Multiplexing and Spectral Microscopy.

Visualization of proteins within a tissue sample via immunohistochemistry (IHC) is a central aspect of pathology. However, such methods are limited to the detection of one or two proteins, due to the overlapping absorption/emission spectra of chromogens and fluorescent dyes. The advent of spectral microscopy has enabled improved visualization of multiple proteins by allowing for the specific light wavelengths/spectral signatures of individual fluorophores and chromogens to be unmixed and analyzed, thus detecting signals that would be indistinguishable with conventional microscopy. Combined with improvements to multiplexed immunohistochemistry (mIHC) protocols, spectral microscopy facilitates the interrogation of spatial relationships between four (enzymatic mIHC) or seven (fluorescent mIHC) proteins, unlocking the wealth of information contained within a single tissue section. Furthermore, the application of linear unmixing for image analysis allows for a reduction in background signal associated with tissue autofluorescence and can distinguish chromogens with similar absorption spectra to identify protein colocalization in brightfield spectral microscopy. While many mIHC protocols have been optimized for spectral microscopy, this chapter will focus in detail on two common methods: enzymatic mIHC and manual fluorescent mIHC using tyramide signal amplification and microwave technology.

Evaluación de las conductas sexuales de riesgo y hábitos anticonceptivos en una muestra de población mexicana / Evaluation of sexual risk behaviors and contraceptive habits in a sample of the Mexican population

Resumen OBJETIVO: Evaluar, a través de una encuesta, la situación actual de las conductas sexuales de riesgo en una muestra de población mexicana de entre 15-60 años, e identificar los métodos de planificación familiar más utilizados y el promedio de inicio de la vida sexual. MATERIALES Y MÉTODOS: Estudio descriptivo, transversal, con muestreo no probabilístico tipo "bola de nieve". Se aplicó una encuesta validada internacionalmente de conductas sexuales de riesgo, entre julio y octubre de 2017, en una muestra mexicana de 15-60 años de edad. Se excluyeron las encuestas incompletas y con discordancias, y personas de otras nacionalidades. Entre las variables estudiadas se incluyeron: sexo, edad y estado socioeconómico. Se calculó un tamaño de muestra de 385, con un intervalo de confianza de 95% (IC95%). Los datos se reunieron en el programa de Excel 2016, donde se analizaron e interpretaron. RESULTADOS: Se encuestaron 689 sujetos, pero se excluyeron 38; de esta forma se registraron 650 casos. El 87.4% de los casos inició su vida sexual entre los 16 y 25 años; un tercio la calificó satisfactoria. El 50% de los encuestados de entre 13 y 15 años utilizó preservativo. El 61% refirió la comunicación de temas sexuales con sus padres poco satisfactoria o inexistente; 6.6% no utiliza o es usuario de algún método de planificación y 40.3% recurre a uno poco efectivo. CONCLUSIÓN: Es importante conocer las prácticas sexuales de la población mexicana, para identificar las áreas de oportunidad, promover la prevención de prácticas sexuales de riesgo y aumentar la difusión de la información.

Abstract OBJECTIVE: Evaluate, through an internationally validated survey, the risky sexual behavior in a Mexican sample between 15-60 years, the average of initiation of sexual activity and the most used contraceptive methods. MATERIALS AND METHODS: Descriptive, cross-sectional study, with non-probabilistic sampling type snowball. We apply an internationally validated survey of Risky Sexual Behavior between July and October 2017 through social networks to Mexican population between 15 and 60 years. Incomplete surveys were excluded, with disagreements in responses and people of other nationalities. The variables studied included sex, age and socioeconomic status. A sample size of 385 was calculated with a confidence level of 95%. The data was gathered in an Excel sheet 2016 where they were analyzed and interpreted. RESULTS: A total of 689 subjects were surveyed, 38 were excluded; In this way, a total of 650 cases were registered. 87.4% started sexual life between 16 and 25 years old, being satisfactory in a third of the cases. 50% of people between 13 and 15 years used condoms and 56% between 16 and 18 years. 61% reported the communication of sexual issues with their parents unsatisfactory or non-existent. 6.6% of people do not have a planning method while 40.3% have a little cash. CONCLUSION: It is important to know the sexual practices in the Mexican population to detect areas of opportunity, make changes in the prevention of risky sexual actions and in the distribution of information.

Implementación y aplicación de la 'Guía de práctica clínica para el diagnóstico, tratamiento y control de la diabetes mellitus tipo 2 en el primer nivel de atención' en una red de establecimientos de salud públicos de Lima / Implementation and application of the 'Clinical practice guideline for diagnosis, treatment and control of type 2 diabetes mellitus at primary care level', in a network of public healthcare facilities in Lima

Objetivo: La diabetes es una enfermedad no transmisible con prevalencia creciente que provoca alta carga de enfermedad por su tardío diagnóstico, complicaciones discapacitantes y elevada mortalidad. Para homogeneizar la atención el Ministerio de Salud peruano publicó la 'Guía de práctica clínica para el diagnóstico, tratamiento y control de la diabetes mellitus tipo 2 en el primer nivel de atención' (GPC). Materiales y métodos: Se elaboró una encuesta para evaluar el equipamiento, así como el conocimiento y uso de GPC en los 32 establecimientos de salud de los 3 distritos de la Dirección de Redes Integrales de Salud de Lima Norte. Resultados: Se evaluó la aplicación de la GPC, el conocimiento de los médicos y el equipamiento en 32 centros de los distritos del Rímac, San Martín de Porres y Los Olivos. Veintisiete médicos (84%) conocían la GPC, diez (28%) han recibido capacitación, 21 (71%) refirieron usarla, 17 (53%) manejaban los conceptos contenidos en la GPC. Solo 4/30 centros disponían de metfomina, ninguno de los centros realizaba hemoglobina glicosilada ni albuminuria y en solo 9 había disponibilidad de glucómetros de manera permanente. Conclusiones: La GPC no ha sido implementada y sólo parcialmente difundida, lo que limita su aplicación, afecta de manera significativa la calidad de atención de pacientes portadores de diabetes mellitus tipo 2 y hace que el esfuerzo y costos invertidos en su elaboración constituyan un dispendio de recursos

Background: Diabetes is a non-communicable disease with increasing prevalence that causes high burden of disease due to its late diagnosis, disabling complications, and high mortality. Aiming to standardize care, the Peruvian Ministry of Health published the 'Clinical practice guideline for diagnosis, treatment and control of type 2 diabetes mellitus at primary care level' (CPG). Materials and methods: A survey was prepared, aiming to assess the equipment as well as knowledge and use of CPG by physicians in 32 healthcare facilities in three districts from the Northern Lima Health Region. Results: The use of CPG, as well as knowledge from physicians and the equipment in 32 healthcare facilities in Rimac, San Martin de Porres, and Los Olivos districts were assessed. Twenty-seven physicians (84%) knew the CPG, 10 (28%) had received training, 21 (71%) declared they use the CPG, and 17 (53%) were familiarized with the concepts delivered by the CPG. Only 4/30 of the facilities had metformin available. None of them had the capability for measuring glycated hemoglobin or albuminuria, and only 9 have permanent availability of blood glucose meters. Conclusions: The GPC has not been implemented and it has been only partially disseminated, which limits its application, this significantly affects the quality of care of patients with type 2 diabetes mellitus; therefore, all effort and costs spent in its elaboration constitute resource waste

Acute Ethanol Increases IGF-I-Induced Phosphorylation of ERKs by Enhancing Recruitment of p52-Shc to the Grb2/Shc Complex.

Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1-24 h) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 h) ethanol (50 mM) enhanced IGF-I-induced phosphorylation of extracellular regulated kinases (ERKs), without affecting IGF-IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF-IR signaling following 3-week ethanol exposure. We then expressed a GFP-tagged IGF-IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently labeled cells, we determined that 50 mM ethanol decreased the half-time of the IGF-IR-associated FRAP, which implied that cell membrane-associated signaling events could be affected. Indeed, co-immunoprecipitation and GST-pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52-Shc to the Grb2-Shc complex, which is known to engage the Ras-Raf-ERKs pathway following IGF-1 stimulation. These experiments indicate that even a short and low-dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. J. Cell. Physiol. 232: 1275-1286, 2017. © 2016 Wiley Periodicals, Inc.

Molecular mechanisms of fenofibrate-induced metabolic catastrophe and glioblastoma cell death.

Fenofibrate (FF) is a common lipid-lowering drug and a potent agonist of the peroxisome proliferator-activated receptor alpha (PPARα). FF and several other agonists of PPARα have interesting anticancer properties, and our recent studies demonstrate that FF is very effective against tumor cells of neuroectodermal origin. In spite of these promising anticancer effects, the molecular mechanism(s) of FF-induced tumor cell toxicity remains to be elucidated. Here we report a novel PPARα-independent mechanism explaining FF's cytotoxicity in vitro and in an intracranial mouse model of glioblastoma. The mechanism involves accumulation of FF in the mitochondrial fraction, followed by immediate impairment of mitochondrial respiration at the level of complex I of the electron transport chain. This mitochondrial action sensitizes tested glioblastoma cells to the PPARα-dependent metabolic switch from glycolysis to fatty acid ß-oxidation. As a consequence, prolonged exposure to FF depletes intracellular ATP, activates the AMP-activated protein kinase-mammalian target of rapamycin-autophagy pathway, and results in extensive tumor cell death. Interestingly, autophagy activators attenuate and autophagy inhibitors enhance FF-induced glioblastoma cytotoxicity. Our results explain the molecular basis of FF-induced glioblastoma cytotoxicity and reveal a new supplemental therapeutic approach in which intracranial infusion of FF could selectively trigger metabolic catastrophe in glioblastoma cells.

Therapeutic efficacy of aldoxorubicin in an intracranial xenograft mouse model of human glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with a median survival of 12 to 15 months after diagnosis. Acquired chemoresistance, high systemic toxicity, and low penetration of the blood brain barrier by many anticancer drugs contribute to the failure of anti-GBM therapies. To circumvent some of these obstacles, we tested a novel prodrug approach to evaluate anti-GBM efficacy by utilizing serum albumin-binding doxorubicin (Doxo), aldoxorubicin (Aldoxo), which is less toxic, is released from albumin in an acidic environment and accumulates in tumor tissues. A human GBM cell line that expresses a luciferase reporter (U87-luc) was stereotactically injected into the left striatum of the brain of immunodeficient mice. Following initial tumor growth for 12 days, mice were injected once a week in the tail-vein with Aldoxo [24 mg/kg or 18 mg/kg of doxorubicin equivalents-3/4 maximum tolerated dose (MTD)], Doxo [6 mg/kg (3/4 MTD)], or vehicle. Aldoxo-treated mice demonstrated significantly slower growth of the tumor when compared to vehicle-treated or Doxo-treated mice. Five out of eight Aldoxo-treated mice remained alive more than 60 days with a median survival of 62 days, while the median survival of vehicle- and Doxo-treated mice was only 26 days. Importantly, Aldoxo-treated mice exhibited high levels of Doxo within the tumor tissue, accompanied by low tumor cell proliferation (Ki67) and abundant intratumoral programmed cell death (cleaved caspase-3). Effective accumulation of Aldoxo in brain tumor tissues but not normal brain, its anti-tumor efficacy, and low toxicity, provide a strong rationale for evaluating this novel drug conjugate as a treatment for patients afflicted with GBM.

“Siempre se vuelve”: análisis del discurso del acto de proclamación como candidata presidencial de Michelle Bachelet / “Always in return”: discourse analysis from Michelle Bachelet’s presidential candidate proclamation event

El presente artículo analiza el discurso realizado por Michelle Bachelet en el Centro Cívico y Artístico de la comuna de El Bosque en Santiago, para presentar su decisión de ser candidata presidencial para las elecciones del año 2013 en Chile. El objetivo de la investigación busca comprender las posiciones de enunciación y las tonalidades discursivas que utiliza la candidata en dicha proclamación, tomando en cuenta la modalidad de liderazgo político femenino que ejerce su figura. Desde el punto de vista metodológico, la herramienta utilizada es el análisis del discurso, considerando conceptos desarrollados por la tradición francesa, desde donde se rescatan los aportes de los trabajos de Foucault (posiciones de enunciación) (Foucault, 1970) y de la escuela rusa, a través de los argumentos desarrollados por Bajtin (tonalidades discursivas) (Voloshinov, 1997). Los resultados exhibieron que, en el discurso de Michelle Bachelet, predomina una posición de sujeto (líder) y tres tonalidades discursivas (familiar, experta y heroica). Las conclusiones apuntan a generar la conexión entre la posición de enunciación hallada y las tonalidades discursivas identificadas a través del análisis del discurso...

This article analyzes Michelle Bachelet’s speech which took place in La Granja’s Civic and Artistic Center in Santiago, Chile to present her decision to run as candidate for the 2013 elections in Chile. This research aims to understand statement positions and discursive tonalities that Bachelet -as candidate used in the mentioned proclamation. Considering the methodological perspective, the analysis tool used was the discourse analysis (DA), considering concepts developed by French tradition -focusing on Foucault’s work constributions (statement positions) (Foucault, 1970) and Russian current with Bajtin’s developed arguments (discursive tonalities (Voloshinov, 1997). Results showed that in Michelle Bachelet’s speech predominate an individual (leading) position and three discursive tonalities (familiar, expert and heroic). These conclusions point to generate connections between the found statement position and the discursive tonalities identified throughout the discourse analysis...

A comunicaçäo como fator relevante para a viabilizaçäo da inclusäo / The relevant role of communication for making inclusion possible

A experiência a ser relatada refere-se ao trabalho de comunicaçäo alternativa e suplementar (CAS), do setor de Fonoaudiologia da AACD, atuante em escola estadual de ensino fundamental, em classes especiais para deficientes físicos, visando a inclusäo do indivíduo, sem fala expressiva, na rede regular de ensino. O desenvolvimento de CAS vem assegurar a aquisiçäo e o desenvolvimento da linguagem, promovendo a integraçäo, a igualdade de oportunidades e maior desenvolvimento cognitivo, afetivo e social

Factores de riesgo para la preeclampsia severa y temprana en el Hospital General de Medellín 1999-2000 / Risk factors for the severe and early pre-eclampsia in the Medellin General Hospital 1999-2000

Objetivo: Identificar los principales factores de riesgo que en nuestro medio se asocian a la presentación de la preeclampsia en sus formas severa y temprana. Metodología: Se realizó un estudio de casos y controles de preeclampsia severa y lejana del término. Lugar: Mujeres que tuvieron su parto en el Hospital General de Medellín (HGM) entre septiembre de 1999 y marzo de 2000.”Conclusión”: Los factores de riesgo que mas se asociaron son factores relacionados con la historia personal y familiar del individuo, que no son susceptibles de modificación o de intervención con la tecnología actual, pero que si le permiten al clínico un diagnóstico más temprano y oportuno del síndrome, además de darle elementos para direccionar la atención de estas embarazadas centro de salud de mayor complejidad. Llama la atención que aquellos factores relacionados con la primipaternidad y con los hábitos sexuales no presentaron ninguna diferencia estadísticamente significativa entre grupos, orientándonos quizás hacia etiologías principalmente de origen genético o hereditario...

Hallazgo citogenético y molecular de un caso de hermafroditismo verdadero / Citogenético and molecular finding of a case of true hermafroditismo

el hermafroditismo verdadero es una condición poco frecuente, en la cual en un mismo individuo coexisten tejido ovárico y testicular. La mayoría de estos individuos presentan un cariotipo normal y alrededor de un 20 por ciento de los hermafroditas verdaderos muestran un cariotiSe valoró un individuo aparentemente de sexo masculino, con cromatina sexual positiva que presentaba genitales externos ambiguos. Sin embargo, la biopsia gonadal por laparotomía mostró tejido ovárico inmaduro; la ecografía pélvica, ovarios sin folículos y un canal hipoecoico que podría corresponder a la vagina; aún más, la cistouretrografía evidenció una vagina elongada y amplia que finalizaba en una cavidad similar a la uterina. Los hallazgos clínicos y paraclínicos condujeron a realizar el estudio citogenético mediante cariotipo y análisis de secuencias microsatélites strs para tratar de esclarecer el posible mecanismo implicado en este caso en particularpo en mosaico 46 xx 46 xy conocido como quimerismo