Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

Do Swiss adolescents perceive the negative effects of their illegal substance use?

This study explores adolescents' perceptions of adverse consequences linked to their illegal psychoactive substance (IPS) use, as they are often thought to minimise them. From a Swiss nationally representative sample of 8740 adolescents aged 16 to 20 pursuing post-mandatory education, 2515 participants reported IPS use in the past month on a self-administered anonymous questionnaire. The percentages of participants reporting problems in four areas (individual, school, relationships and sexual) were assessed, depending on the type of IPS consumption over the last 30 days: occasional cannabis users: ≤2 times; regular cannabis users: ≥3 times; and poly-consumers: cannabis plus at least one other substance used. The percentages varied significantly across these three groups with 26.9% of occasional users, 53.8% of regular users, and 73.3% of poly-consumers reporting at least one problem. Compared to occasional cannabis users, poly-consumers were more likely to report problems in all four categories [relative risk ratio (RRR): 3.38 to 5.44], while regular cannabis users often reported only school and relationship problems [RRR: 2.43 to 3.23]. Thus, many adolescents seem to perceive the negative effects of their IPS use, with heavier consumption being associated with increasing problems. Physicians should feel confident questioning adolescents on the adverse consequences of their IPS consumption, as they are likely to be responsive on this issue.

EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer.

Circulating tumor cells (CTCs) are frequently associated with epithelial-mesenchymal transition (EMT).The objective of this study was to detect EMT phenotype through Vimentin (VIM) and Slug expression in cytokeratin (CK)-negative CTCs in non-metastatic breast cancer patients and to determine the importance of EGFR in the EMT phenomenon. In CK-negative CTCs samples, both VIM and Slug markers were co-expressed in the most of patients. Among patients EGFR+, half of them were positive for these EMT markers. Furthermore, after a systemic treatment 68% of patients switched from CK- to CK+ CTCs. In our experimental model we found that activation of EGFR signaling by its ligand on MCF-7 cells is sufficient to increase EMT phenotypes, to inhibit apoptotic events and to induce the loss of CK expression. The simultaneous detection of both EGFR and EMT markers in CTCs may improve prognostic or predictive information in patients with operable breast cancer.

Incidence of active mycobacterial infections in Brazilian patients with chronic inflammatory arthritis and negative evaluation for latent tuberculosis infection at baseline - A longitudinal analysis after using TNFa blockers

Several studies point to the increased risk of reactivation of latent tuberculosis infection (LTBI) in patients with chronic inflammatory arthritis (CIAs) after using tumour necrosis factor (TNF)a blockers. To study the incidence of active mycobacterial infections (aMI) in patients starting TNFa blockers, 262 patients were included in this study: 109 with rheumatoid arthritis (RA), 93 with ankylosing spondylitis (AS), 44 with juvenile idiopathic arthritis (JIA) and 16 with psoriatic arthritis (PsA). All patients had indication for anti-TNFa therapy. Epidemiologic and clinical data were evaluated and a simple X-ray and tuberculin skin test (TST) were performed. The control group included 215 healthy individuals. The follow-up was 48 months to identify cases of aMI. TST positivity was higher in patients with AS (37.6%) than in RA (12.8%), PsA (18.8%) and JIA (6.8%) (p < 0.001). In the control group, TST positivity was 32.7%. Nine (3.43%) patients were diagnosed with aMI. The overall incidence rate of aMI was 86.93/100,000 person-years [95% confidence interval (CI) 23.6-217.9] for patients and 35.79/100,000 person-years (95% CI 12.4-69.6) for control group (p < 0.001). All patients who developed aMI had no evidence of LTBI at the baseline evaluation. Patients with CIA starting TNFa blockers and no evidence of LTBI at baseline, particularly with nonreactive TST, may have higher risk of aMI.

Predicting response and survival in chemotherapy-treated triple-negative breast cancer.

