Proteomic profiling of the extracellular vesicles and the secretome of "Candida albicans" and functional analysis of cell surface proteins

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Microbiología II, leída el 11-02-2016Candida albicans es un importante patógeno oportunista en humanos, que puede causar distintos tipos de infecciones, desde micosis superficiales hasta sistémicas. La candidiasis invasiva es una enfermedad que puede causar mortalidad en pacientes inmunocomprometidos. Para causar daño en el hospedador, C. albicans cuenta con una serie de factores de virulencia. Entre ellos destaca la capacidad de cambiar su forma de crecimiento de levadura a hifa. La superficie celular es la estructura más externa de la célula y el punto de contacto entre el hongo y el hospedador. Las proteínas de superficie tienen un papel importante en la integridad estructural de la célula y en la adherencia e invasión de células del hospedador. Una de las proteínas localizadas en la superficie celular es Ecm33, una proteína de pared celular con anclaje glicosilfosfatidilinositol (GPI). La deleción de esta proteína afecta a la morfología tanto de levaduras como de hifas, dando como resultado células con la pared celular alterada y virulencia reducida tanto en condiciones in vitro como in vivo. El secretoma o las proteínas secretadas por C. albicans son también relevantes en la interacción patógeno-hospedador. C. albicans secreta muchas proteínas importantes relacionadas con diferentes procesos, entre los que se incluyen la formación de biofilms, la adquisición de nutrientes y el mantenimiento de la integridad de la pared celular. Muchas de estas proteínas secretadas, como las pertenecientes a las familias de aspartil proteasas (Sap) y la familia de fosfolipasas B (Plb), también han sido detectadas en la pared celular, ya que deben pasar a través de ella en su tránsito hacia el medio extracelular. Estas proteínas tienen un péptido señal en el extremo N-terminal que es el responsable de dirigirlas a la ruta clásica de secreción. Sin embargo, cerca de un tercio de las proteínas identificadas en el medio extracelular de C. albicans no poseen dicho péptido señal en su secuencia...Candida albicans is a commensal fungus in humans which causes different infections ranging from superficial to systemic. Invasive candidiasis is an important cause of disease and mortality in immunocompromised patients. The ability to switch from yeast to hypha growth is essential for virulence in C. albicans which express distinct cell surface proteins in these stages. The cell surface is the most external structure and the initial point of contact between the fungus and the host. Surface proteins play an important role in the structural integrity of the cell, adherence and invasion of host cells. One of these proteins is Ecm33, a glycosylphosphatidylinositol (GPI)-linked cell wall protein. The absence of this protein affects both yeast and hypha morphology and results in an aberrant wall structure and reduced virulence in vitro and in vivo. The C. albicans secreted proteins are also relevant in host-pathogen interaction. C. albicans secretes many important proteins involved in different processes, including biofilm formation, cell nutrient acquisition and cell wall integrity maintenance. Some secreted proteins, such as secreted aspartyl proteinases (Sap) and phospholipase B (Plb) families have been detected in the cell wall since they must pass through it to be secreted. These proteins have an amino-terminal signal peptide that is responsible for directing them into the classical secretory pathway. Furthermore, close to one-third of extracellular proteins identified in the C. albicans secretome do not possess a secretion signal. These proteins lacking the N-linked signal peptide should use alternative routes of exportation. Extracellular vesicles (EVs) have been described as a mechanism of molecular traffic across the cell wall to the extracellular space in fungi. All fungal species studied to date were apparently able to use EVs as a general mechanism of molecular traffic to transport intracellular proteins across the cell wall...Depto. de Microbiología y ParasitologíaFac. de FarmaciaTRUEunpu

The external face of Candida albicans: A proteomic view of the cell surface and the extracellular environment.

The cell surface and secreted proteins are the initial points of contact between Candida albicans and the host. Improvements in protein extraction approaches and mass spectrometers have allowed researchers to obtain a comprehensive knowledge of these external subproteomes. In this paper, we review the published proteomic studies that have examined C. albicans extracellular proteins, including the cell surface proteins or surfome and the secreted proteins or secretome. The use of different approaches to isolate cell wall and cell surface proteins, such as fractionation approaches or cell shaving, have resulted in different outcomes. Proteins with N-terminal signal peptide, known as classically secreted proteins, and those that lack the signal peptide, known as unconventionally secreted proteins, have been consistently identified. Existing studies on C. albicans extracellular vesicles reveal that they are relevant as an unconventional pathway of protein secretion and can help explain the presence of proteins without a signal peptide, including some moonlighting proteins, in the cell wall and the extracellular environment. According to the global view presented in this review, cell wall proteins, virulence factors such as adhesins or hydrolytic enzymes, metabolic enzymes and stress related-proteins are important groups of proteins in C. albicans surfome and secretome. BIOLOGICAL SIGNIFICANCE Candida albicans extracellular proteins are involved in biofilm formation, cell nutrient acquisition and cell wall integrity maintenance. Furthermore, these proteins include virulence factors and immunogenic proteins. This review is of outstanding interest, not only because it extends knowledge of the C. albicans surface and extracellular proteins that could be related with pathogenesis, but also because it presents insights that may facilitate the future development of new antifungal drugs and vaccines and contributes to efforts to identify new biomarkers that can be employed to diagnose candidiasis. Here, we list more than 570 C. albicans proteins that have been identified in extracellular locations to deliver the most extensive catalogue of this type of proteins to date. Moreover, we describe 16 proteins detected at all locations analysed in the works revised. These proteins include the glycophosphatidylinositol (GPI)-anchored proteins Ecm33, Pga4 and Phr2 and unconventional secretory proteins such as Eft2, Eno1, Hsp70, Pdc11, Pgk1 and Tdh3. Furthermore, 13 of these 16 proteins are immunogenic and could represent a set of interesting candidates for biomarker discovery

Análisis de una nueva escala de valoración de riesgo para pacientes quirúrgicos

Objetivo: Las escalas de valoración de riesgo quirúrgico son sistemas que sirven para predecir el curso de un paciente sometido a una intervención quirúrgica y mejorar su resultado. Los requisitos que una escala de valoración de riesgo debe cumplir son su simplicidad, su facilidad en la recogida y manejo de datos, ser extrapolable a todo tipo de cirugías y ser recogida en la consulta de preanestesia.Las escalas utilizadas hasta ahora cumplen estas características pero tienen los siguientes inconvenientes. El ASA, tienen el inconveniente de la subjetividad. El Surigical Risk Scale es una escala más objetiva porque incorpora la complejidad y el carácter urgente de la cirugía entre sus variables, pero también incluye el ASA, disminuyendo su objetividad. El indice de Charlson también es utilizado actualmente pero incluye 19 comorbilidades a las cuales se les asigna una puntuación y es cuestionado por algunos autores para su uso en pacientes que van a ser intervenidos. Elaboramos una nueva escala (NE) de valoración de riesgo quirúrgico cuyo objetivo fue comprobar que los pacientes con mayor riesgo para la NE presentaron más complicaciones perioperatorias, y se comparó con las escalas mencionadas anteriormente. Material y Métodos: La NE se construyó en base a los antecedentes cardiovasculares del paciente, hipertensión arterial, diabetes mellitus, insuficiencia cardiaca, cardiopatía isquémica y evidencia clínica de arterioesclerosis, el carácter urgente de la cirugía según la clasificación CEPOD (Confidential enquire into perioperative deaths) y el grado de complejidad de la cirugía según la American Heart Association (AHA).Se analizaron de forma prospectiva 800 pacientes y se recogieron variables preoperatorias y complicaciones intraoperatorias y postoperatorias, así como ingreso en unidades de Críticos, UCI o Reanimación , estancia hospitalaria y mortalidad. .Se halló el punto de corte para la NE en función del cual se dividieron los pacientes en alto y bajo riesgo de sufrir complicaciones perioperatorias, según las curvas R.O.C. de sensibilidad y especificidad, en base a la mitad del tamaño muestral,se aplicó a los otros 400 pacientes y se hallaron los porcentajes de acierto para la NE en comparación con las otras escalas mediante estudios de simulación y validación cruzada. La escala se calibró mediante el modelo GLM con link logit, equivalente a un modelo de regresión logistica lineal . Para la estadística inferencia lse consideró una p<0,05 como significativa aplicando la corrección de Bonferroni para el análisis de test múltiples. Los test estadísticos utilizados son los estándares, el chi-cuadrado para variables cualitativas, la t-de Student para cuantitativas y el test de Kruskal-Wallis para cuantitativas y cualitativas. Resultados: Cuando comparamos pacientes intervenidos de manera urgente versus cirugía programada observamos que la cirugía urgente conlleva más morbimortalidad perioperatoria y mayor estancia hospitalaria. La cirugía más compleja también resultó ser más significativa en cuanto a complicaciones intraoperatorias y postoperatorias,a sí como los pacientes con antecedentes cardiovasculares, que también presentaron mayor morbimortalidad y mayor estancia hospitalaria. El punto de corte para la NE se estableció en 5. El área bajo la curva fué mayor que la de otras escalas, 0,8804 frente a 0,86 para el SRS y 0,6 para ASA y 0,4 para el Charlson .el porcentaje de acierto fue mayor para la nueva escala . Los pacientes con un riesgo alto, es decir, con una puntuación por encima de 5 para la NE presentaron mayor número de complicaciones perioperatorias. Conclusiones: La NE posee mayor sensibilidad y especificidad para predecir morbilidad intraoperatoria y complicaciones postoperatorias en comparación al indice de Charlson, Surgical Risk Scale y clasificación ASA.. La NE es de fácil realización y con alto poder predictivo de desarrollo de complicaciones perioperatorias. La cirugía urgente, la complejidad de la cirugía y los antecedentes cardiovasculares se significaron más en cuanto a morbimortalidad intraoperatoria y postoperatoria se refiere. 1. Tamarit CO. Anestesia intravenosa en el paciente criticamente enfermo. En: Tamariz CO, editor. Alto riesgo perioperatorio. 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Harrison's principles of internal medicine.14ª edicion. San Francisco: Mc Graw Hill, 2001; 334-34

The cell wall protein Ecm33 of Candida albicans is involved in chronological life span, morphogenesis, cell wall regeneration, stress tolerance, and host–cell interaction

Q2Q11-14Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a “veil growth,” never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain

Laparoscopic surgery in 3D improves results and surgeon convenience in sleeve gastrectomy for morbid obesity

Purpose Advanced laparoscopic procedures are still challenging. One critical issue is the lack of stereoscopic vision. The aim of this surgical study is to evaluate whether 3D vision offers any advantages for surgical performance over 2D vision during sleeve gastrectomy for morbid obesity using a laparoscopic system that allows changing between 2D and 3D optics. Methods A total of 78 patients were analyzed, with 37 in the 2D group and 41 in the 3D group. Performance time, hospital stay, complications, and early outcomes were collected. To assess the quality of the 2D and 3D techniques, visual analog scales from 0 to 10 were designed, and image quality, depth of field, precision in performing tasks, and general ergonomics were measured. Results According to the vision system used, the mean duration of surgery was 85 ± 16.8 min for patients operated on with the 2D system and 69 ± 16.9 min for those operated on with the 3D system. There were no significant differences between the overall percentages of complications according to the type of vision used. However, postoperative complications were more severe in the 2D laparoscopy group. The average length of stay was shorter for patients in the 3D group. Regarding the differences perceived by the surgeon, the depth of field and the precision of tasks were better in the 3D vision group. Conclusion The 3D system provided greater depth perception and precision in more complex tasks, enabling safer surgery. This led to a reduction in the operative time and hospital stay. Moreover, the severity of complications was less

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Tooth Enamel and Its Dynamic Protein Matrix

Tooth enamel is the outer covering of tooth crowns, the hardest material in the mammalian body, yet fracture resistant. The extremely high content of 95 wt% calcium phosphate in healthy adult teeth is achieved through mineralization of a proteinaceous matrix that changes in abundance and composition. Enamel-specific proteins and proteases are known to be critical for proper enamel formation. Recent proteomics analyses revealed many other proteins with their roles in enamel formation yet to be unraveled. Although the exact protein composition of healthy tooth enamel is still unknown, it is apparent that compromised enamel deviates in amount and composition of its organic material. Why these differences affect both the mineralization process before tooth eruption and the properties of erupted teeth will become apparent as proteomics protocols are adjusted to the variability between species, tooth size, sample size and ephemeral organic content of forming teeth. This review summarizes the current knowledge and published proteomics data of healthy and diseased tooth enamel, including advancements in forensic applications and disease models in animals. A summary and discussion of the status quo highlights how recent proteomics findings advance our understating of the complexity and temporal changes of extracellular matrix composition during tooth enamel formation