Frequent users of emergency departments present very high prevalence of mental health and substance use disorders, which are largely underdiagnosed by clinicians

Background: The objective of this study was to determine if mental health and substance use diagnoses were equally detected in frequent users (FUs) compared to infrequent users (IUs) of emergency departments (EDs). Methods: In a sample of 399 adult patients (>= 18 years old) admitted to a teaching hospital ED, we compared the mental health and substance use disorders diagnoses established clinically and consigned in the medical files by the ED physicians to data obtained in face-to-face research interviews using the Primary Care Evaluation of Mental Disorders (PRIME-MD) and the Alcohol, Smoking and Involvement Screening Test (ASSIST). Between November 2009 and June 2010, 226 FUs (>4 visits within a year) who attended the ED were included, and 173 IUs (<= 4 visits within a year) were randomly selected from a pool of identified patients to comprise the comparison group. Results: For mental health disorders identified by the PRIME-MD, FUs were more likely than IUs to have an anxiety (34 vs. 16%, Chi2(1) = 16.74, p <0.001), depressive (47 vs. 25%, Chi2(1) = 19.11, p <0.001) or posttraumatic stress (PTSD) disorder (11 vs. 5%, Chi2(1) = 4.87, p = 0.027). Only 3/76 FUs (4%) with an anxiety disorder, 16/104 FUs (15%) with a depressive disorder and none of the 24 FUs with PTSD were detected by the ED medical staff. None of the 27 IUs with an anxiety disorder, 6/43 IUs (14%) with a depressive disorder and none of the 8 IUs with PTSD were detected. For substance use disorders identified by the ASSIST, FUs were more at risk than IUs for alcohol (24 vs. 7%, Chi2(1) = 21.12, p <0.001) and drug abuse/dependence (36 vs. 25%, Chi2(1) = 5.52, p = 0.019). Of the FUs, 14/54 (26%) using alcohol and 8/81 (10%) using drugs were detected by the ED physicians. Of the IUs, 5/12 (41%) using alcohol and none of the 43 using drugs were detected. Overall, there was no significant difference in the rate of detection of mental health and substance use disorders between FUs and IUs (Fisher's Exact Test: anxiety, p = 0.567; depression, p = 1.000; PTSD, p = 1.000; alcohol, p = 0.517; and drugs, p = 0.053). Conclusions: While the prevalence of mental health and substance use disorders was higher among FUs, the rates of detection were not significantly different for FUs vs. IUs. However, it may be that drug disorders among FUs were more likely to be detected.

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Wnt secretion and gradient formation.

Concentration gradients formed by the lipid-modified morphogens of the Wnt family are known for their pivotal roles during embryogenesis and adult tissue homeostasis. Wnt morphogens are also implicated in a variety of human diseases, especially cancer. Therefore, the signaling cascades triggered by Wnts have received considerable attention during recent decades. However, how Wnts are secreted and how concentration gradients are formed remains poorly understood. The use of model organisms such as Drosophila melanogaster has provided important advances in this area. For instance, we have previously shown that the lipid raft-associated reggie/flotillin proteins influence Wnt secretion and spreading in Drosophila. Our work supports the notion that producing cells secrete Wnt molecules in at least two pools: a poorly diffusible one and a reggie/flotillin-dependent highly diffusible pool which allows morphogen spreading over long distances away from its source of production. Here we revise the current views of Wnt secretion and spreading, and propose two models for the role of the reggie/flotillin proteins in these processes: (i) reggies/flotillins regulate the basolateral endocytosis of the poorly diffusible, membrane-bound Wnt pool, which is then sorted and secreted to apical compartments for long-range diffusion, and (ii) lipid rafts organized by reggies/flotillins serve as "dating points" where extracellular Wnt transiently interacts with lipoprotein receptors to allow its capture and further spreading via lipoprotein particles. We further discuss these processes in the context of human breast cancer. A better understanding of these phenomena may be relevant for identification of novel drug targets and therapeutic strategies.

Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells.

Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.

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.

Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux.

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.

Lipids shed into the culture medium by trypomastigotes of Trypanosoma cruzi

Trypomastigote forms of Trypanosoma cruzi were metabolically labeled with [14C]-ethanolamine and [3H]-palmitic acid. Lipids shed to the culture medium were analyzed and compared with the parasite components. Phosphatidylcholine and lysophosphatidylcholine accounted for 53% of the total incorporated precursor. Interestingly, phosphatidylethanolamine and its lyso derivative lysophosphatidylethanolamine, although present in significant amounts in the parasites, could not be detected in the shed material. Shed lipids were highly enriched in the desaturated fatty acids C16:1 and C18:1 when compared to the total fatty acid pool isolated from the parasites.

