Philippe Lefèvre

Mycobacterium tuberculosis with disruption in genes encoding the phosphate binding proteins PstS1 and PstS2 is deficient in phosphate uptake and demonstrates reduced in vivo virulence.

ABSTRACT By measuring phosphate uptake by Mycobacterium tuberculosis strains with the pstS1 and pstS2 genes genetically inactivated, we showed that these pstS genes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosis encounters limiting phosphate concentrations during its intracellular life span.

Molecular Genetics of para-Aminosalicylic Acid Resistance in Clinical Isolates and Spontaneous Mutants of Mycobacterium tuberculosis

ABSTRACT The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.

Evaluation of Combined General Primer-Mediated PCR Sequencing and Type-Specific PCR Strategies for Determination of Human Papillomavirus Genotypes in Cervical Cell Specimens

ABSTRACT A strategy combining human papillomavirus general primer (mainly the PGMY primers)-directed PCR sequencing and type-specific PCR is presented. DNA samples were first tested in general primer-mediated PCR. The amplified fragments of positive samples after ethidium bromide-stained DNA gel analysis were further sequenced, and corresponding DNA samples were further analyzed by PCR using type-specific primers for human papillomavirus (HPV) types 16, 18, 31, and 52. The comparison of the results of 157 samples analyzed by this strategy in parallel with the Hybrid Capture 2 tests and with the HPV INNO-LiPA (Innogenetics line probe assay) shows that this method is suitable for HPV detection and genotyping in cervical cell samples. Although the PCR sequencing method is as sensitive as the HPV INNO-LiPA for HPV detection, our method allows the identification of a broader range of HPV types. In contrast, the HPV INNO-LiPA was less time-consuming and better identified coinfections.

Mass Disaster Victim Identification: The Tsunami Experience

One of the most important reasons to identify unknown persons is because non-identification may result in nu- merous issues at emotional and legal level for the surviving family members and friends. To reach a level of scientific supported positive identification, which can withstand international legal scrutiny, the identi- fication process of mass disaster victims requires a well-structured and pre-planned management based upon the interna- tional Interpol DVI Standing Committee guidelines. The handling of the December 2004 Tsunami disaster, affecting people of different nationalities and ethnic origins, will be used as an example to illustrate this methodology and standard operating protocols which can be used under similar circumstances. The activities of the Belgian DVI Team in the Khao Lak area, as part of the multinational help force bringing assistance to the local Thai law enforcement agencies, is discussed. Taking part in the first operations of identification, we were confronted with the hard reality of the hugeness of this plane- tary tragedy having made on the whole more than 200,000 victims. Differences in composition of the teams and their working philosophies are illustrated, based on the experience of the Belgian multidisciplinary team including scientific forensic experts (pathology, anthropology, odontology), police offi- cers, members of the civil protection (with their technical capabilities in particular working conditions), but also a physi- cian, a nurse and a stress team. Some of the applied methods and techniques used in the identification process were reviewed and suggestions given about how improvements can be made for future reference.

Biochemical and immunological characterization of a cpn60.1 knockout mutant of Mycobacterium bovis BCG

Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (Δcpn60.1) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the Δcpn60.1 mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidentally, with less protein in its outermost cell envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition, the Δcpn60.1 mutant is devoid of phthiocerol dimycocerosates, and its mycolates are two carbon atoms longer than those of the wild-type, a phenotype that is fully reversed by complementation. In addition, Δcpn60.1 bacteria are more sensitive to stress induced by H(2)O(2) but not by SDS, high temperature or acidic pH. Taken together, these data indicate that the cell wall of the Δcpn60.1 mutant is impaired. Analysis by 2D gel electrophoresis and MS reveals the upregulation of a few proteins such as FadA2 and isocitrate lyase in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate, e.g. the well-known Hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the Δcpn60.1 mutant. In mice, the Δcpn60.1 mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific gamma interferon production and protection against Mycobacterium tuberculosis H37Rv challenge as do the parental and complemented BCG strains. Thus, by inactivating the cpn60.1 gene in M. bovis BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to H(2)O(2)-induced stress but is not essential for its vaccine potential.

Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria

ABSTRACT Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing Mycobacterium bovis BCG and M. tuberculosis mutants that lack phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids with wild-type strains, we observed that glycopeptides strongly inhibited PDIM-deprived mycobacteria. Vancomycin together with a drug targeting lipid synthesis inhibited multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential antituberculosis (TB) agents and might provide a new antimycobacterial drug-screening strategy.

Sex determination using the Probabilistic Sex Diagnosis (DSP: Diagnose Sexuelle Probabiliste) tool in a virtual environment.

The hip bone is one of the most reliable indicators of sex in the human body due to the fact it is the most dimorphic bone. Probabilistic Sex Diagnosis (DSP: Diagnose Sexuelle Probabiliste) developed by Murail et al., in 2005, is a sex determination method based on a worldwide hip bone metrical database. Sex is determined by comparing specific measurements taken from each specimen using sliding callipers and computing the probability of specimens being female or male. In forensic science it is sometimes not possible to sex a body due to corpse decay or injury. Skeletalization and dissection of a body is a laborious process and desecrates the body. There were two aims to this study. The first aim was to examine the accuracy of the DSP method in comparison with a current visual sexing method on sex determination. A further aim was to see if it was possible to virtually utilise the DSP method on both the hip bone and the pelvic girdle in order to utilise this method for forensic sciences. For the first part of the study, forty-nine dry hip bones of unknown sex were obtained from the Body Donation Programme of the Université Libre de Bruxelles (ULB). A comparison was made between DSP analysis and visual sexing on dry bone by two researchers. CT scans of bones were then analysed to obtain three-dimensional (3D) virtual models and the method of DSP was analysed virtually by importing the models into a customised software programme called lhpFusionBox which was developed at ULB. The software enables DSP distances to be measured via virtually-palpated bony landmarks. There was found to be 100% agreement of sex between the manual and virtual DSP method. The second part of the study aimed to further validate the method by analysing thirty-nine supplementary pelvic girdles of known sex blind. There was found to be a 100% accuracy rate further demonstrating that the virtual DSP method is robust. Statistically significant differences were found in the identification of sex between researchers in the visual sexing method although both researchers identified the same sex in all cases in the manual and virtual DSP methods for both the hip bones and pelvic girdles.

Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria

ABSTRACT Tuberculosis is still a cause of major concern, partly due to the emergence of multidrug-resistant strains. New drugs are therefore needed. Vancomycin can target mycobacteria with cell envelope deficiency. In this study, we used a vancomycin susceptibility assay to detect drugs hampering lipid synthesis in Mycobacterium bovis BCG and in Mycobacterium tuberculosis. We tested three drugs already used to treat human obesity: tetrahydrolipstatin (THL), simvastatin, and fenofibrate. Only vancomycin and THL were able to synergize on M. bovis BCG and on M. tuberculosis, although mycobacteria could also be inhibited by simvastatin alone. Lipid analysis allowed us to identify several lipid modifications in M. tuberculosis H37Rv treated with those drugs. THL treatment mainly reduced the phthiocerol dimycocerosate (PDIM) content in the mycobacterial cell wall, providing an explanation for the synergy, since PDIM deficiency has been related to vancomycin susceptibility. Proteomic analysis suggested that bacteria treated with THL, in contrast to bacteria treated with simvastatin, tried to recover, inducing, among other reactions, lipid synthesis. The combination of THL and vancomycin should be considered a promising solution in developing new strategies to treat multidrug-resistant tuberculosis.

Biochemical Analysis of the NAD+-Dependent Malate Dehydrogenase, a Substrate of Several Serine/Threonine Protein Kinases of Mycobacterium tuberculosis

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.

The lacertus fibrosus of the biceps brachii muscle: an anatomical study

PurposeThe lacertus fibrosus (LF) is involved in various surgical procedures. However, the anatomy, morphometry, topography and biomechanical involvements of LF are not clear. The purpose of this study was to determine the anatomical and morphometric variations of LF, and to correlate this with anthropometric and morphometric measurements of the upper limb. Furthermore, the presence or absence of a deep layer of LF was verified using forearm cross-sections and dissections.MethodsThis anatomical study was performed by observation of dissections and transverse sections obtained from 50 cadavers. Morphometric analyses [length and width of LF and biceps tendon, stature, length of upper limb, forearm, bi-epicondylar width, forearm perimeter, biceps brachii muscle perimeter (BBm)] were also performed.ResultsThe results demonstrated that there was no significant correlation between LF morphology and morphometric upper limb measurements. The deep layer of LF was observed in all specimens.ConclusionResults of this paper indicate that the LF presents individual characteristics such as length and width. The deeper layer of LF was observed on all specimens. The possible role of LF in force transmission during flexion, BBm moment arm adjustment and supination reduction is discussed in view of these results.