Mycobacterium tuberculosis Infection in Young Children: Analyzing the Performance of the Diagnostic Tests

This study evaluated the performance of the Tuberculin Skin Test (TST) and Quantiferon-TB Gold in-Tube (QFT) and the possible association of factors which may modify their results in young children (0-6 years) with recent contact with an index tuberculosis case. Materials and Methods: A cross-sectional study including 135 children was conducted in Manaus, Amazonas-Brazil. The TST and QFT were performed and the tests results were analyzed in relation to the personal characteristics of the children studied and their relationship with the index case. Results: The rates of positivity were 34.8% (TST) and 26.7% (QFT), with 14.1% of indeterminations by the QFT. Concordance between tests was fair (Kappa = 0.35 P<0.001). Both the TST and QFT were associated with the intensity of exposure (Linear OR = 1.286, P = 0.005; Linear OR = 1.161, P = 0.035 respectively) with only the TST being associated with the time of exposure (Linear OR = 1.149, P = 0.009). The presence of intestinal helminths in the TST+ group was associated with negative QFT results (OR = 0.064, P = 0.049). In the TST- group lower levels of ferritin were associated with QFT+ results (Linear OR = 0.956, P = 0.036). Conclusions: Concordance between the TST and QFT was lower than expected. The factors associated with the discordant results were intestinal helminths, ferritin levels and exposure time to the index tuberculosis case. In TST+ group, helminths were associated with negative QFT results suggesting impaired cell-mediated immunity. The TST-&QFT+ group had a shorter exposure time and lower ferritin levels, suggesting that QFT is faster and ferritin may be a potential biomarker of early stages of tuberculosis infection.

Jurisdictional immunity in tax haven countries

El següent projecte conté informació sobre què són els paradisos fiscals, els seus avantatges, i on s'ubiquen. També s'analitza el procés que algú ha de seguir per anar a un paradís fiscal i avalua com la gent rica i les grans empreses operen els seus negocis a través dels paradisos fiscals i prenen avantatge d'ells reduint les seves obligacions fiscals de manera significativa. El projecte també considera la qüestió del secret bancari, que ha estat un gran conflicte entre els països en els darrers anys.

Whole genome analysis of p38 SAPK-mediated gene expression upon stress

Background: Cells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli.Results: Here, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFα and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38α SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38α the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38α deficient (p38α-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress. Conclusions: Our genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.

Motivational effects of cannabinoids are mediated by μ-opioid and k-opioid receptor

Repeated THC administration produces motivational and somaticadaptive changes leading to dependence in rodents. Toinvestigate the molecular basis for cannabinoid dependenceand its possible relationship with the endogenous opioid system,we explored Δ9-tetrahydrocannabinol (THC) activity in mice lacking μ-, δ- or κ-opioid receptor genes. Acute THCinduced hypothermia, antinociception, and ypolocomotion remained unaffected in these mice, whereas THC tolerance and withdrawal were minimally modified in mutant animals. In contrast, profound phenotypic changes are observed in several place conditioning protocols that reveal both THC rewarding and aversive properties. Absence of μ receptors abolishes THC place preference. Deletion of κ receptors ablates THC place aversion and furthermore unmasks THC place preference. Thus, an opposing activity of μ- and κ-opioid receptors in modulating reward pathways forms the basis for the dual euphoric–dysphoric activity of THC.

A targeted association study of immunity genes and networks suggests novel associations with placental malaria infection

A large proportion of the death toll associated with malaria is a consequence of malaria infection during pregnancy, causing up to 200,000 infant deaths annually. We previously published the first extensive genetic association study of placental malaria infection, and here we extend this analysis considerably, investigating genetic variation in over 9,000 SNPs in more than 1,000 genes involved in immunity and inflammation for their involvement in susceptibility to placental malaria infection. We applied a new approach incorporating results from both single gene analysis as well as gene-gene interactionson a protein-protein interaction network. We found suggestive associations of variants in the gene KLRK1 in the single geneanalysis, as well as evidence for associations of multiple members of the IL-7/IL-7R signalling cascade in the combined analysis. To our knowledge, this is the first large-scale genetic study on placental malaria infection to date, opening the door for follow-up studies trying to elucidate the genetic basis of this neglected form of malaria.

Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Driving rate effects in avalanche-mediated first-order phase transitions

We study the driving-rate and temperature dependence of the power-law exponents that characterize the avalanche distribution in first-order phase transitions. Measurements of acoustic emission in structural transitions in Cu-Zn-Al and Cu-Al-Ni are presented. We show how the observed behavior emerges within a general framework of competing time scales of avalanche relaxation, driving rate, and thermal fluctuations. We confirm our findings by numerical simulations of a prototype model.

Spatial patterns mediated by subcritical and supercritical thermal fluctuations in the splay Fréedericksz transition

The magnetically induced splay Fréedericksz transition is reexamined to look for pattern forming phenomena slightly above or below criticality. By using our traditional scheme of stochastic nematodynamic equations, situations are, respectively, found of transient and permanent predominance of transversal periodicities (wave numbers) along the direction perpendicular to the initial orientation within the sample. The relevance of these predictions in relation with recent observations in the electrically driven splay Fréedericksz transition, and in general with other pattern forming phenomena, is stressed.

Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling

Cells are subjected to dramatic changes of gene expression upon environmental changes. Stresscauses a general down-regulation of gene expression together with the induction of a set of stress-responsivegenes. The p38-related stress-activated protein kinase Hog1 is an important regulator of transcription uponosmostress in yeast. Genome-wide localization studies of RNA polymerase II (RNA Pol II) and Hog1 showed that stress induced major changes in RNA Pol II localization, with a shift toward stress-responsive genes relative to housekeeping genes. RNA Pol II relocalization required Hog1, which was also localized to stress-responsive loci. In addition to RNA Pol II-bound genes, Hog1 also localized to RNA polymerase III-bound genes, pointing to a wider role for Hog1 in transcriptional control than initially expected. Interestingly, an increasing association of Hog1 with stressresponsive genes was strongly correlated with chromatin remodeling and increased gene expression. Remarkably, MNase-Seq analysis showed that although chromatin structure was not significantly altered at a genome-wide level in response to stress, there was pronounced chromatin remodeling for those genes that displayed Hog1 association. Hog1 serves to bypass the general down-regulation of gene expression that occurs in response to osmostress, and does so both by targeting RNA Pol II machinery and by inducing chromatin remodeling at stressresponsive loci.

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells

Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion

The Smc5/6 complex is required for dissolution of DNA-mediated sister chromatid linkages

Mitotic chromosome segregation requires the removal of physical connections between sister chromatids. In addition to cohesin and topological entrapments, sister chromatid separation can be prevented by the presence of chromosome junctions or ongoing DNA replication. We will collectively refer to them as DNA-mediated linkages. Although this type of structures has been documented in different DNA replication and repair mutants, there is no known essential mechanism ensuring their timely removal before mitosis. Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase. Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase. Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation. We propose that Smc5/6 has an essential role in the removal of DNA-mediated linkages to prevent chromosome missegregation and aneuploidy.

The antilipolytic effect of insulin and epidermal growth factor in rat adipocytes are mediated by different mechanisms

Epidermal growth factor (EGF) and insulin induced similar effects in isolated rat adipocytes. To determine whether EGF and insulin produced similar effects through the same mechanisms, we focused on lipolysis. Insulin inhibited the lipolysis stimulated by isoproterenol, glucagon (either alone or in combination with adenosine deaminase), adenosine deaminase itself, or forskolin. In contrast, EGF did not inhibit the lipolysis stimulated by forskolin or by hormones when the cells were also incubated with adenosine deaminase. The effect of insulin, but not that of EGF, on isoproterenol-stimulated lipolysis disappeared when adipocytes were incubated with 1 microM wortmannin. These results indicate that EGF and insulin affected lipolysis through different mechanisms. We observed that EGF, but not insulin, increased cytosolic Ca2+. The effect of EGF, but not that of insulin, disappeared when the cells were incubated in a Ca2+-free medium. We suggest that EGF, but not insulin, mediate its antilipolytic effect through a Ca2+-dependent mechanism which, however, do not involve Ca2+-activated protein kinase C isoforms. This is based on the following: 1) phorbol 12-myristate 13-acetate affected lipolysis in an opposite way to that of EGF; and 2) the protein kinase C inhibitor bisindolylmaleimide GF 109203X did not affect the antilipolytic action of EGF. Our results indicate that the antilipolytic effect of EGF resembles more that of vasopressin than that of insulin.

Role of CYP2E1-mediated metabolism in the acute and vestibular toxicities of nineteen nitriles in the mouse

Allylnitrile, cis-crotononitrile, and 3,3 -iminodipropionitrile are known to cause vestibular toxicity in rodents, and evidence is available indicating that cis-2-pentenenitrile shares this effect. We evaluated nineteen nitriles for vestibular toxicity in wild type (129S1) and CYP2E1-null mice, including all the above, several neurotoxic nitriles, and structurally similar nitriles. A new acute toxicity test protocol was developed to facilitate evaluation of the vestibular toxicity by a specific behavioral test battery at doses up to sub-lethal levels while using a limited number of animals. A mean number of 8.5±0.3 animals per nitrile, strain and sex was necessary to obtain evidence of vestibular toxicity and optionally an estimation of the lethal dose. For several but not all nitriles, lethal doses significantly increased in CYP2E1-null mice. The protocol revealed the vestibular toxicity of five nitriles, including previously identified ototoxic compounds and one nitrile (trans-crotononitrile) known to have a different profile of neurotoxic effects in the rat. In all five cases, both sexes were affected and no decrease in susceptibility was apparent in CYP2E1-null mice respect to 129S1 mice. Fourteen nitriles caused no vestibular toxicity, including six nitriles tested in CYP2E1-null mice at doses significantly larger than the maximal doses that can be tested in wild type animals. We conclude that only a subset of low molecular weight nitriles is toxic to the vestibular system, that species-dependent differences exist in this vestibular toxicity, and that CYP2E1-mediated metabolism is not involved in this effect of nitriles although it has a role in the acute lethality of some of these compounds