Do protein–protein interaction databases identify moonlighting proteins?

One of the most striking results of the human (and mammalian) genomes is the low number of protein-coding genes. To-date, the main molecular mechanism to increase the number of different protein isoforms and functions is alternative splicing. However, a less-known way to increase the number of protein functions is the existence of multifunctional, multitask, or ‘‘moonlighting’’, proteins. By and large, moonlighting proteins are experimentally disclosed by serendipity. Proteomics is becoming one of the very active areas of biomedical research, which permits researchers to identify previously unseen connections among proteins and pathways. In principle, protein–protein interaction (PPI) databases should contain information on moonlighting proteins and could provide suggestions to further analysis in order to prove the multifunctionality. As far as we know, nobody has verified whether PPI databases actually disclose moonlighting proteins. In the present work we check whether well-established moonlighting proteins present in PPI databases connect with their known partners and, therefore, a careful inspection of these databases could help to suggest their different functions. The results of our research suggest that PPI databases could be a valuable tool to suggest multifunctionality.

Actitudes y creencias en los estudiantes del Campus de Bellvitge sobre las medidas higiénicas y las precauciones estándar

Introducción: En los últimos años se han producido avances importantes en el campo de la autoprotección ante las infecciones. Por parte de los docentes que imparten temas relacionados con el campo de la autoprotección del personal de salud y la higiene en las diferentes carreras de ciencias de la salud en el Campus de Bellvitge (CB) de la Universitat de Barcelona (UB) se detectó un desconocimiento y/o confusión de materias relacionadas con la seguridad del paciente y de los profesionales. Objetivo: Mejorar la preparación de los estudiantes de Ciencias de la Salud del CB de la UB en relación con las medidas higiénicas básicas. Material y Método: Estudio de campo, causal y comparativo. Se estableció un grupo formado por los profesores implicados en estas materias para unificar criterios y llevar a cabo un análisis de la situación exhaustiva. Se hicieron entrevistas semiestructuradas a dos estudiantes de cada carrera que fueron registradas y transcritas textualmente por el análisis posterior (Atlas/Ti19). Resultados y Discusión: Del análisis de los discursos surgieron cuatro categorías: 1) El aprendizaje; los estudiantes destacan la falta de consenso entre profesores y la enseñanza demasiado teórica. 2) La experiencia laboral; los estudiantes destacan la divergencia entre la teoría y la práctica. 3) Los argumentos personales; los estudiantes relacionan las medidas con situaciones de excepcionalidad (técnicas muy invasivas) está omnipresente el discurso de"no hacer daño al paciente". 4) Las características propias de las diferentes carreras universitarias; hemos registrado percepciones de los riesgos diferentes entre ellas. Conclusiones: La formación en medidas preventivas y de higiene está contemplada en los planes de estudio como una formación puntual y no lineal en las carreras. Los estudiantes la perciben como una formación necesaria dado su grado de inexperiencia, pero que no es necesaria cuando se adquieren ciertas destrezas. Por lo que respeta a los centros de salud como a los hospitales no hay un consenso en la obligatoriedad que todos los profesionales adopten estas medidas a la práctica diaria y algunas veces los criterios entre teoría y práctica son divergentes.

Actitudes y creencias en los estudiantes del Campus de Bellvitge sobre las medidas higiénicas y las precauciones estándar

Introducción: En los últimos años se han producido avances importantes en el campo de la autoprotección ante las infecciones. Por parte de los docentes que imparten temas relacionados con el campo de la autoprotección del personal de salud y la higiene en las diferentes carreras de ciencias de la salud en el Campus de Bellvitge (CB) de la Universitat de Barcelona (UB) se detectó un desconocimiento y/o confusión de materias relacionadas con la seguridad del paciente y de los profesionales. Objetivo: Mejorar la preparación de los estudiantes de Ciencias de la Salud del CB de la UB en relación con las medidas higiénicas básicas. Material y Método: Estudio de campo, causal y comparativo. Se estableció un grupo formado por los profesores implicados en estas materias para unificar criterios y llevar a cabo un análisis de la situación exhaustiva. Se hicieron entrevistas semiestructuradas a dos estudiantes de cada carrera que fueron registradas y transcritas textualmente por el análisis posterior (Atlas/Ti19). Resultados y Discusión: Del análisis de los discursos surgieron cuatro categorías: 1) El aprendizaje; los estudiantes destacan la falta de consenso entre profesores y la enseñanza demasiado teórica. 2) La experiencia laboral; los estudiantes destacan la divergencia entre la teoría y la práctica. 3) Los argumentos personales; los estudiantes relacionan las medidas con situaciones de excepcionalidad (técnicas muy invasivas) está omnipresente el discurso de"no hacer daño al paciente". 4) Las características propias de las diferentes carreras universitarias; hemos registrado percepciones de los riesgos diferentes entre ellas. Conclusiones: La formación en medidas preventivas y de higiene está contemplada en los planes de estudio como una formación puntual y no lineal en las carreras. Los estudiantes la perciben como una formación necesaria dado su grado de inexperiencia, pero que no es necesaria cuando se adquieren ciertas destrezas. Por lo que respeta a los centros de salud como a los hospitales no hay un consenso en la obligatoriedad que todos los profesionales adopten estas medidas a la práctica diaria y algunas veces los criterios entre teoría y práctica son divergentes.

Actitudes y creencias en los estudiantes del Campus de Bellvitge sobre las medidas higiénicas y las precauciones estándar

Introducción: En los últimos años se han producido avances importantes en el campo de la autoprotección ante las infecciones. Por parte de los docentes que imparten temas relacionados con el campo de la autoprotección del personal de salud y la higiene en las diferentes carreras de ciencias de la salud en el Campus de Bellvitge (CB) de la Universitat de Barcelona (UB) se detectó un desconocimiento y/o confusión de materias relacionadas con la seguridad del paciente y de los profesionales. Objetivo: Mejorar la preparación de los estudiantes de Ciencias de la Salud del CB de la UB en relación con las medidas higiénicas básicas. Material y Método: Estudio de campo, causal y comparativo. Se estableció un grupo formado por los profesores implicados en estas materias para unificar criterios y llevar a cabo un análisis de la situación exhaustiva. Se hicieron entrevistas semiestructuradas a dos estudiantes de cada carrera que fueron registradas y transcritas textualmente por el análisis posterior (Atlas/Ti19). Resultados y Discusión: Del análisis de los discursos surgieron cuatro categorías: 1) El aprendizaje; los estudiantes destacan la falta de consenso entre profesores y la enseñanza demasiado teórica. 2) La experiencia laboral; los estudiantes destacan la divergencia entre la teoría y la práctica. 3) Los argumentos personales; los estudiantes relacionan las medidas con situaciones de excepcionalidad (técnicas muy invasivas) está omnipresente el discurso de"no hacer daño al paciente". 4) Las características propias de las diferentes carreras universitarias; hemos registrado percepciones de los riesgos diferentes entre ellas. Conclusiones: La formación en medidas preventivas y de higiene está contemplada en los planes de estudio como una formación puntual y no lineal en las carreras. Los estudiantes la perciben como una formación necesaria dado su grado de inexperiencia, pero que no es necesaria cuando se adquieren ciertas destrezas. Por lo que respeta a los centros de salud como a los hospitales no hay un consenso en la obligatoriedad que todos los profesionales adopten estas medidas a la práctica diaria y algunas veces los criterios entre teoría y práctica son divergentes.

Actitudes y creencias en los estudiantes del Campus de Bellvitge sobre las medidas higiénicas y las precauciones estándar

Introducción: En los últimos años se han producido avances importantes en el campo de la autoprotección ante las infecciones. Por parte de los docentes que imparten temas relacionados con el campo de la autoprotección del personal de salud y la higiene en las diferentes carreras de ciencias de la salud en el Campus de Bellvitge (CB) de la Universitat de Barcelona (UB) se detectó un desconocimiento y/o confusión de materias relacionadas con la seguridad del paciente y de los profesionales. Objetivo: Mejorar la preparación de los estudiantes de Ciencias de la Salud del CB de la UB en relación con las medidas higiénicas básicas. Material y Método: Estudio de campo, causal y comparativo. Se estableció un grupo formado por los profesores implicados en estas materias para unificar criterios y llevar a cabo un análisis de la situación exhaustiva. Se hicieron entrevistas semiestructuradas a dos estudiantes de cada carrera que fueron registradas y transcritas textualmente por el análisis posterior (Atlas/Ti19). Resultados y Discusión: Del análisis de los discursos surgieron cuatro categorías: 1) El aprendizaje; los estudiantes destacan la falta de consenso entre profesores y la enseñanza demasiado teórica. 2) La experiencia laboral; los estudiantes destacan la divergencia entre la teoría y la práctica. 3) Los argumentos personales; los estudiantes relacionan las medidas con situaciones de excepcionalidad (técnicas muy invasivas) está omnipresente el discurso de"no hacer daño al paciente". 4) Las características propias de las diferentes carreras universitarias; hemos registrado percepciones de los riesgos diferentes entre ellas. Conclusiones: La formación en medidas preventivas y de higiene está contemplada en los planes de estudio como una formación puntual y no lineal en las carreras. Los estudiantes la perciben como una formación necesaria dado su grado de inexperiencia, pero que no es necesaria cuando se adquieren ciertas destrezas. Por lo que respeta a los centros de salud como a los hospitales no hay un consenso en la obligatoriedad que todos los profesionales adopten estas medidas a la práctica diaria y algunas veces los criterios entre teoría y práctica son divergentes.

The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB¿/¿ mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA¿/¿ MEFs, but not FlnB¿/¿ MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Systems medicine and integrated care to combat chronic noncommunicable diseases

We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.

Acylation of chiral alcohols: a simple procedure for chiral GC analysis

The use of iodine as a catalyst and either acetic or trifluoroacetic acid as a derivatizing reagent for determining the enantiomeric composition of acyclic and cyclic aliphatic chiral alcohols was investigated. Optimal conditions were selected according to the molar ratio of alcohol to acid, the reaction time, and the reaction temperature. Afterwards, chiral stability of chiral carbons was studied. Although no isomerization was observed when acetic acid was used, partial isomerization was detected with the trifluoroacetic acid. A series of chiral alcohols of a widely varying structural type were then derivatized with acetic acid using the optimal conditions. The resolution of the enantiomeric esters and the free chiral alcohols was measured using a capillary gas chromatograph equipped with a CP Chirasil-DEX CB column. The best resolutions were obtained with 2-pentyl acetates (α = 3.00) and 2-hexyl acetates (α = 1.95). This method provides a very simple and efficient experimental workup procedure for analyzing chiral alcohols by chiral-phase GC.

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.

CERKL Knockdown Causes Retinal Degeneration in Zebrafish

The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneration

Formoterol treatment downregulates the myostatin system in skeletal muscle of cachectic tumour-bearing rats

Cachexia is a common systemic manifestation. Additionally, myostatin is known to be a negative regulator of skeletal muscle development. The present study aimed to investigate whether formoterol down-regulates the myostatin system in skeletal muscle of tumour-bearing rats. Real-time PCR and Western blotting were used for the analysis. Results showed that rats bearing the Yoshida AH-130 ascites hepatoma, a cachexia-inducing tumour, exhibited marked muscle wasting that affected the mass of the muscles studied. The cachectic animals exhibited a significant increase in the mRNA levels of the myostatin receptor (ActIIB) in gastrocnemius muscles. Notably, the expression of the various forms of follistatin, a protein with the opposite effects to those of myostatin, was significantly reduced as a result of the implantation of the tumour. When the animals were treated with formoterol, a β-agonist with anti-cachectic potential, increases in skeletal muscle weights were observed. The β-agonist significantly increased levels of various follistatin isoforms and significantly decreased the expression levels of the myostatin receptor. In addition, formoterol treatment resulted in a significant decrease of the myostatin protein content of the gastrocnemius muscle. In conclusion, the results presented indicate that certain anabolic actions of formoterol on the skeletal muscle of cachectic animals may be mediated via the myostatin system.

Multilevel control of arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B99 regulatory subunits of protein phosphatase 2A (PP2A), designated B99a and B99b, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B99a and B99b are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B99a and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B99b. Our data indicate that PP2A exerts multilevel control on HMGR through the fivemember B99 protein family during normal development and in response to a variety of stress conditions.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors.