Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus

To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.     

HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.     

Unwrapping the phase portrait features of adventitious crackle for auscultation and classification: a machine learning approach

Journal of Biological Physics - The paper delves into the plausibility of applying fractal, spectral, and nonlinear time series analyses for lung auscultation. The thirty-five sound signals of...     

Screening,statistical optimized production,and application of β‐mannanase from some newly isolated fungi

Eighty‐eight fungi isolated from soil and decaying organic matter were screened for mannanolytic activity. Twenty‐eight fungi produced extracellular mannanase on locust bean gum as evidenced by zone of hydrolysis produced on mannan agar gel. Six prominent producers, including four Fusarium species namely Fusarium fusarioides NFCCI 3282, Fusarium solani NFCCI 3283, Fusarium equiseti NFCCI 3284, Fusarium moniliforme NFCCI 3287 with Cladosporium cladosporioides NFCCI 3285 and Acrophialophora levis NFCCI 3286 produced the β‐mannanase in the range of 84–140 nkat/mL. All these grew well on particulate substrates in solid‐state fermentation (SSF), producing relatively higher titers on mannan‐rich palm kernel cake (PKC) and copra meal. Two high yielding strains, F. equiseti (1747 nkat/gds) and A. levis (897 nkat/gds) were selected for statistical optimization of mannanase on PKC. Interaction of two critical solid state fermentation parameters, pH and moisture on mannanase production by these two molds was studied by response surface method. Optimized production on PKC resulted in three‐ to fourfold enhancement in enzyme yield was observed in case of F. equiseti (5945 nkat/gds) and A. levis (4726 nkat/gds). HPLC analysis of mannan hydrolysate indicated that F. equiseti and A. levis mannanase performed efficient hydrolysis of konjac gum (up to 99%) with exclusive mannooligosaccahride (DP of 4) production. A seminative SDS‐PAGE revealed that A. levis and F. solani produced three isoforms, F. moniliforme produced two isoforms while F. fusarioides, F. equiseti, and C. cladosporioides produced a single enzyme.     

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.     

Stress-dependent Proteolytic Processing of the Actin Assembly Protein Lsb1 Modulates a Yeast Prion

Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI⁺] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI⁺] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI⁺] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.     

Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes

Doxorubicin is a commonly used anti-cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance-associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell-derived cardiomyocytes (hiPSC-CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC-CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.     

Salt-stress-responsive chloroplast proteins in <Emphasis Type="Italic">Brassica juncea</Emphasis> genotypes with contrasting salt tolerance and their quantitative PCR analysis

Brassica juncea is mainly cultivated in the arid and semi-arid regions of India where its production is significantly affected by soil salinity. Adequate knowledge of the mechanisms underlying the salt tolerance at sub-cellular levels must aid in developing the salt-tolerant plants. A proper functioning of chloroplasts under salinity conditions is highly desirable to maintain crop productivity. The adaptive molecular mechanisms offered by plants at the chloroplast level to cope with salinity stress must be a prime target in developing the salt-tolerant plants. In the present study, we have analyzed differential expression of chloroplast proteins in two Brassica juncea genotypes, Pusa Agrani (salt-sensitive) and CS-54 (salt-tolerant), under the effect of sodium chloride. The chloroplast proteins were isolated and resolved using 2DE, which facilitated identification and quantification of 12 proteins that differed in expression in the salt-tolerant and salt-sensitive genotypes. The identified proteins were related to a variety of chloroplast-associated molecular processes, including oxygen-evolving process, PS I and PS II functioning, Calvin cycle and redox homeostasis. Expression analysis of genes encoding differentially expressed proteins through real time PCR supported our findings with proteomic analysis. The study indicates that modulating the expression of chloroplast proteins associated with stabilization of photosystems and oxidative defence plays imperative roles in adaptation to salt stress.     

Design,green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety as potential antithrombotic agents*

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.     

GA3 induced flowering in Podophyllum hexandrum Royle: A rare alpine medicinal herb

Podophyllum hexandrum Royle, an important alpine herb, is the source of highly valued podophyllotoxin. The effect of some plant growth substances (GA₃, BAP & ABA), uniconazole (an inhibitor of GA biosynthesis), and a combination of GA₃ and uniconazole were examined in respect to influence on sprouting in rhizomes of P. hexandrum and on induction of flowering at a lower altitude. Amongst the various chemicals tested, GA₃ had a marked effect resulting in uniform sprouting and also induced flowering in about half of the treated rhizomes. While BAP also promoted early sprouting, delayed sprouting was seen in rhizomes treated with ABA. Uniconazole treatment, either alone or with GA₃ was found to inhibit flowering and also resulted in reduced plant height. GA₃ treatment of rhizomes from plants that was maintained for up to 30 months at a lower altitude also induced flowering thus replacing the normal chilling requirement of plants. These results suggest that treatment of GA₃ could be effectively used for inducing uniform sprouting and flowering in rhizomes of P. hexandrum grown at lower altitudes.