Large analysis of genetic manipulations reveals an inverse correlation between initial alcohol resistance and rapid tolerance phenotypes.

Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce identical behavioral effects. Tolerance is not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we analyzed our own, as well as data published by other labs to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes, thus classifying such mutants as 'secondary' tolerance mutants. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a 'primary' tolerance mutant, where a tolerance phenotype is not solely a consequence of initial resistance, and we offer a framework for understanding the relationship between initial resistance and tolerance.

Iterative assay for transposase-accessible chromatin by sequencing to isolate functionally relevant neuronal subtypes.

The Drosophila brain contains tens of thousands of distinct cell types. Thousands of different transgenic lines reproducibly target specific neuron subsets, yet most still express in several cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we developed an iterative assay for transposase-accessible chromatin (ATAC) approach. Open chromatin regions (OCRs) enriched in neurons, compared to whole bodies, drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCR2s that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression, and we used it to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

Large genetic analysis of alcohol resistance and tolerance reveals an inverse correlation and suggests 'true' tolerance mutants.

Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce the same behavioral effects. Tolerance is historically not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and between labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we have analyzed a large amount of data - our own published and unpublished data and data published by other labs - to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. We show that these residuals provide predictive insight into the likelihood of a mutant being a 'true' tolerance mutant, and we offer a framework for understanding the relationship between initial resistance and tolerance.

Harnessing changes in open chromatin determined by ATAC-seq to generate insulin-responsive reporter constructs.

BACKGROUND: Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed. RESULTS: We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a weak but significant correlation between chromatin regions annotated to enhancers (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate enhancer regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters. CONCLUSIONS: Insulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression.

Optimized assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation from adult Drosophila melanogaster neurons.

Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an untargeted manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation that are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.

Estrategia Pedagógica PICUR: un aporte a la autogestión del conocimiento en la Ingeniería de Sistemas de Uniremington / PICUR Pedagogical Strategy: A contribution to self-management of knowledge in Uniremington Systems Engineering / Estratégia Pedagógica PICUR: Uma contribuição para a autogestão do conhecimento na Engenharia de Sistemas Uniremington

Resumen Introducción: las estrategias y didácticas pedagógicas, se deberán convertir en principales motivantes para el desarrollo de las competencias que se esperan obtener del profesional en ingeniería. Objetivo: ejecutar prototipo de estrategia pedagógica, PICUR (Proyecto Integrador de la Corporación Universitaria Remington), que permita la integralidad de diferentes procesos de formación, para el logro de competencias transversales, específicas y multidisciplinares, que, desde la automotivación, innovación, investigación y creatividad, permitan dar soluciones a situaciones reales en diversos contextos. Materiales y métodos: para alcanzar la meta se propició la generación de condiciones iniciales, para la aplicación de PICUR en relación con recursos físicos, talento humano, actividades, tiempos y espacios, siendo necesario socializar la estrategia con diferentes actores, suministrando parámetros necesarios para su aplicación con el apoyo de guía de trabajo previamente elaborada. De igual forma se generó banco de ideas, para que los equipos de trabajo pudiesen tener acceso a estas para el desarrollo de los proyectos, acorde al nivel de competencias. Resultados: de la aplicación del prototipo PICUR y su respectiva evaluación, se obtuvo hallazgos como: importancia del trabajo en equipo, solución de retos aplicando software para beneficio social, integración de teoría y práctica, entre otros, que impactan positivamente el proceso de formación del ingeniero. Conclusiones: PICUR, como estrategia pedagógica, aplicable a cualquier pregrado, se plantea como una alternativa que permite la dinamización de los procesos académicos, donde el estudiante aparece como actor principal, con capacidad de desarrollar recursos para su autoformación y autoaprendizaje que lo proyectan a la generación de nuevo conocimiento.

Abstract Introduction: the pedagogical strategies and didactics must become the main motivators for the development of the competences that are expected to be obtained from the engineering professional. Objective: execute a prototype of the pedagogical strategy, PICUR (Integrative Project of the Remington University Corporation), which allows the integration of different training processes, for the achievement of transversal, specific and multidisciplinary competences, which from self-motivation, innovation, research and creativity , allow solutions to real situations in various contexts. Materials and methods: to achieve the goal, the generation of initial conditions was propitiated, for the application of PICUR in relation to physical resources, human talent, activities, times and spaces, being necessary to socialize the strategy with different actors, providing necessary parameters for its application with the support of previously developed work guide. In the same way, an idea bank was generated, so that the work teams could have access to them for the development of projects, according to the level of competencies. Results: from the application of the PICUR prototype and its respective evaluation, findings were obtained such as: importance of teamwork, solving challenges by applying software for social benefit, integration of theory and practice, among others, that positively impact the training process of the engineer. Conclusions: PICUR, as a pedagogical strategy, applicable to any undergraduate, is proposed as an alternative that allows the dynamization of academic processes, where the student appears as the main actor, with the ability to develop resources for self-training and self-learning that project him to the generation of new knowledge.

