Comparing Efficiency of Lysis Buffer Solutions and Sample Preparation Methods for Liquid Chromatography–Mass Spectrometry Analysis of Human Cells and Plasma

The use of a proper sample processing methodology for maximum proteome coverage and high-quality quantitative data is an important choice to make before initiating a liquid chromatography–mass spectrometry (LC–MS)-based proteomics study. Popular sample processing workflows for proteomics involve in-solution proteome digestion and single-pot, solid-phaseenhanced sample preparation (SP3). We tested them on both HeLa cells and human plasma samples, using lysis buffers containing SDS, or guanidinium hydrochloride. We also studied the effect of using commercially available depletion mini spin columns before SP3, to increase proteome coverage in human plasma samples. Our results show that the SP3 protocol, using either buffer, achieves the highest number of quantified proteins in both the HeLa cells and plasma samples. Moreover, the use of depletion mini spin columns before SP3 results in a two-fold increase of quantified plasma proteins. With additional fractionation, we quantified nearly 1400 proteins, and examined lower-abundance proteins involved in neurodegenerative pathways and mitochondrial metabolism. Therefore, we recommend the use of the SP3 methodology for biological sample processing, including those after depletion of high-abundance plasma proteins.Research Council of Norway 29591

Análisis macroeconómico del mercado de trabajo

Fil: Salvador, Pablo F.. Universidad Nacional de Cuyo. Facultad de Ciencias EconómicasFil: Valladares, María Victoria. Universidad Nacional de Cuyo. Facultad de Ciencias Económica

A developmental role for the chromatin‑regulating CoREST complex in the cnidarian Nematostella vectensis

Background: Chromatin-modifying proteins are key players in the regulation of development and cell differentiation in animals. Most chromatin modifiers, however, predate the evolution of animal multicellularity, and how they gained new functions and became integrated into the regulatory networks underlying development is unclear. One way this may occur is the evolution of new scaffolding proteins that integrate multiple chromatin regulators into larger complexes that facilitate coordinated deposition or removal of different chromatin modifications. We test this hypothesis by analyzing the evolution of the CoREST-Lsd1-HDAC complex. Results: Using phylogenetic analyses, we show that a bona fide CoREST homolog is found only in choanoflagellates and animals. We then use the sea anemone Nematostella vectensis as a model for early branching metazoans and identify a conserved CoREST complex by immunoprecipitation and mass spectrometry of an endogenously tagged Lsd1 allele. In addition to CoREST, Lsd1 and HDAC1/2 this complex contains homologs of HMG20A/B and PHF21A, two subunits that have previously only been identified in mammalian CoREST complexes. NvCoREST expression overlaps fully with that of NvLsd1 throughout development, with higher levels in differentiated neural cells. NvCoREST mutants, generated using CRISPR-Cas9, fail to develop beyond the primary polyp stage, thereby revealing essential roles during development and for the differentiation of cnidocytes that phenocopy NvLsd1 mutants. We also show that this requirement is cell autonomous using a cell-type-specific rescue approach. Conclusions: The identification of a Nematostella CoREST-Lsd1-HDAC1/2 complex, its similarity in composition with the vertebrate complex, and the near-identical expression patterns and mutant phenotypes of NvCoREST and NvLsd1 suggest that the complex was present before the last common cnidarian-bilaterian ancestor and thus represents an ancient component of the animal developmental toolkit.University of BergenResearch Council of Norway 251185/F20Sars Centre core budgetResearch Council of Norway INFRASTRUKTUR program 29591

La teorización y las técnicas participativas del proceso dialéctico en la educación popular

La educación popular asume una innegable influencia acorde a sus técnicas participativas y de teorización en el actual panorama educativo, donde se llevan a a cabo prácticas únicamente teorizantes y no de participación. Actualmente la educación popular pasa por un proceso de reconceptualización, es decir, un reconocimiento de elementos teóricos y prácticos llevados a cabo por los educadores populares en sus modelos de acción. El artículo precisa componentes básicos como la educación popular, el proceso dialéctico y sus elementos teorizantes y participativos, tomando en cuenta a diferentes autores que se han especializado en educación popular y el proceso dialéctico en la conformación de un aprendizaje didáctico aportado por Paulo FreirePopular education takes over an undeniable influence in agreement with its participative and theorization techniques in the current education overview, where only theorist’s practices are carried out and without participation. At the moment popular education is currently through a process of reconceptualization, ergo, an acknowledgment of theoretical and practical elements carried out by the popular educators in their proceeding models. We will try to precise basic components such as popular education, the dialectic process and its theorists and participative elements, bearing in mind various authors which have specialized in popular education and the dialectic process through the creation of a didactical apprenticeship adduced by Paulo Freir

Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of fasciolosis in sheep and its application under field conditions

[EN] Background: Loop-mediated isothermal amplification (LAMP) is a very specific, efficient, and rapid gene amplification procedure in which the reaction can run at a constant temperature. In the current study we have developed a LAMP assay to improve the diagnosis of Fasciola spp. in the faeces of sheep. Findings: After the optimisation of the LAMP assay we have shown similar results between this technique and the standard PCR using the outer primers of the LAMP reaction. In both cases the limit of detection was 10 pg; also, the diagnosis of fasciolosis was confirmed during the first week post-infection in experimental infected sheep by both techniques. In eight naturally infected sheep, the infection with F. hepatica was confirmed in all animals before a treatment with triclabendazole and on day 30 post treatment in two sheep using the LAMP assay; however, when we carried out the standard PCR with the outer primers, the results before treatment were the same but on day 30 post-treatment the infection was only confirmed in one out of the two sheep. On the other hand, the standard PCR took around 3 h to obtain a result, comparing with 1 h and 10 min for the LAMP assay. Conclusions: The LAMP assay described here could be a good alternative to conventional diagnostic methods to detect F. hepatica in faeces since it solves the drawbacks of the standard PCRSIWe thank Drs Manga-González and González-Lanza (Instituto de Ganadería de Montaña, CSIC-ULE, Spain) for providing DNA samples of D. dendriticum and C. daubneyi. This study has been funded by the national project INIA (Instituto Nacional de Investigaciones Agrarias: RTA2010-00094-C03-02), Ministry of Economy and Competitiveness (Ministerio de Economia y Competitividad), and also co-funded by the European Regional Development Fund (Fondos Feder

Análisis comparativo de la gestión de cuatro áreas litorales protegidas en Puerto Rico

A lo largo de este trabajo, se realiza un análisis de la gestión llevada a cabo en cuatro áreas marinas protegidas de Puerto Rico. Se estudian cuatro de las nueve categorías totales existentes en Puerto Rico: Programa Nacional Estuarino, Reserva Marina, Reserva Natural de Investigación Estuarina y Reserva Natural, que corresponden respectivamente al P.N.E. Estuario de la Bahía de San Juan, R.M. Tres Palmas, R.N.I.E. Bahía de Jobos y R.N. Cabezas de San Juan. Cada una de estas áreas es administrada por diferentes instituciones a nivel federal (EPA), estatal (DRNA), mixto (NOAA y DRNA) y privado (Fideicomiso de Conservación). El presente trabajo hace hincapié en los diferentes enfoques de gestión que utilizan las administraciones anteriormente citadas, además de tratar de estudiar cómo se manejan estas áreas teniendo en cuenta los diferentes ecosistemas que las caracterizan. Para ello, se utiliza la metodología del Decálogo ideada por Barragán (2001), realizando un diagnóstico mediante un análisis de diez aspectos de la gestión. Finalmente, se desarrolla un análisis DAFO y un análisis CAME, que ayudará a esquematizar la situación actual de dicha gestión.Máster Gestión Integrada de Áreas Litorales Universidad de Cádiz, Facultad de Ciencias del Mar Universidad de Puerto Rico, Recinto Ciencias Médicas Beca Fórmula SantanderNúmero de páginas: 9

ZipN is an essential FtsZ membrane tether and contributes to the septal localization of SepJ in the flamentous cyanobacterium Anabaena

