Preservation of a Split Tooth: Nonsurgical Clinical Management.

A tooth is considered to be a split tooth if it contains a fracture line extending from the occlusal surface through both marginal ridges. Currently, the only treatment option for a split tooth is extraction. The present case report describes a novel therapeutic strategy for the preservation of a split tooth (first maxillary premolar) with a fracture in the mesiodistal direction. A systematic nonsurgical procedure involving visualization and slight widening of the fracture line is described. This procedure facilitates sealing of the fracture gap with a biocompatible calcium silicate cement (mineral trioxide aggregate) as well as internal composite resin stabilization and cuspal coverage restoration of the tooth. A 3-year follow-up showed a promising clinical and radiographic outcome. The concept presented here is an alternative treatment option for a split tooth, which allows preservation of the tooth rather than its extraction.

Mycobacterium tuberculosis Rv0560c is not essential for growth in vitro or in macrophages.

Mycobacterium tuberculosis Rv0560c, a putative benzoquinone methyl transferase, is heavily induced in response to salicylate exposure. It has some similarity to Escherichia coli UbiG, although its role in ubiquinone or menaquinone synthesis is not clear, since M. tuberculosis is not known to produce ubiquinone. We constructed an unmarked in-frame deletion of Rv0560c in M. tuberculosis to determine its role in vitro. Deletion of Rv0560c in M. tuberculosis had no effect on growth in medium containing salicylate or in its ability to grow in macrophages. In addition, no change to compound sensitivity, as determined by minimum inhibitory concentrations, for a range of compounds targeting respiration was noted. Plumbagin, ethambutol and CCCP had the same minimum bactericidal concentration against the deletion and wild-type strains. Taken together these data show that Rv0560c is dispensable under in vitro conditions in both axenic and macrophage culture and suggest that the role of Rv0560c may be in an alternate biosynthetic pathway of menaquinone which is only used under specific growth conditions.

Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA.

In Mycobacterium tuberculosis, the genes Rv1954A-Rv1957 form an operon that includes Rv1955 and Rv1956 which encode the HigB toxin and the HigA antitoxin respectively. We are interested in the role and regulation of this operon, since toxin-antitoxin systems have been suggested to play a part in the formation of persister cells in mycobacteria. To investigate the function of the higBA locus, effects of toxin expression on mycobacterial growth and transcript levels were assessed in M. tuberculosis H37Rv wild type and in an operon deletion background. We show that expression of HigB toxin in the absence of HigA antitoxin arrests growth and causes cell death in M. tuberculosis. We demonstrate HigB expression to reduce the abundance of IdeR and Zur regulated mRNAs and to cleave tmRNA in M. tuberculosis, Escherichia coli and Mycobacterium smegmatis. This study provides the first identification of possible target transcripts of HigB in M. tuberculosis.

Mycobacterium tuberculosis ClpP proteases are co-transcribed but exhibit different substrate specificities.

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.

The promoter of Rv0560c is induced by salicylate and structurally-related compounds in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major global health threat. During infection, bacteria are believed to encounter adverse conditions such as iron depletion. Mycobacteria synthesize iron-sequestering mycobactins, which are essential for survival in the host, via the intermediate salicylate. Salicylate is a ubiquitous compound which is known to induce a mild antibiotic resistance phenotype. In M. tuberculosis salicylate highly induces the expression of Rv0560c, a putative methyltransferase. We identified and characterized the promoter and regulatory elements of Rv0560c. P(Rv0560c) activity was highly inducible by salicylate in a dose-dependent manner. The induction kinetics of P(Rv0560c) were slow, taking several days to reach maximal activity, which was sustained over several weeks. Promoter activity could also be induced by compounds structurally related to salicylate, such as aspirin or para-aminosalicylic acid, but not by benzoate, indicating that induction is specific to a structural motif. The -10 and -35 promoter elements were identified and residues involved in regulation of promoter activity were identified in close proximity to an inverted repeat spanning the -35 promoter element. We conclude that Rv0560c expression is controlled by a yet unknown repressor via a highly-inducible promoter.