Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.

Biological control of Biomphalaria tenagophila (Mollusca, Planorbidae), a schistosomiasis vector, using the fish Geophagus brasiliensis (Pisces, Cichlidae) in the laboratory or in a seminatural environment

In order to investigate a possible method of biological control of schistosomiasis, we used the fish Geophagus brasiliensis (Quoy & Gaimard, 1824) which is widely distributed throughout Brazil, to interrupt the life cycle of the snail Biomphalaria tenagophila (Orbigny, 1835), an intermediate host of Schistosoma mansoni. In the laboratory, predation eliminated 97.6% of the smaller snails (3-8 mm shell diameter) and 9.2% of the larger ones (12-14 mm shell diameter). Very promising results were also obtained in a seminatural environment. Studies of this fish in natural snail habitats should be further encouraged.

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease. METHODS: MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells. RESULTS: MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation. CONCLUSIONS/INTERPRETATION: We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.

Effects of stress on economic decision-making: Evidence from laboratory experiments

The ways in which preferences respond to the varying stress of economic environments is a key question for behavioral economics and public policy. We conducted a laboratory experiment to investigate the effects of stress on financial decision making among individuals aged 50 and older. Using the cold pressor task as a physiological stressor, and a series of intelligence tests as cognitive stressors, we find that stress increases subjective discounting rates, has no effect on the degree of risk-aversion, and substantially lowers the effort individuals make to learn about financial decisions.

Preliminary laboratory and field trials of a heated pheromone trap for the sandfly lutzomyia logipalpis (Diptera: Psychodidae)

A heated pheromone trap for the sandfly Lutzomyia longipalpis was tested in the laboratory using filter paper or plastic vial dispensers. Male pheromone extracted from 8 male tergal glands and absorbed on to filter paper dispensers attracted 82/120 (68.3%) of virgin females released in cages. Similarly plastic vial dispensers baited with the extract of 24 males caught 73/120 (61%). In field trials carried out near januária, Minas Gerais, Brazil using the plastic dispensers baited with extracts of 50 male tergal glands only 70 female L. longipalpis were captured. Over 1000 male flies were, however, caught during 6 nights, with greater numbers in the unbaited control traps than in the pheromone baited test traps. It is concluded that at excessive concentrations male L. longipalpis pheromone may act as a repellent to conspecific males.

Susceptibility of laboratory-reared female Lutzomyia longipalpis (Lutz & Neiva, 1912) to infection by different species and strains of Leishmania Ross, 1903

A study was undertaken to compare the susceptibility of laboratory-reared female Lutzomyia longipalpis to infection by different species or strains of New World Leishmania. The sand flies proved to be highly susceptible to infection by a strain of Le. guyanensis, with flagellates developing in all (18/18) of the specimens examined. A lower infection rate of 37 per cents (10/27) was recorded in flies exposed to infection by a strain of Le. amazonensis. Flagellates developed in 13 per cents (6/46) of the sand flies that glood fed on dogs in the earlly stage of experimental infection with an old laboratory strain of Le. chagasi. In contrast, promastigotes did not develop in sand flies that blood fed on dogs with naturally acquired Le. chagasi. The naturally infected dogas were in an advanced stage of disease. Flagellates developed in 9// (3/32) of the sand flies that blood fed on lesions of hamsters infected with a strain of Le. braziliensis and in 9 per cents (3/34) of those that fed on hamsters with lesions due to a parasite fo the mexicana complex (strain MHOM/BR/73/BH121). Sand flies did not develop flagellate infections after blood feeding on hamsters bearing lesions induced by strain MHOM/BR/71/BR49. Factors influencing the susceptibility of Lu. longipalpis to infection by New World species of Leishmania are discussed.

Identification of case complexity and increased health care utilization in patients with rheumatoid arthritis.

OBJECTIVES: To document biopsychosocial profiles of patients with rheumatoid arthritis (RA) by means of the INTERMED and to correlate the results with conventional methods of disease assessment and health care utilization. METHODS: Patients with RA (n = 75) were evaluated with the INTERMED, an instrument for assessing case complexity and care needs. Based on their INTERMED scores, patients were compared with regard to severity of illness, functional status, and health care utilization. RESULTS: In cluster analysis, a 2-cluster solution emerged, with about half of the patients characterized as complex. Complex patients scoring especially high in the psychosocial domain of the INTERMED were disabled significantly more often and took more psychotropic drugs. Although the 2 patient groups did not differ in severity of illness and functional status, complex patients rated their illness as more severe on subjective measures and on most items of the Medical Outcomes Study Short Form 36. Complex patients showed increased health care utilization despite a similar biologic profile. CONCLUSIONS: The INTERMED identified complex patients with increased health care utilization, provided meaningful and comprehensive patient information, and proved to be easy to implement and advantageous compared with conventional methods of disease assessment. Intervention studies will have to demonstrate whether management strategies based on INTERMED profiles can improve treatment response and outcome of complex patients.

Identification and characterization of novel molecular mechanisms involved in initiation and progression of myelination

Schwann cells synthesize a large amount of membrane that form a specialized structure called myelin that surrounds axons and facilitate the transmission of electrical signal along neurons in peripheral nervous system (PNS). Previous studies demonstrated that both Schwann cell differentiation and de-differentiation (in the situation of a nerve injury or demyelinating disease) are regulated by cell-intrinsic regulators including several transcription factors. In particular, the de-differentiation of mature Schwann cells is driven by the activation of multiple negative regulators of myelination including Sox2, c-Jun, Notch and Pax3, all usually expressed in immature Schwann cells and suppressed at the onset of myelination. In order to identify new regulators of myelination involved in the development of the PNS, we analyzed the gene-expression profiling data from developing PNS and from three models of demyelinating neuropathies. This analysis led to the identification of Sox4, a member of the Sox family of transcription factors, as a potential candidate. To characterize the molecular function of Sox4 in PNS, we generated two transgenic lines of mice, which overexpress Sox4 specifically in Schwann cells. Detailed analysis of these mice showed that the overexpression of Sox4 in Schwann cells causes a delay in progression of myelination between post-natal day 2 (P2) and P5. Our in vitro analysis suggested that Sox4 cDNA can be overexpressed while the protein translation is tightly regulated. Interestingly, we observed that Sox4 protein is stabilized in nerves of the CMT4C mouse, a model of the human neuropathy. We therefore crossed Sox4 transgenic mice with CMT4C mice and we observed that Sox4 overexpression exacerbated the neuropathy phenotype in these mice. While recognized as being crucial for the normal function of both neurons and myelinating glial cells, the processes that regulate the beginning of myelination and the nature of the neuro-glial cross-talk remains mostly unknown. In order to gain insight into the molecular pathways involved in the interactions between neurons and associated glial cells, we developed a neuron-glia co-culture system based on microfluidic chambers and successfully induced myelination in this system by ascorbic acid. Importantly, we observed that in addition to acting on Schwann cells, ascorbic acid also modulate neuronal/axonal NRG1/ErbB2-B3 signalling. The experimental setting used in our study thus allowed us to discover a novel phenomena of propagation for myelination in vitro. The further characterization of this event brought us to identify other compounds able to induce myelination: ADAMs secretases inhibitor GM6001 and cyclic-AMP. The results generated during my thesis project are therefore not only important for the advancement of our understanding of how the PNS works, but may also potentially help to develop new therapies aiming at improvement of PNS myelination under disease conditions. - Les cellules de Schwann synthétisent une grande quantité de membrane formant une structure spécialisée appelée myéline qui entoure les axones et facilite la transmission du signal électrique le long des neurones du système nerveux périphérique (SNP). Des études antérieures ont démontré que la différenciation et la dédifférenciation des cellules de Schwann (dans la situation d'une lésion nerveuse ou d'une maladie démyélinisante) sont régulées par des régulateurs cellulaires intrinsèques, incluant plusieurs facteurs de transcription. En particulier, la dédifférenciation des cellules de Schwann matures est contrôlée par l'activation de plusieurs régulateurs négatifs de la myélinisation dont Sox2, c-Jun, Notch et Pax3, tous habituellement exprimés dans des cellules de Schwann immatures et supprimés au début de la myélinisation. Afin d'identifier de nouveaux régulateurs de myélinisation impliqués dans le développement du SNP, nous avons analysé le profil d'expression génique durant le développement du SNP ainsi que dans trois modèles de neuropathies démyélinisantes. Cette analyse a mené à l'identification de Sox4, un membre de la famille des facteurs de transcription Sox, comme étant un candidat potentiel. Dans le but de caractériser la fonction moléculaire de Sox4 dans le SNP, nous avons généré deux lignées transgéniques de souris qui surexpriment Sox4 spécifiquement dans les cellules de Schwann. L'analyse détaillée de ces souris a montré que la surexpression de Sox4 dans les cellules de Schwann provoque un retard dans la progression de la myélinisation entre le jour postnatal 2 (P2) et P5. Notre analyse in vitro a suggéré que l'ADNc de Sox4 peut être surexprimé alors que la traduction des protéines est quand à elle étroitement régulée. De façon intéressante, nous avons observé que la protéine Sox4 est stabilisée dans les nerfs des souris CMT4C, un modèle de neuropathie humaine. Nous avons donc croisé les souris transgéniques Sox4 avec des souris CMT4C et avons observé que la surexpression de Sox4 exacerbe le phénotype de neuropathie chez ces souris. Bien que reconnus comme étant cruciaux pour le fonctionnement normal des neurones et des cellules gliales myélinisantes, les processus qui régulent le début de la myélinisation ainsi que la nature des interactions neurone-glie restent largement méconnus. Afin de mieux comprendre les mécanismes moléculaires impliqués dans les interactions entre les neurones et les cellules gliales leur étant associés, nous avons développé un système de co-culture neurone-glie basé sur des chambres microfluidiques et y avons induit avec succès la myélinisation avec de l'acide ascorbique. Étonnamment, nous avons remarqué que, en plus d'agir sur les cellules de Schwann, l'acide ascorbique module également la voie de signalisation neuronale/axonale NRG1/ErbB2-B3. Le protocole expérimental utilisé dans notre étude a ainsi permis de découvrir un nouveau phénomène de propagation de la myélinisation in vitro. La caractérisation plus poussée de ce phénomène nous a menés à identifier d'autres composés capables d'induire la myélinisation: L'inhibiteur de sécrétases ADAMs GM6001 et l'AMP cyclique. Les résultats obtenus au cours de mon projet de thèse ne sont donc pas seulement importants pour l'avancement de notre compréhension sur la façon dont le SNP fonctionne, mais peuvent aussi potentiellement aider à développer de nouvelles thérapies visant à l'amélioration de la myélinisation du SNP dans des conditions pathologiques.