The organismic unit of heterocyst-forming cyanobacteria is a filament of communicating cells connected by septal junctions, proteinaceous structures bridging the cytoplasms of contiguous cells. This distinct bacterial organization is preserved during cell division. In Anabaena, deletion of the zipN gene could not be segregated. We generated strain CSL109 that expresses zipN from a synthetic regulatable promoter. Under conditions of ZipN depletion, cells progressively enlarged, reflecting restricted cell division, and showed drastic morphological alterations including cell detachment from the filaments, to finish lysing. In contrast to the wild-type localization in midcell Z-rings, FtsZ was found in delocalized aggregates in strain CSL109. Consistently, the proportion of membrane-associated to soluble FtsZ in fractionated cell extracts was lower in CSL109. Bacterial two-hybrid analysis showed that ZipN interacts with FtsZ and other cell-division proteins including cytoplasmic Ftn6 and SepF, and polytopic FtsW, FtsX, FtsQ and FtsI. Additionally, ZipN interacted with the septal protein SepJ, and in CSL109 depletion of ZipN was concomitant with a progressive loss of septal specificity of SepJ. Thus, in Anabaena ZipN represents an essential FtsZ membrane tether and an organizer of the divisome, and it contributes to the conformation of septal structures for filament integrity and intercellular communication.Agencia Estatal de Investigación BFU2013-44686-P, BFU2016-77097-

Mass Spectrometry-Based Proteomics Workflows in Cancer Research: The Relevance of Choosing the Right Steps

The qualitative and quantitative evaluation of proteome changes that condition cancer development can be achieved with liquid chromatography–mass spectrometry (LC-MS). LC-MSbased proteomics strategies are carried out according to predesigned workflows that comprise several steps such as sample selection, sample processing including labeling, MS acquisition methods, statistical treatment, and bioinformatics to understand the biological meaning of the findings and set predictive classifiers. As the choice of best options might not be straightforward, we herein review and assess past and current proteomics approaches for the discovery of new cancer biomarkers. Moreover, we review major bioinformatics tools for interpreting and visualizing proteomics results and suggest the most popular machine learning techniques for the selection of predictive biomarkers. Finally, we consider the approximation of proteomics strategies for clinical diagnosis and prognosis by discussing current barriers and proposals to circumvent them.Research Council of Norway INFRASTRUKTUR-program (project number: 295910

Vacuolar ATPase Is a Possible Therapeutic Target in Acute Myeloid Leukemia: Focus on Patient Heterogeneity and Treatment Toxicity

This work was supported by Kreftforeningen, the Norwegian Cancer Society (grant no. 100933). The Genomics Core Facility (GCF) is supported in part by major grants from the Research Council of Norway (grant no. 245979/F50) and Bergen Research Foundation. The GCF at the University of Bergen, which is part of the NorSeq consortium, provided support in ChIP-Seq bioinformatics analysis.Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jcm12175546/s1Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.Kreftforeningen, the Norwegian Cancer Society 100933Research Council of Norway 245979/F50Bergen Research FoundationNorSeq consortium, University of Berge

La teoría del apego para favorecer el aprendizaje cognitivo a partir del protagonismo a padres tutores y docentes que acompañan a los niños y niñas de 5 años del grupo de párvulos en el proceso educativo.

Aporta instrucciones a padres, tutores y docentes sobre la influencia que tiene el apego en el aprendizaje cognitivo de los niños y niñas parvularios/as. Explicó cómo la disolución entre el afecto y el aprendizaje conlleva a la negligencia y acciones antipedagógicas. Estimuló el área afectiva de los padres, tutores y docentes para aplicar el afecto en el proceso de aprendizaje cognitivo, promover el afecto y la armonía relacional entre padres, tutores y docentes para favorecer el aprendizaje y desarrollo infantil y plantear situaciones afectivas adecuadas en la labor paterna y docente para el acompañamiento académico en el aprendizaje cognitivo. El estudio se llevó a cabo con padres, madres y tutores de niños de párvulos, de la de la Escuela Oficial de Párvulos No. 68., del municipio de Chinautla del Departamento de Guatemala. Para recopilar información se utilizó las técnicas siguientes: cuestionario y observación. Se utilizó como instrumento el cuestionario. Se realizó el análisis de los resultados y se concluyó que, el desconocimiento del vínculo existente entre al afecto y el aprendizaje por parte de los significativos del infante, puede inducirles a incurrir en acciones contraproducentes tales como: castigos, maltrato, negligencia; abusos psicológicos, emocionales y aún físicos en contra de los preescolares. Los padres, madres, tutores y maestras reconocen que no se ha procedido como deberían, con paciencia y afecto, dentro del proceso educativo preescolar; algunos/as por traumas o experiencias negativas de su propia infancia. Sugiere que, como parte de la preparación y formación de las maestras preescolares, de los padres, madres y tutores, se les de capacitación con respecto al vínculo existente entre el afecto y proceso escolar