Microhabitat preferences of Biomphalaria pfeifferi and Lymnaea natalensis in a natural and a man-made habitat in Southeastern Tanzania

Schistosoma mansoni is an important human parasitic disease which is widespread throughout Africa. As Biomphalaria pfeifferi snails act as intermediate host, knowledge of their population ecology is an essential prerequisite towards understanding disease transmission. We conducted a field study and assessed the density and microhabitat preferences of B.pfeifferi in a natural habitat which was a residual pool of a river. Repeated removal collecting revealed a density of 26.6 [95% confidence interval (CI): 24.9-28.3] snails/m2. B.pfeifferi showed microhabitat preferences for shallow water (depths: 0-4cm). They were found most abundantly close to the shoreline (distances: 0-40cm), and preferred either plant detritus or bedrock as substratum. Lymnaea natalensis, a snail which may act as a host for human Fasciola gigantica, also occurred in this habitat with a density of 34.0 (95% CI: 24.7-43.3) snails/m2, and preferred significantly different microhabitats when compared to B.pfeifferi. Microhabitat selection by these snail species was also investigated in a man-made habitat nearby, which consisted of a flat layer of concrete fixed on the riverbed, covered by algae. Here, B.pfeifferi showed no preference for locations close to the shoreline, probably because the habitat had a uniform depth. We conclude that repeated removal collecting in shallow habitats provides reliable estimates of snail densities and that habitat changes through constructions may create favourable microhabitats and contribute to additional disease transmission.

Cardiac dysfunction and impaired compensatory response to pressure overload in mice deficient in stem cell antigen-1.

Stem cell antigen-1 (Sca-1) has been used to identify cardiac stem cells in the mouse heart. To investigate the function of Sca-1 in aging and during the cardiac adaptation to stress, we used Sca-1-deficient mice. These mice developed dilated cardiomyopathy [end-diastolic left ventricular diameter at 18 wk of age: wild-type (WT) mice, 4.2 mm ± 0.3; Sca-1-knockout (Sca-1-KO) mice, 4.6 mm ± 0.1; ejection fraction: WT mice, 51.1 ± 2.7%; Sca-1-KO mice, 42.9 ± 2.7%]. Furthermore, the hearts of mice lacking Sca-1 demonstrated exacerbated susceptibility to pressure overload [ejection fraction after transaortic constriction (TAC): WT mice, 43.5 ± 3.2%; Sca-1-KO mice, 30.8% ± 4.0] and increased apoptosis, as shown by the 2.5-fold increase in TUNEL(+) cells in Sca-1-deficient hearts under stress. Sca-1 deficiency affected primarily the nonmyocyte cell fraction. Indeed, the number of Nkx2.5(+) nonmyocyte cells, which represent a population of cardiac precursor cells (CPCs), was 2-fold smaller in Sca-1 deficient neonatal hearts. In vitro, the ability of CPCs to differentiate into cardiomyocytes was not affected by Sca-1 deletion. In contrast, these cells demonstrated unrestricted differentiation into cardiomyocytes. Interestingly, proliferation of cardiac nonmyocyte cells in response to stress, as judged by BrdU incorporation, was higher in mice lacking Sca-1 (percentages of BrdU(+) cells in the heart after TAC: WT mice, 4.4 ± 2.1%; Sca-1-KO mice, 19.3 ± 4.2%). These data demonstrate the crucial role of Sca-1 in the maintenance of cardiac integrity and suggest that Sca-1 restrains spontaneous differentiation in the precursor population. The absence of Sca-1 results in uncontrolled precursor recruitment, exhaustion of the precursor pool, and cardiac dysfunction.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Estràtegies de gestió per al manteniment de la biodiversitat a la sabana brasilera: control d'especies exòtiques amb foc

Report for the scientific sojourn at the Instituto de Biociências, of the Universidade de Sao Paulo, Brazil, from 2007 to 2009. African exotic grasses, used as forage crops for cattle, found optimal conditions in Brazilian savannas for their spread. They established as invasive grasses in almost all conservation units, becoming a serius threat to native biote and to most ecological processes. Fire is a cyclical event in Brazilian savannas and can be used as a management tool for enhancing competitivity of native grasses and controlling exotic grasses. Seeking for alternatives for the management of these grasses, this study investigates the effect of recurrent burnings in different periods of the year (fire regimes) on: distribution patterns of exotic grasses and their associations wotih native grasses, the local edaphic characteristics, the nutrient pool of the aerial biomass of both native and exotic grasses, and soil seed banks. This project is held on the IBGE Ecological Reserve, in Brasília (Brazil), in an area destined specifically for research of fire effects on brazilian savannas. The project quantifies: aerial biomass of native and exotic grasses, soil seed bank of exotic grasses, nutrient pool in soil and also in grasses tissues. Spatial relationships for any association between species, as well as for nutrient pools in soil and in plant tissues will be established. A better understanding of these processes will provide useful tools for adopting specific policies on the management of exotic grasses in Brazilian savanna.

