Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface

Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and β-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and β-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets

Hemocompatibility of Silicon-Based Substrates for Biomedical Implant Applications

Silicon membranes with highly uniform nanopore sizes fabricated using microelectromechanical systems (MEMS) technology allow for the development of miniaturized implants such as those needed for renal replacement therapies. However, the blood compatibility of silicon has thus far been an unresolved issue in the use of these substrates in implantable biomedical devices. We report the results of hemocompatibility studies using bare silicon, polysilicon, and modified silicon substrates. The surface modifications tested have been shown to reduce protein and/or platelet adhesion, thus potentially improving biocompatibility of silicon. Hemocompatibility was evaluated under four categories—coagulation (thrombin–antithrombin complex, TAT generation), complement activation (complement protein, C3a production), platelet activation (P-selectin, CD62P expression), and platelet adhesion. Our tests revealed that all silicon substrates display low coagulation and complement activation, comparable to that of Teflon and stainless steel, two materials commonly used in medical implants, and significantly lower than that of diethylaminoethyl (DEAE) cellulose, a polymer used in dialysis membranes. Unmodified silicon and polysilicon showed significant platelet attachment; however, the surface modifications on silicon reduced platelet adhesion and activation to levels comparable to that on Teflon. These results suggest that surface-modified silicon substrates are viable for the development of miniaturized renal replacement systems

Transcriptomic analysis of the interaction between <it>Helianthus annuus </it>and its obligate parasite <it>Plasmopara halstedii </it>shows single nucleotide polymorphisms in CRN sequences

<p>Abstract</p> <p>Background</p> <p>Downy mildew in sunflowers (<it>Helianthus annuus </it>L.) is caused by the oomycete <it>Plasmopara halstedii </it>(Farl.) Berlese et de Toni. Despite efforts by the international community to breed mildew-resistant varieties, downy mildew remains a major threat to the sunflower crop. Very few genomic, genetic and molecular resources are currently available to study this pathogen. Using a 454 sequencing method, expressed sequence tags (EST) during the interaction between <it>H. annuus </it>and <it>P. halstedii </it>have been generated and a search was performed for sites in putative effectors to show polymorphisms between the different races of <it>P. halstedii</it>.</p> <p>Results</p> <p>A 454 pyrosequencing run of two infected sunflower samples (inbred lines XRQ and PSC8 infected with race 710 of <it>P. halstedii</it>, which exhibit incompatible and compatible interactions, respectively) generated 113,720 and 172,107 useable reads. From these reads, 44,948 contigs and singletons have been produced. A bioinformatic portal, HP, was specifically created for in-depth analysis of these clusters. Using <it>in silico </it>filtering, 405 clusters were defined as being specific to oomycetes, and 172 were defined as non-specific oomycete clusters. A subset of these two categories was checked using PCR amplification, and 86% of the tested clusters were validated. Twenty putative RXLR and CRN effectors were detected using PSI-BLAST. Using corresponding sequences from four races (100, 304, 703 and 710), 22 SNPs were detected, providing new information on pathogen polymorphisms.</p> <p>Conclusions</p> <p>This study identified a large number of genes that are expressed during <it>H. annuus/P. halstedii </it>compatible or incompatible interactions. It also reveals, for the first time, that an infection mechanism exists in <it>P. halstedii </it>similar to that in other oomycetes associated with the presence of putative RXLR and CRN effectors. SNPs discovered in CRN effector sequences were used to determine the genetic distances between the four races of <it>P. halstedii</it>. This work therefore provides valuable tools for further discoveries regarding the <it>H. annuus/P. halstedii </it>pathosystem.</p