Resumo Introdução: as estratégias pedagógicas e didáticas devem se tornar os principais motivadores para o desenvolvimento das competências que se espera obter do profissional de engenharia. Objetivo: executar um protótipo da estratégia pedagógica, o PICUR (Projeto Integrativo da Remington University Corporation), que permite a integração de diferentes processos de treinamento, para a conquista de competências transversais, específicas e multidisciplinares, a partir de auto-motivação, inovação, pesquisa e criatividade, permita soluções para situações reais em vários contextos. Materiais e métodos: para atingir a meta, propiciou-se a geração de condições iniciais, para a aplicação do PICUR em relação a recursos físicos, talento humano, atividades, tempos e espaços, sendo necessário socializar a estratégia com diferentes atores, fornecendo parâmetros necessários para sua aplicação. aplicação com o apoio do guia de trabalho desenvolvido anteriormente. Da mesma forma, foi gerado um banco de ideias, para que as equipes de trabalho pudessem ter acesso a eles para o desenvolvimento de projetos, de acordo com o nível de competências. Resultados: a partir da aplicação do protótipo PICUR e de suas respectivas avaliações, obtiveram-se resultados como: importância do trabalho em equipe, solução de desafios através da aplicação de software para benefício social, integração de teoria e prática, entre outros, que impactam positivamente o processo de treinamento do engenheiro. Conclusões: O PICUR, como estratégia pedagógica, aplicável a qualquer graduação, é proposto como uma alternativa que permite a dinamização dos processos acadêmicos, onde o aluno aparece como ator principal, com capacidade de desenvolver recursos para auto-treinamento e auto-aprendizagem que o projetam para o geração de novos conhecimentos.

Prevalencia de partos en la adolescencia y factores asociados en el Hospital Universitario de Santander, Bucaramanga (Colombia) 2006: estudio de corte transversal / A cross-sectional study of childbirth prevalence in adolescents and associated factors in the Santander Teaching Hospital, Bucaramanga, Colombia, 2006

Introducción: el embarazo en la adolescencia es la gestación que ocurre entre los 10 y los 19 años de edad. En nuestro medio se ha convertido en un problema de salud pública, evidenciándose en este estudio un aumento de la prevalencia hasta superar las cifras locales y nacionales. Objetivo: determinar la prevalencia de partos y los factores asociados en madres adolescentes que ingresaron al Hospital Universitario de Santander (HUS) durante el año 2006-2007. Metodología: estudio de corte transversal de 273 mujeres seleccionadas de forma aleatoria sistemática que presentaron parto vaginal o por cesárea, en el HUS desde julio de 2006 hasta julio de 2007. Se aplicó una encuesta, donde se incluyeron variables de interés, (sociodemográficas, familiares, psicológicas); se completó la información con la historia clínica y se analizaron en el programa Epi Info® 2000 y Stata® 8.0. Resultados: la prevalencia de partos fue de 31,14% (IC95% 25,8-37,0) en pacientes menores de 20 años. Los factores protectores fueron: tener preocupación por el futuro (OR = 0,5), inicio tardío de relaciones sexuales (OR = 0,51) y no recibir educación sexual por parte de los padres (OR = 0,3). Los factores de riesgo fueron: no planear el embarazo (OR = 2,35) y no planificar (OR = 2,35). Conclusiones: la prevalencia encontrada es muy superior a la hallada en otros estudios locales, nacionales e internacionales.

Objective: adolescent pregnancy refers to gestation occurring between age 10 and 19; this has become a public health problem in Colombia. This study sought to determine the prevalence of births in teenagers admitted to the Santander Teaching Hospital (STH) over a year-long period (2006 and 2007) and the factors associated with the likelihood of teenage pregnancy for this region. Methodology: this was a cross-sectional study of 273 systematically and randomly selected females who underwent vaginal and caesarean births in the STH between July 2006 and July 2007. A survey was applied which included variables of interest (sociodemographic, family, psychological); the information was completed with the females’ clinical history and Epi InfoTM 2000 and StataTM 8.0 were used for analysing the data. Results: there was 31.14% (25.8-37.0 95%CI) prevalence for deliveries in patients aged less than 19. Factors disposing against teenage pregnancy included: being worried about the future (OR = 0.5), late onset of sexual activity (OR = 0.51) and not having received sexual education from parents (OR = 0.3). Risk factors were unplanned pregnancy (OR = 2.35) and not using contraceptive methods (OR = 2.35). Conclusions: the findings revealed a prevalence which was far higher than that found in similar local, national and international studies.

Purification and characterization of hexokinase from Leishmania mexicana.

Hexokinase from Leishmania mexicana was purified to homogeneity from a glycosome-enriched fraction obtained after a differential centrifugation of promastigote form. The kinetic properties of the pure enzyme were determined and the Km values for glucose (Km = 66 microM) and ATP (Km = 303 muM) were comparable to those from hexokinase of Trypanosoma cruzi. L. mexicana hexokinase was able to use fructose (Km = 142 microM), which reflects the condition found in the insect host. In contrast with hexokinases from other trypanosomatids, the enzyme exhibited a moderate sensitivity to inhibition by glucose 6-phosphate. This inhibition was competitive with respect to both ATP and glucose, indicating that an allosteric site for glucose 6-phosphate does not exist in this enzyme. The enzyme was also inhibited by inorganic pyrophosphate, the inhibition being higher than that observed for T. cruzi enzyme. As expected, the enzyme was localized, by immunofluorescence analysis, in glycosomes and is present in both promastigotes and true amastigotes obtained from hamster lesion. Hexokinase specific activity increased with the aging of promastigote culture, and this increment was related to glucose consumption. However, the level of the hexokinase protein remains constant as determined by Western blotting. Several hypotheses are discussed to explain this result.