, 2013     

Over-Expression of 60s Ribosomal L23a Is Associated with Cellular Proliferation in SAG Resistant Clinical Isolates of Leishmania donovani

Background

Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.

Methodology and principal findings

The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode.

Conclusion/Significance

This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.     

Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters

Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.     

3D-QSAR based pharmacophore modeling and virtual screening for identification of novel pteridine reductase inhibitors

Pteridine reductase is a promising target for development of novel therapeutic agents against Trypanosomatid parasites. A 3D-QSAR pharmacophore hypothesis has been generated for a series of L. major pteridine reductase inhibitors using Catalyst/HypoGen algorithm for identification of the chemical features that are responsible for the inhibitory activity. Four pharmacophore features, namely: two H-bond donors (D), one Hydrophobic aromatic (H) and one Ring aromatic (R) have been identified as key features involved in inhibitor-PTR1 interaction. These features are able to predict the activity of external test set of pteridine reductase inhibitors with a correlation coefficient (r) of 0.80. Based on the analysis of the best hypotheses, some potent Pteridine reductase inhibitors were screened out and predicted with anti-PTR1 activity. It turned out that the newly identified inhibitory molecules are at least 300 fold more potent than the current crop of existing inhibitors. Overall the current SAR study is an effort for elucidating quantitative structure-activity relationship for the PTR1 inhibitors. The results from the combined 3D-QSAR modeling and molecular docking approach have led to the prediction of new potent inhibitory scaffolds.     

Fertility Intentions and Use of Contraception among Monogamous Couples in Northern Malawi in the Context of HIV Testing: A Cross-Sectional Analysis

Context

Knowledge of HIV status may influence fertility desires of married men and women. There is little knowledge about the importance of this influence among monogamously married couples and how knowledge of HIV status influences use of contraception among these couples.

Methodology

We carried out a cross-sectional analysis of interview data collected between October 2008 and September 2009 on men aged 15–59 years and women aged 15–49 years who formed 1766 monogamously married couples within the Karonga Prevention Study demographic surveillance study in northern Malawi.

Results

5% of men and 4% of women knew that they were HIV positive at the time of interview and 81% of men and 89% of women knew that they were HIV negative. 73% of men and 83% of women who knew that they were HIV positive stated that they did not want more children, compared to 35% of men and 38% of women who knew they were HIV negative. Concordant HIV positive couples were more likely than concordant negative couples to desire to stop child bearing (odds ratio 11.5, 95%CI 4.3–30.7, after adjusting for other factors) but only slightly more likely to use contraceptives (adjusted odds ratio 1.5 (95%CI 0.8–3.3).

Conclusion

Knowledge of HIV positive status is associated with an increase in the reported desire to cease childbearing but there was limited evidence that this desire led to higher use of contraception. More efforts directed towards assisting HIV positive couples to access and use reproductive health services and limit HIV transmission among couples are recommended.     

Effects of sodium butyrate on proliferation-dependent insulin gene expression and insulin release in glucose-sensitive RIN-5AH cells.

A rat islet tumor subclone, RIN-5AH-T2-B, was cultured with 2 mmol/liter of the proliferation-arresting compound sodium butyrate (NaB). Insulin gene expression and glucose-stimulated insulin release were analyzed and compared with logarithmically proliferating and confluent control cells cultured without NaB. Logarithmically proliferating control cells revealed high insulin gene expression. In the presence of amino acids, these cells showed a dose-dependent insulin response to glucose with a half-maximal and maximal 6.5-fold stimulation by 0.8 and 5.6 mmol/liter D-glucose, respectively. However, as the control cells approached growth arrest, insulin gene expression subsided to below detectability, an occurrence that is associated with decreased insulin release and accumulation of cells in the G1 phase of the cell cycle. In contrast, NaB-arrested cells showed continuous insulin gene expression throughout the experiment. Despite this, insulin release in response to glucose was lost. NaB revealed a biphasic effect on the cell-cycle: after an initial leaky G1 arrest during the first 24 h, the 5AH-B cells were arrested in G2 during the following 3 days. These data suggest that insulin gene expression and glucose-stimulated insulin release are affected by the cell cycle. These glucose-sensitive RIN-5AH-T2-B cells may be useful in studies of insulin secretion and gene regulation.     

Crystal structure of the Bacillus anthracis nucleoside diphosphate kinase and its characterization reveals an enzyme adapted to perform under stress conditions

Nucleoside diphosphate kinases (Ndks) play an important role in a plethora of regulatory and metabolic functions. Inhibition of the B. anthracis Ndk mRNA results in the formation of nonviable aberrant spores. We report the characterization and crystal structure of the enzyme from B. anthracis nucleoside diphosphate kinase (BaNdk), the first from sporulating bacteria. The enzyme, although from a mesophilic source, is active at extremes of pH (3.5–10.5), temperature (10–95°C) and ionic strength (0.25–4.0M NaCl). It exists as a hexamer that is composed of two SDS‐stable trimers interacting in a back‐to‐back association; mutational analysis confirms that the enzyme is a histidine kinase. The high‐resolution crystal structure reported here reveals an unanticipated change in the conformation of residues between 43 and 63 that also regulates substrate entry in other Ndks. A comparative structural analysis involving Ndks from seven mesophiles and three thermophiles has resulted in the delineation of the structure into relatively rigid and flexible regions. The analysis suggests that the larger number of intramolecular hydrogen bonds and to a lesser extent ionic interactions in BaNdk contributes to its high thermostability. Mutational analysis and Molecular Dynamics simulations were used to probe the role of a highly conserved Gly19 (present at the oligomeric interface in most of the Ndks). The results suggest that the mutation leads to a rigidification of those residues that facilitate substrate entry and consequently leads to a large reduction in the kinase activity. Overall, the enzyme characterization helps to understand its apparent adaptation to perform under stress conditions. Proteins 2009. © 2009 Wiley‐Liss, Inc.