BACKGROUND In this study, we evaluated the ability of gene expression profiles to predict chemotherapy response and survival in triple-negative breast cancer (TNBC). METHODS Gene expression and clinical-pathological data were evaluated in five independent cohorts, including three randomised clinical trials for a total of 1055 patients with TNBC, basal-like disease (BLBC) or both. Previously defined intrinsic molecular subtype and a proliferation signature were determined and tested. Each signature was tested using multivariable logistic regression models (for pCR (pathological complete response)) and Cox models (for survival). Within TNBC, interactions between each signature and the basal-like subtype (vs other subtypes) for predicting either pCR or survival were investigated. RESULTS Within TNBC, all intrinsic subtypes were identified but BLBC predominated (55-81%). Significant associations between genomic signatures and response and survival after chemotherapy were only identified within BLBC and not within TNBC as a whole. In particular, high expression of a previously identified proliferation signature, or low expression of the luminal A signature, was found independently associated with pCR and improved survival following chemotherapy across different cohorts. Significant interaction tests were only obtained between each signature and the BLBC subtype for prediction of chemotherapy response or survival. CONCLUSIONS The proliferation signature predicts response and improved survival after chemotherapy, but only within BLBC. This highlights the clinical implications of TNBC heterogeneity, and suggests that future clinical trials focused on this phenotypic subtype should consider stratifying patients as having BLBC or not.

Análisis de contenidos de páginas web sobre servicios de control de plagas en salud pública

Introduction: Our goal was to know the web contents and examine the technical information pest control services available to users through their webpages. Method: A total of 70 webpages from biocides services in the province of Málaga (Spain) were analyzed. We used 15 evaluation indicators grouped into 5 parameters relating to data of the service provider; information’s reliability and services; accuracy of content and writing style; technical resources and interaction with the users. As test instruments were used sectoral legislation, official records of products and deliveries, standards and technical guides. Results: Companies showed a remarkable degree of awareness with the implementation and use of new technologies. Aspects negative that they can have an impact on the confidence of users, relating to the reliability of the information and deficiencies associated with the description of the services portfolio and credentials of the companies were identified. The integration and use of collaborative platforms 2.0 was poorly developed and squandered. Discussion: It is possible to improve the trust of users intervening in those aspects that affect the reliability of the information provided on the web.

The hairy and enhancer of split 1 is a negative regulator of the repressor element silencer transcription factor.

Silencing of the transcriptional repressor REST is required for terminal differentiation of neuronal and beta-cells. In this study, we hypothesized that REST expression is controlled by hairy and enhancer of split 1 (HES-1), a transcriptional repressor that plays an important role in brain and pancreas development. We identified several N elements (CTNGTG) within the promoter of REST and confirmed that HES-1 associates with the endogenous promoter of REST. Moreover, using a cells model that overexpress HES-1 and a combination of experimental approaches, we demonstrated that HES-1 reduces endogenous REST expression. Taken together, these results indicate that HES-1 is an upstream negative regulator of REST expression.

Positive and negative regulation of cellular immune responses in physiologic conditions and diseases.

The immune system has evolved to allow robust responses against pathogens while avoiding autoimmunity. This is notably enabled by stimulatory and inhibitory signals which contribute to the regulation of immune responses. In the presence of a pathogen, a specific and effective immune response must be induced and this leads to antigen-specific T-cell proliferation, cytokines production, and induction of T-cell differentiation toward an effector phenotype. After clearance or control of the pathogen, the effector immune response must be terminated in order to avoid tissue damage and chronic inflammation and this process involves coinhibitory molecules. When the immune system fails to eliminate or control the pathogen, continuous stimulation of T cells prevents the full contraction and leads to the functional exhaustion of effector T cells. Several evidences both in vitro and in vivo suggest that this anergic state can be reverted by blocking the interactions between coinhibitory molecules and their ligands. The potential to revert exhausted or inactivated T-cell responses following selective blocking of their function made these markers interesting targets for therapeutic interventions in patients with persistent viral infections or cancer.

Redox dysregulation and oxidative stress in schizophrenia: genetic and functional anomalies of glutathione synthesis in patients and experimental models involving GABA interneurons and NMDA receptors

Objective: Converging evidence speak in favor of an abnormal susceptibility to oxidative stress in schizophrenia. A decreased level of glutathione (GSH), the principal non-protein antioxidant and redox regulator, was observed both in cerebrospinal-fluid and prefrontal cortex of schizophrenia patients (Do et al., 2000). Results: Schizophrenia patients have an abnormal GSH synthesis most likely of genetic origin: Two independent case-control studies showed a significant association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) catalytic subunit (GCLC) gene. The most common TNR genotype 7/7 was more frequent in controls, whereas the rarest TNR genotype 8/8 was three times more frequent in patients. The disease-associated genotypes correlated with a decrease in GCLC protein expression, GCL activity and GSH content. Such a redox dysregulation during development could underlie the structural and functional anomalies in connectivity: In experimental models, GSH deficit induced anomalies similar to those observed in patients. (a) morphology: In animal models with GSH deficit during the development we observed in prefrontal cortex a decreased dendritic spines density in pyramidal cells and an abnormal development of parvalbumine (but not of calretinine) immunoreactive GABA interneurones in anterior cingulate cortex. (b) physiology: GSH depletion in hippocampal slices induces NMDA receptors hypofunction and an impairment of long term potentiation. In addition, GSH deficit affected the modulation of dopamine on NMDA-induced Ca 2+ response in cultured cortical neurons. While dopamine enhanced NMDA responses in control neurons, it depressed NMDA responses in GSH-depleted neurons. Antagonist of D2-, but not D1-receptors, prevented this depression, a mechanism contributing to the efficacy of antipsychotics. The redox sensitive ryanodine receptors and L-type calcium channels underlie these observations. (c) cognition: Developing rats with low [GSH] and high dopamine lead deficit in olfactory integration and in object recognition which appears earlier in males that females, in analogy to the delay of the psychosis onset between man and woman. Conclusion: These clinical and experimental evidence, combined with the favorable outcome of a clinical trial with N-Acetyl Cysteine, a GSH precursor, on both the negative symptoms (Berk et al., submitted) and the mismatch negativity in an auditory oddball paradigm supported the proposal that a GSH synthesis impairment of genetic origin represent, among other factors, one major risk factor in schizophrenia.

A-8a 2003 Summary of Food Stamp Errors - Active and Negative Cases, April 2003 - September 2003

Summary of food stamp errors.

Combining MAR elements and transposable vectors for more reliable transgene integration and expression

Integration without cytotoxic effects and long-term expression of a transgene constitutes a major challenge in gene therapy and biotechnology applications. In this context, transposons represent an attractive system for gene transfer because of their ability to promote efficient integration of a transgene in a variety of cell lines. However, the transgene integration can lead to insertional mutagenesis and/or unstable transgene expression by epigenetic modifications. These unwanted events may be limited by the use of chromatin control elements called MARs (matrix attachment regions). Indeed, the insertion of these DNA elements next to the transgene usually results in higher and more stable expression by maintaining transgene chromatin in an active configuration and preventing gene silencing. In this study, we tested if the inclusion of the MAR 1-68 in the piggyBac transposon system may lead to efficient and safer transgene integration and ensure reliable stable and long-term expression of a transgene. The MAR-containing transposon construct was tested in CHO cells, for biotechnology applications, and in mesoangioblast cells that can differentiate into muscle cells and are important candidates for potential stem cell therapies of myopathies. We showed that the addition of the MAR 1 -68 in the piggyBac transposon did not interfere with transposition, thereby maintaining high frequency of transgene integrations in these cells. Moreover, the MAR allowed higher transgene expression from fewer transposon integration events. We also found that enriched transgene-expressing cell populations could be obtained without the need of selection pressure. Since antibiotic-enforced selection protocols often result in a higher integrated copy number and mosaic expression patterns, this strategy could benefit many applications in which a low copy number of integrated transgenes and antibiotic-free conditions are desired. In addition, the intramuscular transplantation of mouse tibialis anterior muscles with mesoangioblasts containing the transposon led to widespread and sustained myofiber transgene expression after differentiation of these cells in vivo. These findings indicated that piggyBac vectors may provide a viable approach to achieve stable gene transfer in the context of Duchenne muscular dystrophy therapy. - L'intégration sans effets cytotoxiques et l'expression à long terme d'un transgène constituent un défi majeur en thérapie génique et en biotechnologie. Dans ce contexte, les transposons représentent un système attrayant pour le transfert de gènes en raison de leur capacité à promouvoir l'intégration efficace d'un transgène dans une variété de lignées cellulaires. Toutefois, l'intégration d'un transgène peut conduire à une mutagénèse insertionnelle et/ou à une expression instable due au silençage du transgène suite à des modifications épigénétiques. Ces événements indésirables de silençage génique peuvent être diminués par l'utilisation d'éléments de contrôle de la chromatine appelés MAR (matrix attachment region). En effet, l'insertion de ces éléments d'ADN à proximité du transgène se traduit généralement par une expression plus élevée et plus stable de celui-ci, en permettant le maintien d'une chromatine dans une configuration active autour du transgène et en empêchant l'inactivation du gène. Dans cette étude, nous avons testé si l'inclusion du MAR 1-68 dans le système transposon piggyBac peut améliorer l'efficacité d'intégration de façon sécuritaire et l'expression à long terme d'un transgène. Le transposon contenant l'élément MAR a été testé dans les cellules CHO, couramment utilisées en biotechnologie, et dans des cellules progénitrices appelées mésoangioblastes, qui peuvent se différencier en cellules musculaires, et qui constituent ainsi des candidats prometteurs pour la thérapie à partir de cellules souches de patients souffrant de myopathie. Nous avons montré que l'addition du MAR 1-68 dans le transposon piggyBac n'interfère pas avec la transposition et permet de maintenir une fréquence élevée d'intégration du transgène dans ces deux types cellulaires. De plus, il semble que cette association mène à une meilleure expression du transgène à partir de peu d'événements d'intégration du transposon. En outre, ces populations enrichies en cellules exprimant de façon stable le transgène ont pu être obtenues sans avoir recours à une pression de sélection. Etant donné que les protocoles de sélection basée sur l'utilisation d'antibiotiques conduisent souvent à un nombre plus élevé de copies intégrées et à la variégation de l'expression du transgène et qu'ils impliquent une longue culture in vitro, cette stratégie pourrait profiter à des applications pour lesquelles on souhaite un faible nombre de copies intégrées et/ou l'utilisation d'antibiotiques n'est pas souhaitable. De plus, la transplantation intramusculaire de mésoangioblastes contenant le transposon dans le muscle tibial antérieur de souris a conduit, après la différentiation de ces cellules in vivo, à une expression constante et étendue du transgène dans les myofibres. Ces résultats indiquent que les vecteurs piggyBac pourraient fournir une approche viable pour assurer un transfert de gènes stables dans le contexte d'un traitement de la dystrophic musculaire de Duchenne.

