¿Qué puede hacer la bioinformática para responder preguntas microbiológicas?

Motivation: Bioinformatics now makes it possible to analyze thousands of bacterial genomes and answer biological questions such as how many different genes we can associate with a species and how to discover gene markers linked to resistance and other bacterial defense systems.Methods: In this talk I showed how to perform bacterial pangenomes using several public and home-made bioinformatics tools, including the Sma3s gene annotator, or the Roary pangenome software.Results: With this methodology, we have analyzed groups of genomes of human pathogenic bacteria with CRISPR-Cas-acquired immunity systems and have discovered that they are highly specialized in phages, especially in variants called phagoplasmids. In addition, we have found that some of the genomes carrying these systems may have been forced to acquire them because they have genes that encode possible receptors for entry of specific phages

Use of bioinformatics tools to find new genes involved in rare diseases

Motivation: Rare diseases are a big challenge of our community and it is important to find answers and put forward a cure in the medicine field. Thanks to the huge amount of data that transcriptomic researches provide to public databases, we can use bioinformatics tools to analyse and seek new paths to understand better their molecular mechanisms and find new molecules that are related to the disease in order to make a future drug discovery process to this kind of research.Methods: We use transcriptomic data from the Expression Atlas repository, searching for experiments where the gene related to rare diseases is differently expressed.. We use the fold change data to choose those proteins that the expression are correlated to the expression of our gene of interest (R2>= 0.95). Using enrichment tools from Reactome database, or DAVID computational tool, we can stablish a Gene Ontology (GO) study among which we we can choose those that belong to the same biological process and path. This first step means an approach to select from thousands of genes a few gene cluster that may be highly related with the gene that cause the disease. The use of analysis tool R with bioinformatics packages, such as Bioconductor, CompGo, RDavidWebService or Clusterprofiler, allow us to keep improving the methodology making a deep analysis of Gene Ontology of our gene cluster, crafting relationships between them.Results: The current status of this research consists in the analysis of all GO terms that are belonged to our genes of interest that were crossed with the terms of the gene related to the studied disease. This step is crucial in order to find genes that are also affected by rare diseases in their metabolic path. This methodology could discover new biomarkers or, in another case, new strategies to understand the correct operation of the biological process of rare diseases and most importantly, the possibility to find a possible cure for these conditions

Search of phage-plasmids and analysis of their role in CRISPR-Cas mediated phage-bacteria interaction in Acinetobacter baumannii.

Motivation: The emergence of new antibiotic-resistant bacterial strains represents one of the top 10 global health threats, with A. baumannii as a species of special concern. The use of bacteriophages as a treatment for infectious diseases is a potential solution for this issue. Making this therapy a viable alternative requires a deep knowledge of interaction between phages and bacteria. Some special elements in these context are sequences known as phage-plasmids (PPs), wich carry sometimes defense system such as CRISPR-Cas.Methods: Identification of PPs was carried out by similarity searching between phage and plasmid sequences from two databases. Results obtained in this search were confirmed by using the phage prediction tool PHASTER, and comparison with previous studies. CRISPRCasTyper tool was used to search CRISPR arrays in sequences.Results: Results obtained from previous PPs studies were confirmed, also providing a larger quantity of new plasmids identified as PPs. It remains clear that PPs show a great diversity throughout a variety of bacteria species, being more represented in gram-negative species rather than gram-positive. Hence, we propose a method of identification of PPs elements. CRISPR-Cas arrays were found in a small quantity of identified PPs, although none were found in PPs from Acinetobacter baumannii. Analysis of protospacers of PPs in A. baumannii show that they are targeted by CRISPR-Cas defence system, finding a higher percentage of protospacers identified as IF-a1 type in PPs, comparing with simple phages or plasmids.Conclusions: The results obtained confirm the idea that PPs have a prominent presence throughout strains of different bacteria species. The protocol proposed in this work provides a fast way of finding new phage-plasmid when databases information is updated. Previous studies reported the presence of CRISPR-Cas defence systems in a phage-plasmid from A. baumannii. However, no CRISPR arrays were found in the present work in that species. Future searches applying this method with actualized databases may find the presence of CRISPR arrays in A. baumannii PPs. The higher presence of IF-a1 in PPs than in other elements suggest a specialization of this type of CRISPR array on PPs. Given that IF-a1 arrays have been found in phages and PPs, these results show some insight into the process of competition between phages

