Sequence analysis and homology modeling of peroxidase from Medicago sativa

Plant peroxidases are one of the most extensively studied group of enzymes which find applications in the environment, health, pharmaceutical, chemical and biotechnological processes. Class III secretary peroxidase from alfalfa (Medicago sativa) has been characterized using bioinformatics approach Physiochemical properties and topology of alfalfa peroxidase were compared with that of soybean and horseradish peroxidase, two most popular commercially available peroxidase preparations. Lower value of instability index as predicted by ProtParam and presence of extra disulphide linkages as predicted by Cys_REC suggested alfalfa peroxidase to be more stable than either of the commercial preparations. Multiple Sequence Alignment (MSA) with other functionally similar proteins revealed the presence of highly conserved catalytic residues. Three dimensional model of alfalfa peroxidase was constructed based on the crystal structure of soybean peroxidase (PDB Id: 1FHF A) by homology modelling approach. The model was checked for stereo chemical quality by PROCHECH, VERIFY 3D, WHAT IF, ERRAT, 3D MATCH AND ProSA servers. The best model was selected, energy minimized and used to analyze structure function relationship with substrate hydrogen peroxide by Autodock 4.0. The enzyme substrate complex was viewed with Swiss PDB viewer and one residue ASP43 was found to stabilize the interaction by hydrogen bonds. The results of the study may be a guiding point for further investigations on alfalfa peroxidase.     

Comparative Whole-Genome Analysis of Clinical Isolates Reveals Characteristic Architecture of Mycobacterium tuberculosis Pangenome

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.     

A Geographic Mosaic of Plant–Pollinator Interactions in the Eastern Caribbean Islands

The interplay between generalized and specialized plant–animal interactions is a core concept in understanding the evolution of mutualisms. Within the Eastern Caribbean, Heliconia bihai is a dominant forest species in the southern island of St. Vincent where H. caribaea is virtually absent. Heliconia caribaea is most common on the northern island of St. Kitts where H. bihai is restricted to the tops of the highest peaks. Both species are abundant on the central island of Dominica. We compared flowering patterns, nectar characteristics, and visitation frequency of hummingbirds in the two heliconias on the three islands to determine the extent of geographic variations in this plant–pollinator mutualism. The peak flowering season of the two heliconias was observed to be in April–May on all three islands with little within‐ and between‐island variations. Nectar production significantly varied between species and between islands. Visitation patterns by the principal hummingbird pollinators also varied between the islands: (1) on Dominica, only females of a single species of hummingbird pollinated the flowers of H. bihai (sexual specialization), whereas both sexes of the same hummingbird pollinated the flowers of H. caribaea (species specialization); (2) on St. Vincent, both sexes of the same hummingbird pollinated the flowers of H. bihai (species specialization); and (3) on St. Kitts, only females pollinated the flowers of H. bihai (sexual specialization), whereas several species of hummingbird visited the flowers of H. caribaea (species generalization). We propose that the Heliconia–hummingbird interactions in the Eastern Caribbean represent a geographically variable coevolutionary mosaic of plant–pollinator interactions.     

Direct Expression and Validation of Phage-selected Peptide Variants in Mammalian Cells

Phage display is a key technology for the identification and maturation of high affinity peptides, antibodies, and other proteins. However, limitations of bacterial expression restrict the range and sensitivity of assays that can be used to evaluate phage-selected variants. To address this problem, selected genes are typically transferred to mammalian expression vectors, a major rate-limiting step in the iterative improvement of peptides and proteins. Here we describe a system that combines phage display and efficient mammalian expression in a single vector, pDQ1. This system permits immediate expression of phage-selected genes as IgG1-Fc fusions in mammalian cells, facilitating the rapid, sensitive characterization of a large number of library outputs for their biochemical and functional properties. We demonstrate the utility of this system by improving the ability of a CD4-mimetic peptide to bind the HIV-1 envelope glycoprotein and neutralize HIV-1 entry. We further improved the potency of the resulting peptide, CD4mim6, by limiting its ability to induce the CD4-bound conformation of the envelope glycoprotein. Thus, CD4mim6 and its variants can be used to investigate the properties of the HIV-1 envelope glycoprotein, and pDQ1 can accelerate the discovery of new peptides and proteins through phage display.     

Prevention of aggregation and autocatalysis for sustaining biological activity of recombinant BoNT/A-LC upon long-term storage

Protein aggregation during expression, purification, storage, or transfer into requisite assay buffers hampers the use of proteins for in vitro studies. The formation of these aggregates represents a major obstacle in the study of biological activity and also restricts the spectrum of protein products being available for the biomedical applications. The catalytic light chain of botulinum neurotoxin type A undergoes autocatalysis and aggregation after purification upon long-term storage and freeze-thawing. In present study the conditions for the high level expression and purification of biologically active light chain protein of botulinum neurotoxin were optimized from a synthetic gene. Several co-solvents were screened in order to prevent autocatalysis and aggregation of rBoNT/A-LC. The effect of the co-solvents is studied on endopeptidase activity during long term storage of the recombinant protein. The purified rBoNT/A-LC was also evaluated for its immunogenicity.     

Synthesis and biological evaluation of substituted 4-arylthiazol-2-amino derivatives as potent growth inhibitors of replicating Mycobacterium tuberculosis H₃₇Rv

In search of potential therapeutics for tuberculosis, we describe herein synthesis and biological evaluation of some substituted 4-arylthiazol-2-amino derivatives as modified analogues of the antiprotozoal drug Nitazoxanide (NTZ), which has recently been reported as potent inhibitor of Mtb H(37)Rv (Mtb MIC=52.12 μM) with an excellent ability to evade resistance. Among the synthesized derivatives, the two compounds 7a (MIC=15.28 μM) and 7c (MIC=17.03 μM) have exhibited about three times better Mtb growth inhibitory activity over NTZ and are free from any cytotoxicity (Vero CC(50) of 244 and 300 μM respectively). These two compounds represent promising leads for further optimization.     

Human sodium iodide symporter (hNIS) in fibroadenoma breast--a immunohistochemical study

Human sodium iodide symporter (hNIS), responsible for the active transport of iodine is an integral plasma membrane glycoprotein present in the thyroid cells and extrathyroid tissues like breast and salivary glands. If its functional form is unequivocally shown in benign or malignant breast tissues, then it may serve as a basis for diagnosis and treatment using radioactive iodine. With an aim to analyze the hNIS expression in a distinct benign breast condition of fibroadenoma, biopsy proven fibroadenoma tissues, normal non-lactating breast tissue and biopsy proven infiltrating duct carcinoma tissues were examined for hNIS expression using immunohistochemistry. Out of 20 biopsy proven fibroadenoma tissues, 19 (95%) showed positivity for hNIS protein and only one was negative. Of these 10% were mildly positive, 50% cases were moderately positive and 35% showed intense positivity. None of the control tissue obtained from reduction mammoplasty specimens or normal breast tissues samples (5 cms away from the tumor) were positive, hNIS was also intensely positive in 9 out of 10 (90%) infiltrating duct carcinoma tissues and moderately positive in one case. These preliminary results show that hNIS was present in high frequency as demonstrated by immunohistochemistry in fibroadenoma breast.     

Design, synthesis and antitubercular activity of diarylmethylnaphthol derivatives

A new series of diarylmethylnapthyloxy ethylamines were synthesized by aminoalkylation of diarylmethylnaphthols which were obtained by Friedel-Crafts alkylation on 1- and 2-naphthols using diarylcarbinols as the alkylating agents. The title compounds were evaluated for antitubercular activity against Mycobacterium tuberculosis H(37)R(v) in vitro and showed MIC in the range of 3.12-25 microg/ml.     

Development and characterization of microsatellite loci for two Caribbean Heliconia (Heliconiaceae: H. bihai and H. caribaea)

? Premise of the study: Microsatellite loci were developed to characterize genetic variation and population subdivision in Heliconia bihai and H. caribaea from the Caribbean Islands. ? Methods and Results: A total of 13 new microsatellite markers were developed and characterized in the two Caribbean heliconias. Di-, tri-, and tetranucleotide repeats were identified with one to 17 alleles per locus, and the observed heterozygosity ranged from 0.13 to 0.87. Additionally, cross-species amplification was successful in eight out of 13 loci. ? Conclusions: The microsatellite loci developed have discriminatory potential to be used in genetic characterizations of Caribbean Heliconia. Both H. bihai and H. caribaea are known to have adaptive interactions with their hummingbird pollinators, and the characterized microsatellite markers will be used to study mating system, genetic structure, and phylogeographic patterns in Caribbean Heliconia.