Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Diagnostic value of lipopolysaccharide-binding protein and procalcitonin for sepsis diagnosis in forensic pathology.

The aims of this study were twofold. The first was to investigate the diagnostic performance of two biochemical markers, procalcitonin (PCT) and lipopolysaccharide-binding protein (LBP), considering each individually and then combined, for the postmortem diagnosis of sepsis. We also tested the usefulness of pericardial fluid for postmortem LBP determination. Two study groups were formed, a sepsis-related fatalities group of 12 cases and a control group of 30 cases. Postmortem native CT scans, autopsy, histology, neuropathology, and toxicology as well as other postmortem biochemical investigations were performed in all cases. Microbiological investigations were also carried out in the septic group. Postmortem serum PCT and LBP levels differed between the two groups. Both biomarkers, individually considered, allowed septic states to be diagnosed, whereas increases in both postmortem serum PCT and LBP levels were only observed in cases of sepsis. Similarly, normal PCT and LBP values in postmortem serum were identified only in non-septic cases. Pericardial fluid LBP levels do not correlate with the presence of underlying septic states. No relationship was observed between postmortem serum and pericardial fluid LBP levels in either septic or non-septic groups, or between pericardial fluid PCT and LBP levels.

Adolescent substance-use assessment: methodological issues in the use of the Adolescent Drug Abuse Diagnosis (ADAD).

During the past twenty years, various instruments have been developed for the assessment of substance use in adolescents, mainly in the United States. However, few of them have been adapted to, and validated in, French-speaking populations. Consequently, although increasing alcohol and drug use among teenagers has become a major concern, the various health and social programs developed in response to this specific problem have received little attention with regard to follow-up and outcome assessment. A standardized multidimensional assessment instrument adapted for adolescents is needed to assess the individual needs of adolescents and assign them to the most appropriate treatment setting, to provide a single measurement within and across health and social systems, and to conduct treatment outcome evaluations. Moreover, having an available instrument makes it possible to develop longitudinal and transcultural research studies. For this reason, a French version of the Adolescent Drug Abuse Diagnosis (ADAD) was developed and validated at the University Child and Adolescent Psychiatric Clinic in Lausanne, Switzerland. This article aims to discuss the methodological issues that we faced when using the ADAD instrument in a 4-year longitudinal study including adolescent substance users. Methodological aspects relating to the content and format of the instrument, the assessment administration and the statistical analyses are discussed.

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions.

Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal specific sequence motifs; however, CCG and CGC sequences seem to be overrepresented. Nonrandom association (multiple correspondence analysis, p < 0.0001) between silent genes, DNA methylation and MBD2 binding was observed. The association between MBD2 binding and transcriptional repression weakened as the distance between binding site and TSS increased, suggesting that MBD2 represses transcriptional initiation. This hypothesis may represent a functional explanation for the preferential binding of MBD2 at methyl-CpG in TSS regions.

HIV Specific Humoral Immune Response in Rio de Janeiro, Brazil

Efforts to characterize HIV-1 polymorphism and anti-HIV immune response are being made in areas where anti-HIV/AIDS vaccines are to be employed. Anti-HIV-1 humoral immune response is being studied in infected individuals resident in Rio de Janeiro, in distinct cohorts involving recent seroconvertors, pregnant women or intravenous drug users (IDU). Comparative analyses of specificity of antibody response towards epitopes important for anti-HIV-1 immune response indicate quantitative differences between cohorts, with an exceptionally strong response in IDUs and weakest response in pregnant women. However, a comparative analysis between pregnant women cohorts from Rio de Janeiro and Rio Grande do Sul indicated an even lower response (with exception of the anti-V3-C clade peptide recognition) for the southern cohort. Studies analysing the immune function of the humoral response indicate a quite elevated occurrence of antibodies capable of neutralizing heterologous primary HIV-1 isolates from Rio de Janeiro. Attempts to correlate seroreactivity with HIV-1 neutralization with respect to HIV-1 polymorphism were not very successfull: while the Brazilian B clade B" variant could be recognized by binding assays, no significant distinction of HIV-1 clades/variants was observed in viral neutralization assays.

Suivi thérapeutique des concentrations d'inhibiteurs de tyrosine kinase et d'autres médicaments oncologiques = Therapeutisches Drug-Monitoring der Konzentration von Tyrosinkinase-Inhibitoren und anderen Onkologika

Les médicaments anticancéreux sont souvent caractérisés par une importante variabilité pharmacocinétique interindividuelle, des relations entre concentration et réponse clinique et une marge thérapeutique étroite. Pourtant, le suivi thérapeutique des concentrations de ces médicaments (TDM) est encore rare en oncologie. Les bases scientifiques justifiant un TDM des nouvelles thérapies ciblées orales sont encore très hétérogènes. Cependant, d'assez solides évidences existent pour l'imatinib et certaines apparaissent progressivement pour d'autres composés. A côté de cela, le TDM est aussi pratiqué dans des situations spécifiques de traitement par certaines chimiothérapies conventionnelles. Des efforts considérables restent toutefois à réaliser pour mieux caractériser la pharmacocinétique de ces médicaments, pour préciser leurs relations concentration-effet et pour conduire des études prospectives randomisées évaluant le bénéfice clinique de l'approche TDM en oncologie.

Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo.

OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.

HIV type 1 drug resistance among naive patients from Venezuela.

In this study, we characterize proviral DNA of 20 HIV-1 asymptomatic antiretroviral-naive patients from Venezuela in env, gag, and pol genes regions. Results from both env/gag HMA subtyping and phylogenetic analysis of pol partial sequences led to the description of clade B in all cases. Nevertheless, the high prevalence of polymorphisms was particularly evident among the protease sequences. A 10% prevalence of major resistance mutations to RTIs was found. Our data also suggested that the protease polymorphisms I62T and V77T could be considered as molecular markers of the subtype B local epidemic. In addition, we show how proviral DNA can be used as a reliable tool to follow trends of resistance mutation transmission.

Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis.

PURPOSE: Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death. METHODS: Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA. RESULTS: In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death. CONCLUSIONS: These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

Molecular Genetic Analysis of Multi-drug Resistance in Indian Isolates of Mycobacterium tuberculosis

A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo b, gyr A, kat G were studied for mutation(s) by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) technique. The SSCP positive samples were sequenced to characterize the mutations in rpo b, and gyr A loci. While previously reported mutations in the gyr A and rpo b loci were found to be present, several novel mutations were also scored in the rpo b locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s) that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR) in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis one from India, highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

Antimalarial Drug Resistance: Surveillance and Molecular Methods for National Malaria Control Programmes

National malaria control programmes have the responsibility to develop a policy for malaria disease management based on a set of defined criteria as efficacy, side effects, costs and compliance. These will fluctuate over time and national guidelines will require periodic re-assessment and revision. Changing a drug policy is a major undertaking that can take several years before being fully operational. The standard methods on which a decision can be taken are the in vivo and the in vitro tests. The latter allow a quantitative measurement of the drug response and the assessment of several drugs at once. However, in terms of drug policy change its results might be difficult to interpret although they may be used as an early warning system for 2nd or 3rd line drugs. The new WHO 14-days in vivo test addresses mainly the problem of treatment failure and of haematological parameters changes in sick children. It gives valuable information on whether a drug still `works'. None of these methods are well suited for large-scale studies. Molecular methods based on detection of mutations in parasite molecules targeted by antimalarial drugs could be attractive tools for surveillance. However, their relationship with in vivo test results needs to be established

Selective and powerful stress gene expression in Arabidopsis in response to malondialdehyde.

The provenance, half-life and biological activity of malondialdehyde (MDA) were investigated in Arabidopsis thaliana. We provide genetic confirmation of the hypothesis that MDA originates from fatty acids containing more than two methylene-linked double bonds, showing that tri-unsaturated fatty acids are the in vivo source of up to 75% of MDA. The abundance of the combined pool of free and reversibly bound MDA did not change dramatically in stress, although a significant increase in the free MDA pool under oxidative conditions was observed. The half-life of infiltrated MDA indicated rapid metabolic turnover/sequestration. Exposure of plants to low levels of MDA using a recently developed protocol powerfully upregulated many genes on a cDNA microarray with a bias towards those implicated in abiotic/environmental stress (e.g. ROF1 and XERO2). Remarkably, and in contrast to the activities of other reactive electrophile species (i.e. small vinyl ketones), none of the pathogenesis-related (PR) genes tested responded to MDA. The use of structural mimics of MDA isomers suggested that the propensity of the molecule to act as a cross-linking/modifying reagent might contribute to the activation of gene expression. Changes in the concentration/localisation of unbound MDA in vivo could strongly affect stress-related transcription.

Drug uptake in a rodent sarcoma model after intravenous injection or isolated lung perfusion of free/liposomal doxorubicin.

The distribution of free and liposomal doxorubicin (Liporubicin) administered by intravenous injection (IV) or isolated lung perfusion (ILP) was compared in normal and tumor tissues of sarcoma bearing rodent lungs. A single sarcomatous tumor was generated in the left lung of 35 Fischer rats, followed 10 days later by left-sided ILP (n=20) or IV drug administration (n=12), using 100 microg and 400 microg free or liposomal doxorubicin, respectively. The tumor and lung tissue drug concentration was measured by HPLC. Free doxorubicin administered by ILP resulted in a three-fold (100 microg) and 10-fold (400 microg) increase of the drug concentration in the tumor and normal lung tissue compared to IV administration. In contrast, ILP with Liporubicin resulted in a similar drug uptake in the tumor and lung tissue compared to IV injection. For both drug formulations and dosages, ILP resulted in a higher tumor to lung tissue drug ratio but also in a higher spatial heterogeneity of drug distribution within the lung compared to IV administration. ILP resulted in a higher tumor to lung tissue drug ratio and in a more heterogeneous drug distribution within the lung compared to IV drug administration.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.