Src function in a unicellular relative of metazoans

Motivación: Tyrosine kinases play important roles in different cell signaling cascades in metazoans (1). The non-receptor Src family has been found to be involved in the regulation of cell-cell interaction, cell adhesion and migration (2). For this reason, its malfunctioning is implicated in diseases, such as cancer. Tyrosine kinases were thought to be specific to Metazoa, but recent comparative genomic analyses showed that they are also present in some of the closest unicellular relatives of metazoans (3). The role played by these kinases in these unicellular organisms remains unknown. The goal of this project is to decipher the function of Src in unicellular organisms.Métodos: The src gene was predicted in the genome of Creolimax fragrantissima. The gene was cloned from cDNA by PCR. A dominant negative (DN) version of the gene was designed introducing an amino acid change in the two conserved Tyr that are important for the regulation of the protein, blocking the downstream cascade (4). Both the wild-type version  and DN were cloned in a vector under the beta-tubulin promoter (b-tub) with constitutive expression, and fused to the mCherry fluorescent marker. Transformation was done by electroporation using the Neon® transfection system by Invitrogen (1μg each vector). The experiment consisted on transforming C. fragrantissima with either the normal Src or the DN  constructs plus b-tub::venus and b-tub::H2B-mCherry as controls for the transformation. Negative control had the two lasts constructs but none of the src constructs. The cells were examined using a Nikon fluorescence microscope, and time-lapse was performed when needed.Resultados: We know that the constitutive expression of src affects cell-cell interaction in cell culture experiments. C.fragrantissima has a colonial stage during its cell cycle that “explodes” giving rise to free-living amoebas (see http://www.youtube.com/watch?v=7Gvrg1I8jBA). We assume that cell-cell interaction may have some relevance during this stage. For this reason we expected to see a resulting phenotype either during the colony formation or during its development. The preliminary results showed some different phenotypes although they are not significant enough. Experiments on transformation and time-lapse need to be repeated, in order to be sure that differences are found between the overexpressed and the DN src transformants, in the colonies or amoebas

Búsqueda de reguladores de ciclo celular en Schizosaccharomyces pombe

En el presente trabajo se llevó a cabo un escrutinio genético de sinteticidad letal con el objetivo de encontrar nuevos reguladores de ciclo celular que coordinasen crecimiento y división en el organismo modelo Schizosaccharomyces pombe. Para ello se realizaron cruces entre una colección de mutantes nulos viables y el mutante termosensible wee1.50, un mutante que a 36ºC da lugar a un fenotipo wee, caracterizadas por una fase G2 de mitosis más corta, entrando en ella con menor tamaño que la cepa silvestre. Al comparar el crecimiento de estos dobles mutantes con la cepa silvestre se asignó una puntuación a cada doble mutante. De esta forma, se identificaron mutantes que presentaban muerte celular (sintéticos letales), otros con un fenotipo enfermo o sick y otros que suprimían el fenotipo de wee1.50 a 360C. Mediante el análisis de las anotaciones GO (Gene Ontology) se clasificaron a todos los mutantes en diferentes categorías funcionales y se realizó un estudio combinado de morfología y citología, dinámica de ciclo celular y sensibilidad a distintos fármacos.Wee1 es una proteína quinasa que inhibe la entrada en mitosis por fosforilación de Cdc2 en un residuo tirosina localizado en  el sitio de unión del ATP. Cdc2 es la proteína quinasa que junto con la ciclina Cdc13, induce la entrada en mitosis mediante la fosforilación de sus sustratos mitóticos.Uno de los objetivos de este trabajo ha sido la caracterización de tres mutantes cuya función es desconocida o poco estudiada en S. pombe: dml1Δ y pi040Δ, son mutantes sintéticos letales con wee1.50, y zds1Δ suprime el fenotipo de células pequeñas de wee1.50. Además, también se va continuar el estudio de Rnc1, que es una proteína de unión a ARN cuya función es estabilizar mensajeros de proteínas de la ruta de las MAP quinasas, una importante ruta de señalización intracelular. El mutante rnc1Δ también pertenece al grupo de los sintéticos letales con wee1.50.Los primeros pasos del proyecto han consistido en rehacer estos mutantes mediante transformaciones, cruzarlos para obtener los dobles mutantes con wee1.50, comprobar el fenotipo del doble mutante y caracterizar el fenotipo de los mutantes sencillos. Para caracterizar el fenotipo de los mutantes se emplean técnicas que nos permiten determinar el contenido en ADN (citometría de flujo), la morfología celular (tinciones con DAPI y calcoflúor) y ensayos de viabilidad con diluciones en gotas en diferentes condiciones de medios y temperaturas. El objetivo final es encontrar cuál es su función y dentro de qué ruta bioquímica participa, así como estudiar si son importantes para la regulación del ciclo celular y, por tanto, susceptibles de estudios más avanzados como dianas terapéuticas en cáncer