Performance of classic electrocardiographic criteria for left ventricular hypertrophy in an African population.

ECG criteria for left ventricular hypertrophy (LVH) have been almost exclusively elaborated and calibrated in white populations. Because several interethnic differences in ECG characteristics have been found, the applicability of these criteria to African individuals remains to be demonstrated. We therefore investigated the performance of classic ECG criteria for LVH detection in an African population. Digitized 12-lead ECG tracings were obtained from 334 African individuals randomly selected from the general population of the Republic of Seychelles (Indian Ocean). Left ventricular mass was calculated with M-mode echocardiography and indexed to body height. LVH was defined by taking the 95th percentile of body height-indexed LVM values in a reference subgroup. In the entire study sample, 16 men and 15 women (prevalence 9.3%) were finally declared to have LVH, of whom 9 were of the reference subgroup. Sensitivity, specificity, accuracy, and positive and negative predictive values for LVH were calculated for 9 classic ECG criteria, and receiver operating characteristic curves were computed. We also generated a new composite time-voltage criterion with stepwise multiple linear regression: weighted time-voltage criterion=(0.2366R(aVL)+0.0551R(V5)+0.0785S(V3)+ 0.2993T(V1))xQRS duration. The Sokolow-Lyon criterion reached the highest sensitivity (61%) and the R(aVL) voltage criterion reached the highest specificity (97%) when evaluated at their traditional partition value. However, at a fixed specificity of 95%, the sensitivity of these 10 criteria ranged from 16% to 32%. Best accuracy was obtained with the R(aVL) voltage criterion and the new composite time-voltage criterion (89% for both). Positive and negative predictive values varied considerably depending on the concomitant presence of 3 clinical risk factors for LVH (hypertension, age >/=50 years, overweight). Median positive and negative predictive values of the 10 ECG criteria were 15% and 95%, respectively, for subjects with none or 1 of these risk factors compared with 63% and 76% for subjects with all of them. In conclusion, the performance of classic ECG criteria for LVH detection was largely disparate and appeared to be lower in this population of East African origin than in white subjects. A newly generated composite time-voltage criterion might provide improved performance. The predictive value of ECG criteria for LVH was considerably enhanced with the integration of information on concomitant clinical risk factors for LVH.