Mecanismos moleculares y celulares de la acción del Rab18 en adipositos

El mantenimiento de la homeostasis lipídica en adipocitos está regulado por la combinación de síntesis, tráfico y almacenamiento de ácidos grasos en gotas lipídicas. Estudios previos han mostrado que las gotas lipídicas presentan una gran variedad de proteínas asociadas a su superficie. Una de estas proteínas es Rab18, un miembro de la familia de GTPasas de bajo peso molecular Rab, para la que se ha sugerido que participa en el control de la movilización de lípidos de gotas lipídicas. Sin embargo, la función específica de esta proteína en relación a la homeostasis lipídica en adipocitos aún se desconoce. En este sentido, el objetivo general de la Tesis consiste en ampliar el conocimiento acerca de la regulación y función que ejerce la proteína Rab18 en adipocitos en condiciones pato-fisiológicas. Los resultados principales obtenidos en el presente estudio son los siguientes: La obesidad y el ayuno alteran la expresión de Rab18 en tejido adiposo en modelos animales. Asimismo, la expresión de Rab18 también se ve modificada en humanos obesos, con resistencia a insulina y con diabetes mellitus de tipo II. La expresión de Rab18 en adipocitos está regulada por hormonas hipofisarias, por TSH directamente o a través de la acción de las hormonas tiroideas en el tejido adiposo. La expresión de Rab18 en adipocitos 3T3-L1 está regulada por catecolaminas e insulina, estimuladores de lipólisis y lipogénesis, respectivamente. Además, ambas sustancias incrementan la localización de Rab18 alrededor de las gotas lipídicas y la colocalización de Rab18 con marcadores de retículo endoplasmático y de otras proteínas de la cubierta de la gota lipídica. La sobreexpresión de Rab18 en 3T3-L1 aumenta el tamaño de las gotas lipídicas y la actividad lipolítica y lipogénica basal en dichas células. Por otra parte, el silenciamiento de esta GTPasa bloquea la respuesta a catecolaminas e insulina. En conclusión, los resultados anteriores sugieren que Rab18 participa de forma activa en la regulación del metabolismo lipídico en adipocitos, lo que es interesante en la búsqueda de nuevas dianas terapéuticas para combatir la obesidad y las enfermedades relacionadas

Effect of disinfectants used in clinical facilities on the induction of the SOS response and mutation frequency in Escherichia coli

Motivation: The development of antibiotic resistance is one of the mechanism used to study adaptive evolution. Antibiotics not only select for preexisting resistant strains in a population, but they can also promote the appearance of resistant strains (1, 2). Antibiotics at high concentrations can be lethal to bacteria, but it has been shown that sub-inhibitory concentrations of some antibiotics can stimulate horizontal transfer of DNA and mutation in different chromosomal loci (1, 3). These antibiotics can increase the mutation rate through several mechanisms, such as oxidative stress and the SOS response, which is triggered by DNA damage (2, 3). In this context, we wanted to determine if commonly-used disinfectants also promote mutation. Methods: The disinfectants used were ethanol, sodium hypochlorite, chlorhexidine, silver nitrate, benzalkonium chloride, triclosan and povidone-iodine complex. The E.coli strain used was IBDS1, which includes a tetracycline gene regulated by the cI (Ind-) repressor gene, that allows us to measure the mutation rate.To study the effect of these disinfectants, the strain IBDS1 was tested with a window of concentrations very close to the minimum inhibitory concentration (MIC) by evaluating the appearance of mutants resistant to rifampicin or tetracycline (3). To determine if this effect could be linked to the induction of the SOS system, we used a plasmid which expresses the "Green Fluorescence Protein" (GFP) under the control of the promoter of the recA gene to detect when the SOS system is activated by measuring fluorescence.Results: Three of the disinfectants tested increased mutation frequency. Concentrations of 0.013 μg/ml and 0.026 μg/ml of NaClO (1/4x CMI y 1/2x MIC) increased the mutation frequency approximatly 4 fold and 0.5 μg/ml of clorhexidine (1/4x MIC) and 0.125 μg/ml of triclosan (1/4x MIC) 3 fold, approximately. In relation to the SOS system, none of the concentrations tested induced the SOS response. Conclusions: These results show that certain concentrations of sodium hypochlorite, chlorhexidine and triclosan increase the mutation frequency and therefore may facilitate the appearance of resistant strains, although it appears that this mutagenic effect is not related to the induction of the SOS system. Therefore, it would be interesting to study whether this mutagenic effects is due to the reactive oxygen species (ROS) produced by disinfectants.