Nutritional improvement of Manihot esculenta roots by boosting the lipids storage

Motivation: The human population continues to grow and it is necessary to produce more and better quality food to meet the world population's demand. Genetic engineering opens great possibilities to improve the quantity of available food. The cassava (Manihot esculenta) plant is the basis of food for more than 1 billion people in the world. All plants have genes coding for oil biosynthetic pathways and transcription factors that activate the expression of these genes. If these transcription factors are activated in other tissues, like roots, the conversion of sucrose to oil could increase. In this way, crops that accumulate sugars and starch could become more nutritious (2.2 times more energy than carbohydrates). It has been shown that starch and oil can accumulate in the same cell, as is the case for oats (Ekman et al., 2008). In this project, cassava has been chosen as a model plant because it has a high starch content in their edible roots.Methods: Two somatic embryos were obtained from mother plants with ecotype 60444. The in-vitro plants will be transformed by the vector via Agrobacterium tumefaciens. The functional annotation of the cassava proteome was carried out using Sma3s (Casimiro-Soriguer et al., 2017). This annotation will allow us to know the function of the protein-coding genes present in cassava. To know those genes involved in the synthesis of fatty acids they must be filtered. The expression of these genes in different tissues was comparated with ArrayExpress. Posible candidates will be examined in order to choose the most suitable ones to be transformed and expressed in the cassava plants.Results: From 41,381 cassava predicted proteins, 35,889 were scored, meaning Sma3s annotated 86% of the proteome. The list of possible candidates is currently around 600 genes and their expression wilI checked with public database. In vitro plants are growing and the second phase of the transformation will be begun.Conclusions: The project will (i) expand knowledge on cassava, particularly on the development of their storage organs, such as roots and seeds, as well as carbohydrate and lipid metabolism, and (ii) develop a cassava crop modification platform using genetic engineering techniques. This work aims to cover two demands of society, try to mitigate hunger, and on the other hand be able to extrapolate the scientific knowledge generated to other crops of interest to cover the current demand for vegetable oil

Análisis del efecto de las modificaciones post-traduccionales inducidas por óxido nítrico en células troncales embrionarias de ratón.

Motivación: La nitrosilación es un tipo de modificación postraduccional de las proteínas derivada de la presencia de óxido nítrico (NO) en el medio, y cuya acción se centra en residuos de tirosina y cisteína. Sin embargo, no se sabe con certeza como afectan estas modificaciones a la expresión génica en células embrionarias cuando se ven afectados factores de transcripción, histonas o determinados complejos proteicos. De esta forma, podría jugar un papel relevante en procesos de mantenimiento de pluripotencia o de diferenciación celular.En este proyecto se pretende analizar la influencia a nivel genómico de este patrón de expresión diferencial, para así discenir el papel que desempeña la nitrosilación en los mecanismos de regulación transcripcional de células embrionarias. Métodos: Se realizó un ensayo ChIP on chip, para visualizar qué regiones génicas se ven afectadas por el NO en forma de sus dos modificaciones principales (Schnetz MP 2010). Este ensayo permite determinar qué regiones génicas se han visto afectadas por factores de transcripción modificados respecto a una muestra control. Con los resultados de este ensayo, se han iniciado diferentes procedimientos bioinformáticos para la normalización, cribado y visualización de las regiones a las que se unen factores de transcripción nitrosilados, así como identificación de motivos modificados y factores implicados en el proceso. Resultados: Resultados preeliminares demostraron que las proteínas nitrosiladas en residuos de cisteína ocupan regiones reguladoras de genes implicados en procesos de autorenovación y diferenciación celular. Posteriormente, se ha procedido al análisis de genes concretos implicados, motivos y factores de transcripción afectados. Conclusiones: Estamos a la espera de obtener resultados definitivos para realizar las valoraciones oportunas

In vitro characterization of solute transport in the spinal canal

This paper presents results of an experimental investigation of solute transport in a simplified model of the spinal canal. The work aims to provide increased understanding of the mechanisms responsible for drug dispersion in intrathecal drug delivery (ITDD) procedures. The model consists of an annular channel bounded externally by a rigid transparent tube of circular section, representing the dura mater, and internally by an eccentric cylindrical compliant insert, representing the spinal cord. The tube, closed at one end, is connected to a rigid acrylic reservoir, representing the cranial cavity. The system is filled with water, whose properties are almost identical to those of the cerebrospinal fluid. A programmable peristaltic pump is employed to generate oscillatory motion at frequencies that are representative of those induced by the cardiac and respiratory cycles. Laser induced fluorescence is used to characterize the dispersion of fluorescent dye along the canal and into the cranial cavity for different values of the relevant Womersley number and different eccentricities of the annular section. The present work corroborates experimentally, for the first time, the existence of a steady bulk flow, associated with the mean Lagrangian motion, which plays a key role in the transport of the solute along the spinal canal. The measurements of solute dispersion are found to be in excellent agreement with theoretical predictions obtained using a simplified transport equation derived earlier on the basis of a two-timescale asymptotic analysis. The experimental results underscore the importance of the eccentricity and its variations along the canal and identifies changes in the flow topology associated with differences in the Womersley number, with potential implications in guiding future designs of ITDD protocols.This work was supported by the coordinated project, PID2020-115961RB-C31, PID2020-115961RB-C32, and PID2020-115961RA-C33, financed by MCIN/AEI/10.13039/501100011033, and by the Junta de Andalucia and European Funds, Project No. P18-FR-4619. F. Moral-Pulido wants to thank the Spanish Ministry of Universities for the financial support provided by the Fellowship FPU18/05694. The work of A. L. Sánchez was supported by the U.S. National Science Foundation through Grant No. 1853954

Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation

Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited neurologic disorders in which iron accumulates in the basal ganglia resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration. The most prevalent form of NBIA is pantothenate kinase-associated neurodegeneration (PKAN) associated with mutations in the gene of pantothenate kinase 2 (PANK2), which is essential for coenzyme A (CoA) synthesis. There is no cure for NBIA nor is there a standard course of treatment. In the current work, we describe that fibroblasts derived from patients harbouring PANK2 mutations can reproduce many of the cellular pathological alterations found in the disease, such as intracellular iron and lipofuscin accumulation, increased oxidative stress, and mitochondrial dysfunction. Furthermore, mutant fibroblasts showed a characteristic senescent morphology. Treatment with pantothenate, the PANK2 enzyme substrate, was able to correct all pathological alterations in responder mutant fibroblasts with residual PANK2 enzyme expression. However, pantothenate had no effect on mutant fibroblasts with truncated/incomplete protein expression. The positive effect of pantothenate in particular mutations was also confirmed in induced neurons obtained by direct reprograming of mutant fibroblasts. Our results suggest that pantothenate treatment can stabilize the expression levels of PANK2 in selected mutations. These results encourage us to propose our screening model as a quick and easy way to detect pantothenate-responder patients with PANK2 mutations. The existence of residual enzyme expression in some affected individuals raises the possibility of treatment using high dose of pantothenate.Instituto de Salud Carlos III FIS PI16/00786Junta de Andalucía CTS-5725, BIO-122Dirección General de Investigación Científica y Técnica BFU2015-64536-

Análisis de Costes y de Coste/Eficacia de las Pautas Recomendadas por GESIDA/Plan Nacional sobre el Sida en 2018 Para el Tratamiento Antirretroviral Inicial en Adultos Infectados Por el VIH

[Background]: The GESIDA/National AIDS Plan expert panel recommended preferred regimens (PR), alternative regimens (AR) and other regimens (OR) for antiretroviral treatment (ART) as initial therapy in HIV-infected patients for 2018. The objective of this study was to evaluate the costs and the efficiency of initiating treatment with PR and AR. [Methods]: Economic assessment of costs and efficiency (cost-effectiveness) based on decision tree analyses. Effectiveness was defined as the probability of reporting a viral load <50 copies/mL at week 48, in an intention-to-treat analysis. Cost of initiating treatment with an ART regimen was defined as the costs of ART and its consequences (adverse effects, changes of ART regimen, and drug-resistance studies) over the first 48 weeks. The payer perspective (National Health System) was applied considering only differential direct costs: ART (official prices), management of adverse effects, studies of resistance, and HLA B*5701 testing. The setting was Spain and the costs correspond to those of 2018. A deterministic sensitivity analysis was conducted, building three scenarios for each regimen: base case, most favourable and least favourable. [Results]: In the base-case scenario, the cost of initiating treatment ranges from 6788 euros for TAF/FTC/RPV (AR) to 10,649 euros for TAF/FTC + RAL (PR). The effectiveness varies from 0.82 for TAF/FTC + DRV/r (AR) to 0.91 for TAF/FTC + DTG (PR). The efficiency, in terms of cost-effectiveness, ranges from 7814 to 12,412 euros per responder at 48 weeks, for ABC/3TC/DTG (PR) and TAF/FTC + RAL (PR), respectively. [Conclusion]: Considering ART official prices, the most efficient regimen was ABC/3TC/DTG (PR), followed by TAF/FTC/RPV (AR) and TAF/FTC/EVG/COBI (AR).[Introducción]: El panel de expertos de GESIDA/Plan Nacional del Sida ha recomendado pautas preferentes (PP), pautas alternativas (PA) y otras pautas (OP) para el tratamiento antirretroviral (TAR) como terapia de inicio en pacientes infectados por VIH para 2018. El objetivo de este estudio es evaluar los costes y la eficiencia de iniciar tratamiento con PP y PA. [Métodos]: Evaluación económica de costes y eficiencia (coste/eficacia) mediante construcción de árboles de decisión. Se definió eficacia como la probabilidad de tener carga viral <50 copias/ml en la semana 48 en análisis por intención de tratar. Se definió coste de iniciar tratamiento con una pauta como los costes del TAR y de todas sus consecuencias (efectos adversos, cambios de pauta y estudio de resistencias) que se producen en las siguientes 48 semanas. Se utilizó la perspectiva del Sistema Nacional de Salud, considerando solo costes directos diferenciales: TAR (a precio oficial), manejo de efectos adversos, estudios de resistencias y determinación de HLA-B*5701. El ámbito es España, con costes de 2018. Se realizó un análisis de sensibilidad determinista construyendo 3 escenarios para cada pauta: basal, más favorable y más desfavorable. [Resultados]: En el escenario basal, los costes de iniciar tratamiento oscilaron entre 6.788 para TAF/FTC/RPV (PA) y 10.649 para TAF/FTC + RAL (PP). La eficacia osciló entre 0,82 para TAF/FTC + DRV/r (PA) y 0,91 para TAF/FTC + DTG (PP). La eficiencia, en términos de coste/eficacia, osciló entre 7.814 y 12.412 por respondedor a las 48 semanas, para ABC/3TC/DTG (PP) y TAF/FTC + RAL (PP), respectivamente. [Conclusión]: Considerando el precio oficial del TAR, la pauta más eficiente fue ABC/3TC/DTG (PP), seguida de TAF/FTC/RPV (PA) y TAF/FTC/EVG/COBI (PA)