Automated systems in the identification and determination of methicillin resistance among coagulase negative staphylococci

Coagulase-negative staphylococci (CoNS) are an important cause of nosocomial bacteremia, specially in patients with indwelling devices or those submitted to invasive medical procedures. The identification of species and the accurate and rapid detection of methicillin resistance are directly dependent on the quality of the identification and susceptibility tests used, either manual or automated. The objective of this study was to evaluate the accuracy of two automated systems MicroScan and Vitek - in the identification of CoNS species and determination of susceptibility to methicillin, considering as gold standard the biochemical tests and the characterization of the mecA gene by polymerase chain reaction, respectively. MicroScan presented better results in the identification of CoNS species (accuracy of 96.8 vs 78.8%, respectively); isolates from the following species had no precise identification: Staphylococcus haemolyticus, S. simulans, and S. capitis. Both systems were similar in the characterization of methicillin resistance. The higher discrepancies for gene mec detection were observed among species other than S. epidermidis (S. hominis, S. saprophyticus, S. sciuri, S. haemolyticus, S. warneri, S. cohnii), and those with borderline MICs.

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Identification and functional characterization of a monofunctional peroxisomal enoyl-CoA hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

More about the role of 2,6-dichlorophenol in tick courtship: identification and olfactometer bioassay in Amblyomma cajennense and Rhipicephalus sanguineus

This study aimed to identify 2,6-dichlorophenol (2,6-DCP) in Amblyomma cajennense and to evaluate its role in A. cajennense and Rhipicephalus sanguineus courtship. Hexanic extract from attractive females was purified by solid phase extraction and the phenol was identified by the single ion monitoring method using GC/MS. In an olfactometer, the responses of A. cajennense and R. sanguineus males to females, control rubber septa or rubber septa impregnated with 2,6-DCP at 50, 500, and 5000 ng, respectively, were studied. 2,6-DCP was identified in A. cajennense extract and the males oriented themselves toward the concentration of 500 ng. These septa and the females were recognized as copula partners. The septa treated with 2,6-DCP did not attract and were not even recognized by the R. sanguineus males, whereas the females were recognized. Due to the presence of 2,6-DCP in A. cajennense and the results of biological bioassays, it was concluded that this compound acts as an attractant and mounting sex pheromone in this tick, but it does not play any role in R. sanguineus courtship.

Pathophysiology and classification of the vibration white finger.

Skin, arteries and nerves of the upper extremities can be affected by vibration exposure. Recent advances in skin and vascular biology as well as new investigative methods, have shown that neurovascular symptoms may be due to different vascular and neurological disorders which should be differentiated if proper management is to be evaluated. Three types of vascular disorder can be observed in the vibration white finger: digital organic microangiopathy, a digital vasospastic phenomenon and arterial thrombosis in the upper extremities. An imbalance between endothelin-1 and calcitonin-gene-related peptide is probably responsible for the vasospastic phenomenon. Moreover, paresthesiae can be due to either a diffuse vibration neuropathy or a carpal tunnel syndrome. A precise diagnosis is then necessary to adapt the treatment to individual cases. A classification describing the type and severity of the vascular lesions is presented. Asymptomatic lesions are included for adequate epidemiological studies and risk assessment of vibrating tools. Monitoring of vibration exposed workers should include not only a questionnaire about symptoms, but also a clinical evaluation including diagnostic tests for the screening of early asymptomatic neurovascular injuries.

Identification and Semiactive Control of Smart Structures Equipped with Magnetorheological Actuators

This paper deals with the problem of identification and semiactive control of smart structures subject to unknown external disturbances such as earthquake, wind, etc. The experimental setup used is a 6-story test structure equipped with shear-mode semiactive magnetorheological actuators being installed in WUSCEEL. The experimental results obtained have verified the effectiveness of the proposed control algorithms

Identification and characterization of a caspase-2 activating complex

Résumé Les caspases sont des protéases essentielles lors de l'induction de l'apoptose ou pour la maturation de certaines cytokines. Elles peuvent être divisées en deux groupes: les caspases initiatrices, qui sont les premières activées lors d'un signal pro-apoptotique, et les caspases effectrices, qui sont activées par les caspases initiatrices et sont responsables du clivage et de la dégradation des substrats cellulaires. Les caspases initiatrices sont activées dans des complexes de haut poids moléculaire: l'apoptosome pour la caspase-9 et le DISC pour la caspase-8. La caspase-2 est également une caspase initiatrice qui contient un domaine CARD. Cependant son mécanisme d'activation n'est pas encore connu. Lors de cette étude, nous avons découvert et caractérisé le complexe qui permet l'activation de la caspase-2. Ce complexe, appelé le PIDDosome, est composé de PIDD/LRDD, de la protéine adaptatrice RAIDD et de la protéase caspase-2. L'expression forcée de PIDD induit l'activation constitutive de la caspase-2. Cela entraîne la mort ou la sensibilisation à la mort des cellules selon la lignée étudiée. Cet effet est expliqué par une perte du potentiel de membrane de la mitochondrie, certainement dû à un effet direct de la caspase-2. Peu de choses sont connues sur PIDD: c'est une protéine contenant un domaine DD qui peut être induite par p53. Nous avons caractérisé PIDD et montré qu'elle est exprimée de façon ubiquitaire. PIDD est constitutivement auto-clivée environ au milieu de la protéine, ce qui génère deux fragments qui restent liés l'un à l'autre. Le fragment N-terminal a une activité régulatrice et le C-terminal une activité effectrice. De plus, PIDD peut se déplacer entre le cytoplasme et le noyau. Enfin, nous avons découvert que PIDD est également impliquée dans l'induction de NF¬ -κB en réponse à des dommages à l'ADN. PIDD est responsable de la modification par sumo de NEMO, étape nécessaire à l'induction de NF-κB après des dommages à l'ADN. Ainsi PIDD semble être à l'intersection de la décision que prend la cellule entre survivre et réparer les dommages, ou entrer en apoptose. Summary Caspases are a family of proteases that fulfill varied and often critical roles in mammalian apoptosis or proteolytic activation of cytokines. Caspases can be divided into two sub-groups: initiator caspases, which are the first activated after a pro-apoptotic signal, and effector caspases, which are activated by initiator caspases and that are responsible for the cleavage and degradation of cellular components. Initiator caspases are activated in high molecular weight platforms such as the apoptosome for caspase-9 or the DISC for caspase-8. Caspase-2 is a CARD-containing initiator caspase whose mechanism of activation was not yet known. In this study we have identified an activating platform for caspase-2. This high molecular weight complex, called the PIDDosome, is composed of PIDD/LRDD, the adaptor protein RAIDD and caspase-2. Constitutive expression of PIDD led to constitutive activation of caspase-2, which in some cell lines was sufficient to induce cell death while in others it merely sensitizes. Active caspase-2 was found to disturb directly the mitochondria by inducing a partial loss of the transmembrane potential. Very little was known on PIDD. It can be induce by p53 and inhibition of its expression by antisense oligonucleotides diminishes p53-dependent apoptosis. We decided to further characterize PIDD function and expression. PIDD possesses seven LRR, two Zu5 domains and one DD. It is ubiquitously expressed and appears to be constitutively cleaved by auto- processing into two main fragments equal in size. The two fragments remain bound to one another and constitute a regulatory N-terminal fragment and an active C-terminal fragment. In addition, PIDD can shuttle between the cytoplasm and the nucleus. Finally, investigating the possible relevance of new interaction partners, we found that PIDD is implicated in DNA damage-induced NF- κB. PIDD binds to RIP1 and to NEMO. In response to DNA damage, PIDD translocates to the nucleus and mediates sumo- modification of NEMO, a necessary step in DNA damage-induced NF-κB. All together these results raise the possibility that PIDD acts as a molecular switch between proliferation and repair, and apoptosis following DNA damage.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

Identification and in vitro reactivity of celiac immunoactive peptides in an apparent gluten-free beer.

Gluten content from barley, rye, wheat and in certain oat varieties, must be avoid in individuals with celiac disease. In most of the Western countries, the level of gluten content in food to be considered as gluten-free products is below 20 parts per million measured by ELISA based on specific anti-gluten peptide antibody. However, in beverages or food suffering complex hydrolytic processes as beers, the relative proportion of reactive peptides for celiac patients and the analytical techniques may differ, because of the diversity of the resulting peptide populations after fermentations. A beer below 20 parts per million of gluten but yet detectable levels of gluten peptides by anti-gliadin 33-mer antibodies (G12 and A1) was analyzed. We identified and characterized the relevant peptides for either antibody recognition or immunoactivity in celiac patients. The beer was fractionated by HPLC. The relative reactivity of the different HPLC fractions to the G12/A1 antibodies correlated to the reactivity of peripheral blood mononuclear cells isolated from 14 celiac individuals. Peptides from representative fractions classified according to the relative reactivity to G12/A1 antibodies were identified by mass spectrometry. The beer peptides containing sequences with similarity to those of previously described G12 and A1 epitopes were synthesized and confirmed significant reactivity for the antibodies. The most reactive peptides for G12/A1 also confirmed the highest immunogenicity by peripheral blood mononuclear cell activation and interferon γ production from celiac patients. We concluded that preparative HPLC combined with anti-gliadin 33-mer G12/A1 antibodies were very sensitive and specific methods to analyze the relevant immunogenic peptides in hydrolyzed gluten.

Revised nomenclature and classification of inherited ichthyoses: results of the First Ichthyosis Consensus Conference in Sorèze 2009.

BACKGROUND: Inherited ichthyoses belong to a large, clinically and etiologically heterogeneous group of mendelian disorders of cornification, typically involving the entire integument. Over the recent years, much progress has been made defining their molecular causes. However, there is no internationally accepted classification and terminology. OBJECTIVE: We sought to establish a consensus for the nomenclature and classification of inherited ichthyoses. METHODS: The classification project started at the First World Conference on Ichthyosis in 2007. A large international network of expert clinicians, skin pathologists, and geneticists entertained an interactive dialogue over 2 years, eventually leading to the First Ichthyosis Consensus Conference held in Sorèze, France, on January 23 and 24, 2009, where subcommittees on different issues proposed terminology that was debated until consensus was reached. RESULTS: It was agreed that currently the nosology should remain clinically based. "Syndromic" versus "nonsyndromic" forms provide a useful major subdivision. Several clinical terms and controversial disease names have been redefined: eg, the group caused by keratin mutations is referred to by the umbrella term, "keratinopathic ichthyosis"-under which are included epidermolytic ichthyosis, superficial epidermolytic ichthyosis, and ichthyosis Curth-Macklin. "Autosomal recessive congenital ichthyosis" is proposed as an umbrella term for the harlequin ichthyosis, lamellar ichthyosis, and the congenital ichthyosiform erythroderma group. LIMITATIONS: As more becomes known about these diseases in the future, modifications will be needed. CONCLUSION: We have achieved an international consensus for the classification of inherited ichthyosis that should be useful for all clinicians and can serve as reference point for future research.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Identification and antifungal susceptibility of a large collection of yeast strains isolated in Tunisian hospitals.

Abstract In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.

Identification and modelling of human breast cancer subtypes

Résumé Le cancer du sein est le cancer le plus commun chez les femmes et est responsable de presque 30% de tous les nouveaux cas de cancer en Europe. On estime le nombre de décès liés au cancer du sein en Europe est à plus de 130.000 par an. Ces chiffres expliquent l'impact social considérable de cette maladie. Les objectifs de cette thèse étaient: (1) d'identifier les prédispositions et les mécanismes biologiques responsables de l'établissement des sous-types spécifiques de cancer du sein; (2) les valider dans un modèle ín vivo "humain-dans-souris"; et (3) de développer des traitements spécifiques à chaque sous-type de cancer du sein identifiés. Le premier objectif a été atteint par l'intermédiaire de l'analyse des données d'expression de gènes des tumeurs, produite dans notre laboratoire. Les données obtenues par puces à ADN ont été produites à partir de 49 biopsies des tumeurs du sein provenant des patientes participant dans l'essai clinique EORTC 10994/BIG00-01. Les données étaient très riches en information et m'ont permis de valider des données précédentes des autres études d'expression des gènes dans des tumeurs du sein. De plus, cette analyse m'a permis d'identifier un nouveau sous-type biologique de cancer du sein. Dans la première partie de la thèse, je décris I identification des tumeurs apocrines du sein par l'analyse des puces à ADN et les implications potentielles de cette découverte pour les applications cliniques. Le deuxième objectif a été atteint par l'établissement d'un modèle de cancer du sein humain, basé sur des cellules épithéliales mammaires humaines primaires (HMECs) dérivées de réductions mammaires. J'ai choisi d'adapter un système de culture des cellules en suspension basé sur des mammosphères précédemment décrit et pat décidé d'exprimer des gènes en utilisant des lentivirus. Dans la deuxième partie de ma thèse je décris l'établissement d'un système de culture cellulaire qui permet la transformation quantitative des HMECs. Par la suite, j'ai établi un modèle de xénogreffe dans les souris immunodéficientes NOD/SCID, qui permet de modéliser la maladie humaine chez la souris. Dans la troisième partie de ma thèse je décris et je discute les résultats que j'ai obtenus en établissant un modèle estrogène-dépendant de cancer du sein par transformation quantitative des HMECs avec des gènes définis, identifiés par analyse de données d'expression des gènes dans le cancer du sein. Les cellules transformées dans notre modèle étaient estrogène-dépendantes pour la croissance, diploïdes et génétiquement normales même après la culture cellulaire in vitro prolongée. Les cellules formaient des tumeurs dans notre modèle de xénogreffe et constituaient des métastases péritonéales disséminées et du foie. Afin d'atteindre le troisième objectif de ma thèse, j'ai défini et examiné des stratégies de traitement qui permettent réduire les tumeurs et les métastases. J'ai produit un modèle de cancer du sein génétiquement défini et positif pour le récepteur de l'estrogène qui permet de modéliser le cancer du sein estrogène-dépendant humain chez la souris. Ce modèle permet l'étude des mécanismes impliqués dans la formation des tumeurs et des métastases. Abstract Breast cancer is the most common cancer in women and accounts for nearly 30% of all new cancer cases in Europe. The number of deaths from breast cancer in Europe is estimated to be over 130,000 each year, implying the social impact of the disease. The goals of this thesis were first, to identify biological features and mechanisms --responsible for the establishment of specific breast cancer subtypes, second to validate them in a human-in-mouse in vivo model and third to develop specific treatments for identified breast cancer subtypes. The first objective was achieved via the analysis of tumour gene expression data produced in our lab. The microarray data were generated from 49 breast tumour biopsies that were collected from patients enrolled in the clinical trial EORTC 10994/BIG00-01. The data set was very rich in information and allowed me to validate data of previous breast cancer gene expression studies and to identify biological features of a novel breast cancer subtype. In the first part of the thesis I focus on the identification of molecular apacrine breast tumours by microarray analysis and the potential imptìcation of this finding for the clinics. The second objective was attained by the production of a human breast cancer model system based on primary human mammary epithelial cells {HMECs) derived from reduction mammoplasties. I have chosen to adopt a previously described suspension culture system based on mammospheres and expressed selected target genes using lentiviral expression constructs. In the second part of my thesis I mainly focus on the establishment of a cell culture system allowing for quantitative transformation of HMECs. I then established a xenograft model in immunodeficient NOD/SCID mice, allowing to model human disease in a mouse. In the third part of my thesis I describe and discuss the results that I obtained while establishing an oestrogen-dependent model of breast cancer by quantitative transformation of HMECs with defined genes identified after breast cancer gene expression data analysis. The transformed cells in our model are oestrogen-dependent for growth; remain diploid and genetically normal even after prolonged cell culture in vitro. The cells farm tumours and form disseminated peritoneal and liver metastases in our xenograft model. Along the lines of the third objective of my thesis I defined and tested treatment schemes allowing reducing tumours and metastases. I have generated a genetically defined model of oestrogen receptor alpha positive human breast cancer that allows to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.

Multiuser detection in a dynamic environment — Part II: Joint user identification and parameter estimation

The problem of jointly estimating the number, the identities, and the data of active users in a time-varying multiuser environment was examined in a companion paper (IEEE Trans. Information Theory, vol. 53, no. 9, September 2007), at whose core was the use of the theory of finite random sets on countable spaces. Here we extend that theory to encompass the more general problem of estimating unknown continuous parameters of the active-user signals. This problem is solved here by applying the theory of random finite sets constructed on hybrid spaces. We doso deriving Bayesian recursions that describe the evolution withtime of a posteriori densities of the unknown parameters and data.Unlike in the above cited paper, wherein one could evaluate theexact multiuser set posterior density, here the continuous-parameter Bayesian recursions do not admit closed-form expressions. To circumvent this difficulty, we develop numerical approximationsfor the receivers that are based on Sequential Monte Carlo (SMC)methods (“particle filtering”). Simulation results, referring to acode-divisin multiple-access (CDMA) system, are presented toillustrate the theory.

Applause identification and its relevance to archival of Carnatic music

A Carnatic music concert is made up of a sequence of pieces, where each piece corresponds to a particular genre and ra¯aga (melody). Unlike a western music concert, the artist may be applauded intra-performance inter-performance. Most Carnatic music that is archived today correspond to a single audio recordings of entire concerts.The purpose of this paper is to segment single audio recordings into a sequence of pieces using thecharacteristic features of applause and music. Spectral flux, spectral entropy change quite significantly from music to applause and vice-versa. The characteristics of these features for a subset of concerts was studied. A threshold based approach was used to segment the pieces into music fragments and applauses. Preliminary resultson recordings 19 concerts from matched microphones show that the EER is about 17% for a resolution of 0.25 seconds. Further, a parameter called CUSUM is estimatedfor the applause regions. The CUSUM values determine the strength of the applause. The CUSUM is used to characterise the highlights of a concert.