Design, solvent free synthesis, and antimicrobial evaluation of 1,4 dihydropyridines

Here in, we report the usage of cellulose sulfuric acid as a heterogeneous eco friendly catalyst for the synthesis of 1,4 dihydropyridines under solvent free conditions via Hantzsch three component reaction of an aldehyde, ethyl acetoacetate and ammonium acetate at 100°C for 2-5h. In silico studies were performed on twenty two possible 1,4 dihydropyridines (DHPs) analogues against K(+) channel receptor (KcsA). In order to validate in silico studies, thirteen compounds were synthesized and evaluated as antibacterials against twenty seven ESBL isolates of Klebsiella pneumoniae and Escherichia coli.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

Interleukin-21 enhances NK cell activation in response to antibody-coated targets

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.     

In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin

Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% alpha-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 microg/ml. Refolded His-rMcnapin exhibited approximately 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.     

Biological evaluation of a multi-targeted small molecule inhibitor of tumor-induced angiogenesis

RO4396686 is a small molecule KDR, FGFR, and PDGFR inhibitor with good pharmacokinetic properties in rodents. In a mouse corneal neovascularization assay, this compound inhibited VEGF-induced angiogenesis. Tested in a H460a xenograft tumor model this agent effected significant tumor growth inhibition at doses as low as 50mg/kg.     

Reduced Fertility of Drosophila melanogaster Hybrid male rescue (Hmr) Mutant Females Is Partially Complemented by Hmr Orthologs From Sibling Species

The gene Hybrid male rescue (Hmr) causes lethality in interspecific hybrids between Drosophila melanogaster and its sibling species. Hmr has functionally diverged for this interspecific phenotype because lethality is caused specifically by D. melanogaster Hmr but not by D. simulans or D. mauritiana Hmr. Hmr was identified by the D. melanogaster partial loss-of-function allele Hmr¹, which suppresses hybrid lethality but has no apparent phenotype within pure-species D. melanogaster. Here we have investigated the possible function of Hmr in D. melanogaster females using stronger mutant alleles. Females homozygous for Hmr mutants have reduced viability posteclosion and significantly reduced fertility. We find that reduced fertility of Hmr mutants is caused by a reduction in the number of eggs laid as well as reduced zygotic viability. Cytological analysis reveals that ovarioles from Hmr mutant females express markers that distinguish various stages of wild-type oogenesis, but that developing egg chambers fail to migrate posteriorly. D. simulans and D. mauritiana Hmr⁺ partially complement the reduced fertility of a D. melanogaster Hmr mutation. This partial complementation contrasts with the complete functional divergence previously observed for the interspecific hybrid lethality phenotype. We also investigate here the molecular basis of hybrid rescue associated with a second D. melanogaster hybrid rescue allele, In(1)AB. We show that In(1)AB is mutant for Hmr function, likely due to a missense mutation in an evolutionarily conserved amino acid. Two independently discovered hybrid rescue mutations are therefore allelic.     

Susceptibility of the Mediterranean fruit fly (Ceratitis capitata) and the Natal fruit fly (Ceratitis rosa) to entomopathogenic nematodes

The potential of entomopathogenic nematodes, Heterorhabditis bacteriophora, Heterorhabditis zealandica and Steinernema khoisanae, to infect pupariating larvae, pupae and adults of Ceratitis capitata and Ceratitis rosa was investigated in laboratory bioassays. Pupariating larvae and adult flies were susceptible to nematode infection, with no infection recorded for the pupae. Pupariating larvae of C. capitata were generally more susceptible to infection than those of C. rosa. Significantly more larvae of C. capitata were infected by H. bacteriophora. For C. rosa, highest infectivity of larvae was obtained with H. zealandica. In contrast, adults of both species were highly infected by S. khoisanae.     

The first SSR-based genetic linkage map for cultivated groundnut (Arachis hypogaea L.)

Molecular markers and genetic linkage maps are pre-requisites for molecular breeding in any crop species. In case of peanut or groundnut (Arachis hypogaea L.), an amphidiploid (4X) species, not a single genetic map is, however, available based on a mapping population derived from cultivated genotypes. In order to develop a genetic linkage map for tetraploid cultivated groundnut, a total of 1,145 microsatellite or simple sequence repeat (SSR) markers available in public domain as well as unpublished markers from several sources were screened on two genotypes, TAG 24 and ICGV 86031 that are parents of a recombinant inbred line mapping population. As a result, 144 (12.6%) polymorphic markers were identified and these amplified a total of 150 loci. A total of 135 SSR loci could be mapped into 22 linkage groups (LGs). While six LGs had only two SSR loci, the other LGs contained 3 (LG_AhXV) to 15 (LG_AhVIII) loci. As the mapping population used for developing the genetic map segregates for drought tolerance traits, phenotyping data obtained for transpiration, transpiration efficiency, specific leaf area and SPAD chlorophyll meter reading (SCMR) for 2 years were analyzed together with genotyping data. Although, 2–5 QTLs for each trait mentioned above were identified, the phenotypic variation explained by these QTLs was in the range of 3.5–14.1%. In addition, alignment of two linkage groups (LGs) (LG_AhIII and LG_AhVI) of the developed genetic map was shown with available genetic maps of AA diploid genome of groundnut and Lotus and Medicago. The present study reports the construction of the first genetic map for cultivated groundnut and demonstrates its utility for molecular mapping of QTLs controlling drought tolerance related traits as well as establishing relationships with diploid AA genome of groundnut and model legume genome species. Therefore, the map should be useful for the community for a variety of applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Screen for Chemical Modulators of Autophagy Reveals Novel Therapeutic Inhibitors of mTORC1 Signaling

Background

Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties.

Methodology/Principal Findings

Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.

Conclusion/Significance

The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.     

Dense and sparse aggregations in complex motion: Video coupled with simulation modeling

Investigations into the complex behaviors of aggregations of highly mobile animals have not used the link between image processing technology and simulation modeling fruitfully to address many fundamental ecological issues. Examples include population censusing, which remains difficult despite the demonstrated ecological importance of assessing abundance and density of organisms. We introduce a theoretical framework that connects thermal infrared video imaging with an individual-based simulation model—an approach that appears to be applicable to a diverse set of aggregated, highly mobile, nocturnal animals. To demonstrate the framework two applications are presented. The first is a dense aggregation of Brazilian free-tailed bats (Tadarida brasiliensis) that exhibits an emergence pattern that has complex dynamics and the second is a sparse local aggregation of agricultural pest moths whose dynamics are insipid. The first application uses individual-based modeling to mimic the behavior in the video of bats during a nightly emergence from a cave and to provide reliable estimates of the numbers, and associated error bounds. The second application uses video recordings of sparse aggregations to provide consistent estimates of the numbers of flying noctuid moths recorded over a corn and cotton-dominated agroecosystem in south-central Texas. This does not use a mathematical model because error estimates tend to be small.