Identification and characterization of sex-linked proteins of Schistosoma mansoni

The proteins of adults worms (male and female) of two isolates (BH and RJ) of Shistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of mael and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 KDa, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females protins were detected by Western vlotting using a sera from infected Nectomys squamipes.

Distinctions between promastigotes of Leishmania species developing in the digestive tract of laboratory reared Lutzomyia longipalpis

Female Lutzomya longipalpis were exposed to infection by three different species/strains of Leishmania. When the insects were dissected four days after exposure, stained preparations were made of the flagellates contained in the digestive tract. Using traditional morphometric methods, L. amazonensis, L. guyanensis and an unnamed species of the mexicana complex could be distinguished from one another.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Summary : Internal ribosome entry sites (IRES) are used by viruses as a strategy to bypass inhibition of cap-dependent translation that commonly results from viral infection. IRES are also used in eukaryotic cells to control mRNA translation under conditions of cellular stress (apoptosis, heat shock) or during the G2 phase of the cell cycle when general protein synthesis is inhibited. Variation in cellular expression levels has been shown to be inherited. Expression is controlled, among others, by transcriptional factors and by the efficiency of cap-mediated translation and ribosome activity. We aimed at identifying genomic determinants of variability in IRES-mediated translation of two representative IRES [Encephalomyocarditis virus (EMCV) and X-linked Inhibitor-of-Apoptosis (XIAP) IRES]. We used bicistronic lentiviral constructions expressing two fluorescent reporter transgenes. Lentiviruses were used to transduce seven different laboratory cell lines and B lymphoblastoid cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209); representing an in vitro approach to family structure allowing genome scan analyses. The relative expression of the two markers was assessed by FACS. IRES efficiency varies according to cellular background, but also varies, for a same cell type, among individuals. The control of IRES activity presents an inherited component (h2) of 0.47 and 0.36 for EMCV and XIAP IRES, respectively. A genome scan identified a suggestive Quantitative Trait Loci (LOD 2.35) involved in the control of XIAP IRES activity. Résumé : Les sites internes d'entrée des ribosomes (IRES = internal ribosome entry sites) sont utilisés par les virus comme une stratégie afin d'outrepasser l'inhibition de traduction qui résulte communément d'une infection virale. Les IRES sont également utilisés par les cellules eucaryotes pour contrôler la traduction de l'ARN messager dans des conditions de stress cellulaire (apoptose, choc thermique) ou durant la phase G2 du cycle cellulaire, situations durant lesquelles la synthèse générale des protéines est inhibée. La variation des niveaux d'expression cellulaire de transcription est un caractère héréditaire. L'expression des gènes est contrôlée entre autre par les facteurs de transcription et par l'efficacité de la traduction initiée par la coiffe ainsi que par l'activité des ribosomes. Durant cette étude nous avons eu pour but d'identifier les déterminants génomiques responsables de la variabilité de la traduction contrôlée par l'IRES. Ceci a été effectué en étudiant deux IRES représentatifs : l'IRES du virus de l'encéphalomyocardite (EMCV) et l'IRES de l'inhibiteur de l'apoptose XIAP (X-linked Inhibitor-of-Apoptosis). Nous avons utilisés des lentivirus délivrant un transgène bicistronique codant pour deux gènes rapporteurs fluorescents. Ces lentivirus ont été utilisés pour transduire sept différentes lignées cellulaires de laboratoire et des lignées cellulaires lymphoblastoïdes B du Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209) qui représentent une approche in vitro de la structure familiale et qui permettent des analyses par balayage du génome. L'expression relative des deux marqueurs fluorescents a été analysée par FACS. Nos résultats montrent que l'efficacité des IRES varie en fonction du type de cellules. Il varie aussi, pour le même type de cellules, selon les individus. Le contrôle de l'activité de l'IRES est un caractère héritable (héritabilité h2) de 0.47 et 0.36 pour les IRES de EMCV et XIAP respectivement. Le balayage du génome a permis l'identification d'un locus à effets quantitatifs [QTL Quantitative Trait Loci (LOD 2.35)] impliqué dans le contôle de l'activité de l'IRES de XIAP.

