Errors are inherent in all biological systems. Errors in protein translation are particularly frequent giving rise to a collection of protein quasi-species, the diversity of which will vary according to the error rate. As mistranslation rates rise, these new proteins could produce new phenotypes, although none have been identified to date. Here, we find that mycobacteria substitute glutamate for glutamine and aspartate for asparagine at high rates under specific growth conditions. Increasing the substitution rate results in remarkable phenotypic resistance to rifampicin, whereas decreasing mistranslation produces increased susceptibility to the antibiotic. These phenotypic changes are reflected in differential susceptibility of RNA polymerase to the drug. We propose that altering translational fidelity represents a unique form of environmental adaptation.drug tolerance | persisters

The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [ 14 C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.T he rise of multidrug-resistant (MDR) and extensively drugresistant (XDR) Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), makes the validation of new antitubercular agents a major global priority. Since tubercular drug resistance is chromosomally encoded (17), chemotherapeutic agents directed against new cellular targets are likely to be effective against both drug-sensitive and drug-resistant M. tuberculosis strains (5,12,13,18). Target identification and validation are usually achieved by either genetic or chemical approaches. The former has the advantage of identifying a likely cellular target a priori but yields no information with regard to the druggability of the target and the access of the drug to the target (a particular problem in mycobacteria [23]). It is therefore not surprising that no current antitubercular agents have been identified through rational drug design (23). Alternatively, the identification of a cellular target candidate through chemical screening has the advantage of knowing that the compound can bind and affect the cellular target in vivo. The identification of the target for an active compound allows the rational modification of lead candidates through medicinal chemistry while ensuring that the compound retains activity against its primary target. However, finding which proteins are inhibited by a compound can be quite challenging.We randomly screened a library of compounds to identify structures of interest for further development. Several azole compounds containing imidazole, pyrrole, toluidine, or methanamine groups were tested for antimycobacterial activity. Among them, 1-{[1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl]methyl}-4-methylpiperazine (BM212) (Fig. 1) proved to be active against multidrug-resistant clinical isolates, against M. tuberculosis residing within macrophages, and against Mycobacterium avium as well as other nontuberculous mycobacteria (7). The identification of BM21...

In normally functioning prosthetic heart valves, subclinical hemolysis is a frequent finding. A low incidence of hemolysis is found in stented biologic prostheses, and it is absent in stentless aortic valves. Modifications of valve design may contribute to minimize the occurrence of hemolysis in mechanical prostheses.

A significant number of patients submitted to mitral repair for functional MR present > or =3+ TR at follow-up as consequence of progression of untreated TR or failure of tricuspid repair. A more aggressive and effective treatment of functional TR in this setting should be pursued.

Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.T uberculosis (TB) caused by Mycobacterium tuberculosis affects 9.0 million people annually, with 1.5 million deaths in 2013 (1). Standard TB treatment involves a regimen of four antibiotics taken daily for 6 to 9 months. However, the long treatment duration, toxicity, and interaction with antiretrovirals lead to poor patient compliance and treatment failure. Novel TB drug regimens are therefore urgently needed to treat both standard and drug-resistant forms of TB. Two new drugs, bedaquiline (2) and delamanid (3), were recently approved for the treatment of multidrug-resistant (MDR) TB, and other compounds are in the clinical development pipeline (4). Yet, the search for new TB drug candidates with different modes of action seeks to increase the chances of finding new drugs.Screening of chemical libraries is the first crucial step in the antimicrobial discovery process. Potential antimycobacterial agents are identified by testing chemicals for the ability to inhibit M. tuberculosis growth under in vitro growth conditions in culture medium. However, in vitro screening results are often misleading, as the culture broth does not reflect the environment M. tuberculosis encounters in vivo during the natural course of the disease, neglecting important factors such as compound activation, membrane permeability, removal by efflux pump, and toxicity to mammalian cells (4). Furthermore, adaptive metabolic changes that M. tuberculosis undergoes within the host may affect compound activity (5). Ex vivo screening, in the macrophage, may represent physiological conditions that mimic disease and take into consideration the favorable contribution of host cells in the process of eradicating M. tuberculosis.M. tuberculosis's intracellular lifestyle presents an attractive area for new drug discovery programs. A successful example is the intracellular high-content screening campaign that led to the discovery of Q203 (6). Image-based high-content screening technologies are being adopted more frequently to evaluate the activities of compounds against M. tuberculosis by using various cell types (7-9) or the granuloma infection model (10).High-content screening against M. tuberculosis is a robust and informative assay; however, it is still lacking in terms of speed and simplicity since the endpoint assay requires multiple steps for staining, image acquisition, and cumbersome data analysis. In addition, most of the intracellular compound screening done so far was performed inside e...