Pathogenic Acinetobacter species have a functional type I secretion system and contact-dependent inhibition systems

Pathogenic Acinetobacter species, including Acinetobacter baumannii and Acinetobacter nosocomialis, are opportunistic human pathogens of increasing relevance worldwide. Although their mechanisms of drug resistance are well studied, the virulence factors that governAcinetobacter pathogenesis are incompletely characterized. Here we define the complete secretome of A. nosocomialis strain M2 in minimal medium and demonstrate that pathogenicAcinetobacter species produce both a functional type I secretion system (T1SS) and a contact-dependent inhibition (CDI) system. Using bioinformatics, quantitative proteomics, and mutational analyses, we show that Acinetobacter uses its T1SS for exporting two putative T1SS effectors, an Repeatsin-Toxin (RTX)-serralysin-like toxin, and the biofilm-associated protein (Bap). Moreover, we found that mutation of any component of the T1SS system abrogated type VI secretion activity under nutrient-limited conditions, indicating a previously unrecognized cross-talk between these two systems. We also demonstrate that the Acinetobacter T1SS is required for biofilm formation. Last, we show that both A. nosocomialis and A. baumannii produce functioning CDI systems that mediate growth inhibition of sister cells lacking the cognate immunity protein. The Acinetobacter CDI systems are widely distributed across pathogenicAcinetobacter species, with manyA. baumannii isolates harboring two distinct CDI systems. Collectively, these data demonstrate the power of differential, quantitative proteomics approaches to study secreted proteins, define the role of previously uncharacterized protein export systems, and observe cross-talk between secretion systems in the pathobiology of medically relevant Acinetobacter speciesSubprograma Sara Borrell from the Instituto de Salud Carlos IIISubdirección General de Evaluación y Fomento de la InvestigaciónMinisterio de Economía y Competitividad de España CD14/0001

Highly versatile polyelectrolyte complexes for improving the enzyme replacement therapy of lysosomal storage disorders

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically crosslinked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and -galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity and hemocompatibility of RGD-targeted and un-targeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freezedrying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows keeping intact the activity of the therapeutic protein. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders

Rab18 Dynamics in Adipocytes in Relation to Lipogenesis, Lipolysis and Obesity

Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed

Complement component 3 (C3) expression in the hippocampus after excitotoxic injury: role of C/EBPβ

[Background] The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor implicated in the control of proliferation, differentiation, and inflammatory processes mainly in adipose tissue and liver; although more recent results have revealed an important role for this transcription factor in the brain. Previous studies from our laboratory indicated that CCAAT/enhancer-binding protein β is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. More recently, we have shown that the complement component 3 gene (C3) is a downstream target of CCAAT/enhancer-binding protein β and it could be a mediator of the proinflammatory effects of this transcription factor in neural cells.[Methods] Adult male Wistar rats (8–12 weeks old) were used throughout the study. C/EBPβ+/+ and C/EBPβ–/– mice were generated from heterozygous breeding pairs. Animals were injected or not with kainic acid, brains removed, and brain slices containing the hippocampus analyzed for the expression of both CCAAT/enhancer-binding protein β and C3.[Results] In the present work, we have further extended these studies and show that CCAAT/enhancer-binding protein β and C3 co-express in the CA1 and CA3 regions of the hippocampus after an excitotoxic injury. Studies using CCAAT/enhancer-binding protein β knockout mice demonstrate a marked reduction in C3 expression after kainic acid injection in these animals, suggesting that indeed this protein is regulated by C/EBPβ in the hippocampus in vivo.[Conclusions] Altogether these results suggest that CCAAT/enhancer-binding protein β could regulate brain disorders, in which excitotoxic and inflammatory processes are involved, at least in part through the direct regulation of C3.This work was supported by MINECO, Grant SAF2014-52940-R and partially financed with FEDER funds. CIBERNED is funded by the Instituto de Salud Carlos III. JAM-G was supported by CIBERNED. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts