Impact of IRS: Four-years of entomological surveillance of the Indian Visceral Leishmaniases elimination programme

BackgroundIn 2005, Bangladesh, India and Nepal agreed to eliminate visceral leishmaniasis (VL) as a public health problem. The approach to this was through improved case detection and treatment, and controlling transmission by the sand fly vector Phlebotomus argentipes, with indoor residual spraying (IRS) of insecticide. Initially, India applied DDT with stirrup pumps for IRS, however, this did not reduce transmission. After 2015 onwards, the pyrethroid alpha-cypermethrin was applied with compression pumps, and entomological surveillance was initiated in 2016.MethodsEight sentinel sites were established in the Indian states of Bihar, Jharkhand and West Bengal. IRS coverage was monitored by household survey, quality of insecticide application was measured by HPLC, presence and abundance of the VL vector was monitored by CDC light traps, insecticide resistance was measured with WHO diagnostic assays and case incidence was determined from the VL case register KAMIS.ResultsComplete treatment of houses with IRS increased across all sites from 57% in 2016 to 70% of houses in 2019, rising to >80% if partial house IRS coverage is included (except West Bengal). The quality of insecticide application has improved compared to previous studies, average doses of insecticide on filters papers ranged from 1.52 times the target dose of 25mg/m² alpha-cypermethrin in 2019 to 1.67 times in 2018. Resistance to DDT has continued to increase, but the vector was not resistant to carbamates, organophosphates or pyrethroids. The annual and seasonal abundance of P. argentipes declined between 2016 to 2019 with an overall infection rate of 0.03%. This was associated with a decline in VL incidence for the blocks represented by the sentinel sites from 1.16 per 10,000 population in 2016 to 0.51 per 10,000 in 2019.ConclusionThrough effective case detection and management reducing the infection reservoirs for P. argentipes in the human population combined with IRS keeping P. argentipes abundance and infectivity low has reduced VL transmission. This combination of effective case management and vector control has now brought India within reach of the VL elimination targets.     

Kinetic stability plays a dominant role in the denaturant-induced unfolding of Erythrina indica lectin

The urea-induced denaturation of dimeric Erythrina indica lectin (EIL) has been studied at pH 7.2 under equilibrium and kinetic conditions in the temperature range of 40-55 degrees C. The structure of EIL is largely unaffected in this temperature range in absence of denaturant, and also in 8 M urea after incubation for 24 h at ambient temperature. The equilibrium denaturation of EIL exhibits a monophasic unfolding transition from the native dimer to the unfolded monomer as monitored by fluorescence, far-UV CD, and size-exclusion FPLC. The thermodynamic parameters determined for the two-state unfolding equilibrium show that the free energy of unfolding (DeltaGu, aq) remains practically same between 40 and 55 degrees C, with a value of 11.8 +/- 0.6 kcal mol(-1) (monomer units). The unfolding kinetics of EIL describes a single exponential decay pattern, and the apparent rate constants determined at different temperatures indicate that the rate of the unfolding reaction increases several fold with increase in temperature. The presence of probe like external metal ions (Mn2+, Ca2+) does not influence the unfolding reaction thermodynamically or kinetically; however, the presence of EDTA affects only kinetics. The present results suggest that the ability of EIL to preserve the structural integrity against the highly denaturing conditions is linked primarily to its kinetic stability, and the synergic action of heat and denaturant is involved in the unfolding of the protein.     

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein

Compared with other mammalian tissues, retina is highly enriched in PUFA. Long-chain PUFA (LC-PUFA; C18-C24) are essential FAs that are enriched in the retina and are necessary for maintenance of normal retinal development and function. The retina, brain, and sperm also contain very LC-PUFA (VLC-PUFA; >C24). Although VLC-PUFA were discovered more than two decades ago, very little is known about their biosynthesis and functional roles in the retina. This is due mainly to intrinsic difficulties associated with working on these unusually long polyunsaturated hydrocarbon chains and their existence in small amounts. Recent studies on the FA elongase elongation of very long chain fatty acids-4 (ELOVL4) protein, however, suggest that VLC-PUFA probably play some uniquely important roles in the retina as well as the other tissues. Mutations in the ELOVL4 gene are found in patients with autosomal dominant Stargardt disease. Here, we review the recent literature on VLC-PUFA with special emphasis on the elongases responsible for their synthesis. We focus on a novel elongase, ELOVL4, involved in the synthesis of VLC-PUFA, and the importance of these FAs in maintaining the structural and functional integrity of retinal photoreceptors.     

Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.     

Disease-causing mutations in proteins: structural analysis of the CYP1B1 mutations causing primary congenital glaucoma in humans

In this communication, we report an in-depth structure-based analysis of the human CYP1b1 protein carrying disease-causing mutations that are discovered in patients suffering from primary congenital glaucoma (PCG). The "wild-type" and the PCG mutant structures of the human CYP1b1 protein obtained from comparative modeling were subjected to long molecular dynamics simulations with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of time evolution as well as time averaged values of various structural properties--especially of those of the functionally important regions: the heme binding region, substrate binding region, and substrate access channel--gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type the core regions in the mutant structures are associated with subtle but significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.     

A triterpenoid saponin possessing antileishmanial activity from the leaves of Careya arborea

Bioguided-fractionation of the methanol extract of the leaves of Careya arborea led to isolation of a triterpenoid saponin, designated arborenin, and characterized as 3-O-beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl-2 alpha,3beta-dihydroxy-taraxast-20-en-28-oic acid (1), together with desacylescin III (2). The structures were determined on the basis of extensive 2D NMR spectroscopic analysis. The saponin showed in vitro antileishmanial activity against Leishmania donovani (strain AG 83).     

Copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate act as inhibitors of Ribonuclease A

Green tea polyphenols, which have the ability to inhibit angiogenesis, form complexes with Cu(II), a known potent stimulator of blood vessel proliferation. Copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate were found to inhibit the enzymatic activity of Ribonuclease A (RNase A) as revealed by an agarose gel based assay and urea denatured gel electrophoresis. The copper complexes were found to be non-competitive inhibitors of RNase A with inhibition constants in the micromolar range. Changes in the secondary structure of the protein are found to occur due to the interaction as revealed from Fourier transform infrared and circular dichroism studies.