A glutathione-specific aldose reductase of Leishmania donovani and its potential implications for methylglyoxal detoxification pathway

Methylglyoxal is mainly catabolized by two major enzymatic pathways. The first is the ubiquitous detoxification pathway, the glyoxalase pathway. In addition to the glyoxalase pathway, aldose reductase pathway also plays a crucial role in lowering the levels of methylglyoxal. The gene encoding aldose reductase (ALR) has been cloned from Leishmania donovani, a protozoan parasite causing visceral leishmaniasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 855 bp encoding a putative protein of 284 amino acids with a calculated molecular mass of 31.7 kDa and a predicted isoelectric point of 5.85. The sequence identity between L. donovani ALR (LdALR) and mammals and plants is only 36-44%. The ORF is a single copy gene. A protein with a molecular mass that matched the estimated approximately 74 kDa according to the amino acid composition of LdALR with a maltose binding tag present at its N-terminal end was induced by heterologous expression of LdALR in Escherichia coli. In the presence of glutathione, recombinant LdALR reduced methylglyoxal with a K(m) of approximately 112 microM. Comparative structural analysis of the human ALR structure with LdALR model suggests that the active site anchoring the N-terminal end of the glutathione is highly conserved. However, the C-terminal end of the glutathione backbone is expected to be exposed in LdALR, as the residues anchoring the C-terminal end of the glutathione backbone come from the three loop regions in human, which are apparently shortened in the LdALR structure. Thus, the computational analysis provides clues about the expected mode of glutathione binding and its interactions with the protein. This is the first report of the role of an ALR in the metabolic disposal of methylglyoxal in L. donovani and of thiol binding to a kinetoplastid aldose reductase.     

Synthesis of acryloyl guar gum and its hydrogel materials for use in the slow release of l-DOPA and l-tyrosine

Acryloyl guar gum (AGG) and its hydrogel materials were synthesized for use as carriers and slow release devices of two pro-drugs, l-tyrosine and 3,4-dihydroxy phenylalanine (l-DOPA). To evaluate their structure-properties relationship, these were characterized by scanning electron micrography (SEM), FTIR spectroscopy and swelling studies. The hydrogel materials responded to the change of pH of the swelling medium, and exhibited reversible transitions in 0.9% saline solution. These were loaded with two pro-drugs, and their cumulative release behavior was studied at pH 2.2 and pH 7.4. The hydrogel materials exhibited structure-property relationship in the release of these pro-drugs. The % cumulative release of l-tyrosine was the maximum from the AGG-g-poly(methacrylic acid), while the maximum release of l-DOPA was observed from AGG-g-poly(AAc) in both the media. On the other hand, the AGG-g-poly(2-hydroxyethyl methacrylate) and AGG-g-poly(2-hydroxypropyl methacrylate) retained 42.33% and 49.05% of the drug even after 12 h.     

In vitro studies on degradation of synthetic dye mixture by Comamonas sp. VS-MH2 and evaluation of its efficacy using simulated microcosm

Reactive azo dyes are considered as one of the most detrimental pollutants from industrial effluents and therefore their biodegradation is receiving constant scientific consideration. A bacterial isolate VS-MH2, originating from dye contaminated sites of Gujarat, India, was exploited for its ability to degrade a synthetic dye mixture (SDM) (comprising of four azo reactive dyes) under static conditions. The identification of the isolate by 16S rRNA gene sequencing revealed it to be Comamonas sp. The biodegradation of the SDM was analyzed by UV-vis spectroscopy, IR spectroscopy and GC-MS analysis. The isolate showed high metabolic activity towards SDM and degraded it completely (100 mg L(-1)) within 30 h at pH 7 and 35 °C. Simulated microcosm studies in the presence and absence of indigenous microflora confirmed the ability of Comamonas sp. VS-MH2 for dye degradation and to colonize the soil. This is the first investigation reporting the degradation of SDM by Comamonas sp. under simulated soil microcosms.     

Mechanism of action of novel synthetic dodecapeptides against Candida albicans

Background

Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,β-didehydrophenylalanine (?Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated.

Methods

The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy.

Results and conclusions

The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250 μM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans.

General significance

The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.     

Microsatellite markers based genetic diversity and bottleneck studies in Zanskari pony

Genetic diversity in Zanskari pony breed was evaluated at 48 microsatellite loci using fifty adult, healthy and unrelated animals. Allele frequency data was used to detect genetic diversity and bottleneck. The estimated average number of alleles (±s.e.) was 8.5208±2.5010 with a total of 409 alleles. A high level of genetic diversity within this breed was observed in terms of number of alleles, observed heterozygosity (0.6763±0.1704), expected Leven's heterozygosity (0.7724±0.795), expected Nei's heterozygosity (0.7644±0.0787) and polymorphism information content (>0.5). In-breeding coefficient (F(is)) was 0.115±0.0209, suggesting moderately high in-breeding in Zanskari breed. Although analysis of bottleneck revealed no bottleneck in recent past but population of Zanskari ponies has decreased drastically and only a few thousand pure-bred animals are left. The information is useful for proposing effective population management strategies for future.     

