Synthesis of (R)-norbgugaine and its potential as quorum sensing inhibitor against Pseudomonas aeruginosa

(R)-Bgugaine is a natural pyrrolidine alkaloid from Arisarum vulgare, which shows antifungal and antibacterial activity. In this Letter, we have accomplished the simple synthesis of norbgugaine (demethylated form of natural bgugaine) employing Wittig olefination and cat. hydrogenation as the key steps and its biological studies are reported for the first time. The synthesized norbgugaine was evaluated for inhibition of quorum sensing mediated virulence factors (motility, biofilm formation, pyocyanin pigmentation, rhamnolipid production and LasA protease) in Pseudomonas aeruginosa wherein swarming motility is reduced by 95%, and biofilm formation by 83%.     

Zinc regulates the activity of kinase-phosphatase pair (BasPrkC/BasPrpC) in Bacillus anthracis

Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn²⁺ metal ions can alter their activities. Zn²⁺ promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn²⁺ in growing B. anthracis cells was found to vary with growth phase. Zn²⁺ was found to be lowest in log phase cells while it was highest in spores. This variation in Zn²⁺ concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn²⁺ as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation.     

Total soluble protein profiles of Beauveria bassiana and their relationship with virulence against Helicoverpa armigera

Intracellular total soluble proteins of Beauveria bassiana are believed to play an important role in virulence against insect hosts. Thirty B. bassiana isolates collected from different geographical regions and host ranges were characterised by total soluble proteins present in cells, using the SDS–PAGE technique to differentiate the isolates based on virulence and host insect origin. In vitro analysis of total soluble protein profiles of 30 isolates was studied to understand the relationship of isolates with their host of origin and virulence against Helicoverpa armigera. There was a positive relationship between virulence and host origin. All the non-virulent isolates are grouped together. Similarly, highly virulent isolates against H. armigera were grouped together. The relationship between total soluble proteins and pathogenicity was positively correlated. Thirty isolates shared only 22% similarity in their protein profiles.     

Synthesis of Benzothiazolylguanidines as Antituberculars and Antibacterials

The synthesis of twenty three new N-p-tolyl-N′-2-(substituted)benzothiazolyl-N″-alkyl-guanidines has been reported. Several of these including some N-p-tolyl-N′-2-(substitued)- benzothiazolylguanidines* have been evaluated for their antitubercular activity against M. tuberculosis (H 37 Rv) and also for their antibacterial activity. Among these N-p-tolyl-N′-2-(6-methoxy)benzothiazolylguanidine and N-p-tolyl-N′2-(6-methyUbenzothiazolyl-N″-methyl-guanidine are the most active as antituberculars but all the tested compounds are inactive as antibacterials.     

Mechanism of action of the tuberculosis and Crohn disease risk factor IRGM in autophagy

Polymorphisms in the IRGM gene, associated with Crohn disease (CD) and tuberculosis, are among the earliest identified examples documenting the role of autophagy in human disease. Functional studies have shown that IRGM protects against these diseases by modulating autophagy, yet the exact molecular mechanism of IRGM's activity has remained unknown. We have recently elucidated IRGM's mechanism of action. IRGM functions as a platform for assembling, stabilizing, and activating the core autophagic machinery, while at the same time physically coupling it to conventional innate immunity receptors. Exposure to microbial products or bacterial invasion increases IRGM expression, which leads to stabilization of AMPK. Specific protein-protein interactions and post-translational modifications such as ubiquitination of IRGM, lead to a co-assembly with IRGM of the key autophagy regulators ULK1 and BECN1 in their activated forms. IRGM physically interacts with 2 other CD risk factors, ATG16L1 and NOD2, placing these 3 principal players in CD within the same molecular complex. This explains how polymorphisms altering expression or function of any of the 3 factors individually can affect the same process—autophagy. Furthermore, IRGM's interaction with NOD2, and additional pattern recognition receptors such as NOD1, RIG-I, and select TLRs, transduces microbial signals to the core autophagy apparatus. This work solves the long-standing enigma of how IRGM controls autophagy.     