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.

Comparison of the reproductive behavior between isolated Peckia chrysostoma (Wiedemann, 1830) and Adiscochaeta ingens (Walker, 1849) (Diptera: Sarcophagidae) females reared in laboratory

In both species, maintained under laboratory environmental conditions, anautogeny was comproved and all females that had free access to proteic source were fertiles. We obtained the following average values for Peckiachrysostoma: 59.7 ± 15.6 and 81.8 ± 15.4 days of longevity in the respective cases of free access and no access to proteic source, 21.4 ± 4.3 days of pre-larviposition period and 35.2 ± 16.5 days of larviposition period, 5.3 ± 1.8 larvipositions female with 7.0 ± 1.1 days of periodicity, 35.7 ± 6.1 larvae per larviposition leading to a total number of 183.8 ± 69.2 viable larvae per female and 94.8% ± 5.3% of productivity. The mean number of ovarioles per female was 56.4 ± 9.8, resulting in a reproductive potential of 63.3%. For Adiscochaeta ingens, the obtained average values were: 41.3 ± 6.3 and 52 ± 13.1 days of longevity in the respective cases of free access and no access to proteic source, 15.3 ± 1.7 days of pre-larviposition period and 21.5 ± 7.5 days of larviposition period, 3 ± 0.7 larvipositions per female with 10.4 ± 0.8 days of periodicity, 30.3 ± 8.2 larvae per larviposition leading to a total number of 78.5 ± 21.7 viable larvae per female and 90.1% ± 16% of productivity. The mean number of ovarioles per female was 54.6 ± 5.2, resulting in a reproductive potential of 55.5%. Within applied parameters, the values obtained for P. chrysostoma demonstrate its superior productivity in comparison with A. ingens

Radio frequency identification (RFID) of dentures in long-term care facilities.

STATEMENT OF PROBLEM: The difficulty of identifying the ownership of lost dentures when found is a common and expensive problem in long term care facilities (LTCFs) and hospitals. PURPOSE: The purpose of this study was to evaluate the reliability of using radiofrequency identification (RFID) in the identification of dentures for LTCF residents after 3 and 6 months. MATERIAL AND METHODS: Thirty-eight residents of 2 LTCFs in Switzerland agreed to participate after providing informed consent. The tag was programmed with the family and first names of the participants and then inserted in the dentures. After placement of the tag, the information was read. A second and third assessment to review the functioning of the tag occurred at 3 and 6 months, and defective tags (if present) were reported and replaced. The data were analyzed with descriptive statistics. RESULTS: At the 3-month assessment of 34 residents (63 tags) 1 tag was unreadable and 62 tags (98.2%) were operational. At 6 months, the tags of 27 of the enrolled residents (50 tags) were available for review. No examined tag was defective at this time period. CONCLUSIONS: Within the limits of this study (number of patients, 6-month time span) RFID appears to be a reliable method of tracking and identifying dentures, with only 1 of 65 devices being unreadable at 3 months and 100% of 50 initially placed tags being readable at the end of the trial.