Pharmacokinetics and biodistribution of genetically engineered antibodies

The engineering of monoclonal antibodies has created a new generation of pharmaceuticals with the desired pharmacokinetics and biodistribution properties. For radioimmunotherapy and radioscintigraphy, optimum tumor targeting can be achieved using engineered constructs that provide high antigen affinity and specificity, effective tumor penetration, circulation properties that allow high tumor uptake with acceptable doses to the normal tissues, and fast clearance allowing low background. Recent advances have made possible the development of antibodies with these properties.     

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.     

Evaluation of antibody serology to determine current helminth and Plasmodium falciparum infections in a co-endemic area in Southern Mozambique

BackgroundSoil-transmitted helminths (STH), Schistosoma spp. and Plasmodium falciparum are parasites of major public health importance and co-endemic in many sub-Saharan African countries. Management of these infections requires detection and treatment of infected people and evaluation of large-scale measures implemented. Diagnostic tools are available but their low sensitivity, especially for low intensity helminth infections, leaves room for improvement. Antibody serology could be a useful approach thanks to its potential to detect both current infection and past exposure.MethodologyWe evaluated total IgE responses and specific-IgG levels to 9 antigens from STH, 2 from Schistosoma spp., and 16 from P. falciparum, as potential markers of current infection in a population of children and adults from Southern Mozambique (N = 715). Antibody responses were measured by quantitative suspension array Luminex technology and their performance was evaluated by ROC curve analysis using microscopic and molecular detection of infections as reference.Principal findingsIgG against the combination of EXP1, AMA1 and MSP2 (P. falciparum) in children and NIE (Strongyloides stercoralis) in adults and children had the highest accuracies (AUC = 0.942 and AUC = 0.872, respectively) as markers of current infection. IgG against the combination of MEA and Sm25 (Schistosoma spp.) were also reliable markers of current infection (AUC = 0.779). In addition, IgG seropositivity against 20 out of the 27 antigens in the panel differentiated the seropositive endemic population from the non-endemic population, suggesting a possible role as markers of exposure although sensitivity could not be assessed.ConclusionsWe provided evidence for the utility of antibody serology to detect current infection with parasites causing tropical diseases in endemic populations. In addition, most of the markers have potential good specificity as markers of exposure. We also showed the feasibility of measuring antibody serology with a platform that allows the integration of control and elimination programs for different pathogens.     

Crystal structure and conformation of a highly constrained linear tetrapeptide Boc-Leu-dehydro Phe-Ala-Leu-OCH3.

The conformation of a tetrapeptide containing a dehydro amino acid, delta ZPhe, in its sequence has been determined in the crystalline state using X-ray crystallographic techniques. The tetrapeptide, Boc-Leu-delta ZPhe-Ala-Leu-OCH3, crystallizes in the orthorhombic space group P2(1)2(1)2(1) with four molecules in a unit cell of dimensions a = 11.655(1) A, b = 15.698(6) A and c = 18.651(3) A V = 3414.9 A and Dcalc = 1.12 g/cm-3. The asymmetric unit contains one tetrapeptide molecule, C30H46N4O7, a total of 41 nonhydrogen atoms. The structure was determined using the direct methods program SHELXS86 and refined to an R-factor of 0.049 for 3347 reflections (I3.0(I). The linear tetrapeptide in the crystal exhibits a double bend of the Type III-I, with Leu1 (phi = -54.1 degrees, psi = -34.5 degrees) and delta ZPhe2 (phi = -59.9 degrees, psi = -17.1 degrees) as the corner residues of Type III turn and delta ZPhe2 (phi = -59.9 degrees, psi = -17.1 degrees) and Ala3 (phi = -80.4 degrees, psi = 0.5 degrees) residues occupying the corners of Type I turn, with delta ZPhe as the common residue in the double bend. The turn structures are further stabilized by two intramolecular 4----1 type hydrogen bonds.     

The Lower Paleolithic of the Indian subcontinent

This broad overview highlights the Indian subcontinent as an important and exciting source of new discoveries regarding Lower Paleolithic hominins and their biological and behavioral evolution. Broadly situated in the center of the Old World, the region arbitrarily encompasses Pakistan, India, Nepal, Sri Lanka, Bangladesh, and Bhutan; it represents the richest easternmost domain of classic Acheulean bifaces in the Old World. 1 The region comprises diverse ecological zones with complex geological and climatic histories, including a bi‐annual monsoon prevalent since the Miocene, all of which had major impacts on faunal and floral distributions and associated hominin adaptations.