Effect of Methamphetamine on Spectral Binding,Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA_max and K_D of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δA_max (0.004±0.0003 vs. 0.0068±0.0001) and K_D (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δA_max (0.0038±0.0003 vs. 0.0055±0.0003) and K_D (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced K_D in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.     

Molecular Identification of Hookworm Isolates in Humans,Dogs and Soil in a Tribal Area in Tamil Nadu,India

BackgroundHookworms (Necator americanus and Ancylostoma duodenale) remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense) are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored.Conclusions/SignificanceIn our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.     

Evaluation of QTLs for Shoot Fly (<Emphasis Type="Italic">Atherigona soccata</Emphasis>) Resistance Component Traits of Seedling Leaf Blade Glossiness and Trichome Density on Sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis>) Chromosome SBI-10L

Shoot fly is a major insect pest of sorghum damaging early crop growth, establishment and productivity. Host plant resistance is an efficient approach to minimize yield losses due to shoot fly infestation. Seedling leaf blade glossiness and trichome density are morphological traits associated with shoot fly resistance. Our objective was to identify and evaluate QTLs for glossiness and trichome density using- i) 1894 F₂s, ii) a sub-set of 369 F₂-recombinants, and iii) their derived 369 F_2:3 progenies, from a cross involving introgression lines RSG04008-6 (susceptible)?×?J2614-11 (resistant). The QTLs were mapped to a 37–72 centimorgan (cM) or 5–15 Mb interval on the long arm of sorghum chromosome 10 (SBI-10L) with flanking markers Xgap001 and Xtxp141. One QTL each for glossiness (QGls10) and trichome density (QTd10) were mapped in marker interval Xgap001-Xnhsbm1044 and Xisep0630-Xtxp141, confirming their loose linkage, for which phenotypic variation accounted for ranged from 2.29 to 11.37 % and LOD values ranged from 2.03 to 24.13, respectively. Average physical map positions for glossiness and trichome density QTLs on SBI-10 from earlier studies were 4 and 2 Mb, which in the present study were reduced to 2 Mb and 800 kb, respectively. Candidate genes Glossy15 (Sb10g025053) and ethylene zinc finger protein (Sb10g027550) falling in support intervals for glossiness and trichome density QTLs, respectively, are discussed. Also we identified a sub-set of recombinant population that will facilitate further fine mapping of the leaf blade glossiness and trichome density QTLs on SBI-10.     

Revisiting cobalt chloride preconditioning to prevent hypobaric hypoxia-induced damage: identification of global proteomic alteration and key networks

Several studies have supported the hypoxia mimetic roles and cytoprotective properties of cobalt chloride in vitro and in vivo. However, a clear understanding of biological process-based mechanism that integrates the available information remains unknown. This study was aimed to explore the potential mechanism of cobalt chloride deciphering its benefits and well-known physiological challenge caused by hypobaric hypoxia that reportedly affects nearly 24 % of the global population. In order to explore the mechanism of CoCl₂, we used global proteomic and systems biology approach in rat model to provide a deeper insight into molecular mechanisms of preconditioning. Furthermore, key conclusions were drawn based on biological network analysis and their enrichment with ontological overlaps. The study was further strengthened by consistent identification of validation of proteins using immunoblotting. CoCl₂-pretreated animals exposed to hypoxia showed two significant networks, one lipid metabolism and other cell cycle associated, with a total score of 23 and eight focus molecules. In this study, we delineated two primary routes: one, by direct modulation of reactive oxygen species metabolism and, second, by regulation of lipid metabolism which was not known until now. The previously known benefits of cobalt chloride during physiological challenge by hypobaric hypoxia are convincing and could be explained by some basic set of metabolic and molecular reorganization within the hypoxia model. Interestingly, we also observed some of the completely unknown roles of cobalt chloride such as regulation of lipid that could undulate the translational roles of cobalt chloride supplementation beyond hypoxia preconditioning.