Registre isotòpic d'alquenones i lípids terrestres en sediment marí: una reconstrucció paleoclimàtica a l'oceà Índic

Projecte de recerca elaborat a partir d’una estada a la Institute of mineralogy and geochemistry de la University of Lausanne, Suïssa, entre 2007 i 2009. Durant l’última dècada, la comunitat científica ha reconegut que les zones tropicals juguen un paper clau en els processos dinàmics que controlen el canvi climàtic global, probablement com a desencadenant dels canvis succeïts en altes latituds. A més a més, els sediments dels oceans tropicals, en trobar-se fora de l’impacte directe de les plaques de gel continentals creades durant les glaciacions, proporcionen un registre continu de les variacions climàtiques del planeta. Malgrat tot, encara hi ha moltes incògnites sobre el paper específic de les zones tropicals, especialment pel que fa a les variacions brusques suborbitals, degut als pocs registres d’alta resolució estudiats en aquestes àrees que abastin varis cicles glacial/interglacial. Per tal d’ajudar a clarificar el paper de les zones tropicals de l’hemisferi sud en el control del clima a escala mil•lenària s’ha estudiat la distribució i la composició isotòpica de biomarcadors moleculars marins i terrestres, a baixa resolució, en el testimoni MD98-2165 (9º39’S, 118º20’E, 2100 m de profunditat d’aigua, 42.3 m de llarg) està situat al sud-oest d’Indonèsia, on s’enregistren les temperatures superficials del mar més elevades del planeta i una elevada activitat convectiva, que té una influència en la distribució de la humitat atmosfèrica en una extensa superfície de la Terra. Les distribucions observades de biomarcadors terrígens (C23-C33 n-alcans i C20-C32 n-alcan-1-ols) són típiques del lipids de plantes superiors que arriben a l’oceà principalment per via eòlica. L’alcà de 31 àtoms de carboni i els alcohols de 28 o 32 àtoms de carboni són els homòlegs més abundants en ambdós testimonis. Cal destacar l’alcohol C32 com a homòleg principal durant les èpoques glacials, tot suggerint una expansió de les plantes tropicals C4 associada a unes condicions més àrides. La procedència d’aquests lipids queda corroborada mitjançant la seva composició isotòpica de carboni, que ens permet diferenciar la ruta fotosintètica emprada i per tant, entre el tipus de plantes.

Diethyldithiocarbamic acid inhibits the octadecanoid signaling pathway for the wound induction of proteinase-inhibitors in tomato leaves

The induction of proteinase inhibitor I synthesis in tomato (Lycopersicon esculentum) leaves in response to wounding is strongly inhibited by diethyldithiocarbamic acid (DIECA). DIECA also inhibits the induction of inhibitor I synthesis by the 18-amino acid polypeptide systemin, polygalacturonic acid (PCA), and linolenic acid, but not by jasmonic acid, suggesting that DIECA interferes with the octadecanoid signaling pathway. DIECA only weakly inhibited tomato lipoxygenase activity, indicating that DIECA action occurred at a step after the conversion of linolenic acid to 13(S)-hydroperoxylinolenic acid (HPOTrE). DIECA was shown to efficiently reduce HPOTrE to 13-hydroxylinolenic acid (HOTrE), which is not a signaling intermediate. Therefore, in vivo, DIECA is likely inhibiting the signaling pathway by shunting HPOTrE to HOTrE, thereby severely reducing the precursor pool leading to cyclization and eventual synthesis of jasmonic acid. Phenidone, an inhibitor of lipoxygenase, inhibited proteinase inhibitor I accumulation in response to wounding, further supporting a role for its substrate, linolenic acid, and its product, HPOTrE, as components of the signal-transduction pathway that induces proteinase inhibitor synthesis in response to wounding, systemin, and PCA.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

An efficient use of virtualization in grid/cloud environments

Grid is a hardware and software infrastructure that provides dependable, consistent, pervasive, and inexpensive access to high-end computational resources. Grid enables access to the resources but it does not guarantee any quality of service. Moreover, Grid does not provide performance isolation; job of one user can influence the performance of other user’s job. The other problem with Grid is that the users of Grid belong to scientific community and the jobs require specific and customized software environment. Providing the perfect environment to the user is very difficult in Grid for its dispersed and heterogeneous nature. Though, Cloud computing provide full customization and control, but there is no simple procedure available to submit user jobs as in Grid. The Grid computing can provide customized resources and performance to the user using virtualization. A virtual machine can join the Grid as an execution node. The virtual machine can also be submitted as a job with user jobs inside. Where the first method gives quality of service and performance isolation, the second method also provides customization and administration in addition. In this thesis, a solution is proposed to enable virtual machine reuse which will provide performance isolation with customization and administration. The same virtual machine can be used for several jobs. In the proposed solution customized virtual machines join the Grid pool on user request. Proposed solution describes two scenarios to achieve this goal. In first scenario, user submits their customized virtual machine as a job. The virtual machine joins the Grid pool when it is powered on. In the second scenario, user customized virtual machines are preconfigured in the execution system. These virtual machines join the Grid pool on user request. Condor and VMware server is used to deploy and test the scenarios. Condor supports virtual machine jobs. The scenario 1 is deployed using Condor VM universe. The second scenario uses VMware-VIX API for scripting powering on and powering off of the remote virtual machines. The experimental results shows that as scenario 2 does not need to transfer the virtual machine image, the virtual machine image becomes live on pool more faster. In scenario 1, the virtual machine runs as a condor job, so it easy to administrate the virtual machine. The only pitfall in scenario 1 is the network traffic.