Analysis of chromatin accessibility to characterize transcription factor activities in malignancies

In the context of the study of human disorders, exploring the chromatin accessibility applying the Assay for Transposase Accessible Chromatin followed by sequencing (ATAC-seq) in combination with the study of certain histone markers via chromatin-immunoprecipita-tion followed by sequencing (ChIP-seq) has proven to be a robust approach. It allows to unravel essential transcriptional regulatory circuitry, as well as to identify genes or genetic pathways, not necessarily presenting mutations, but relevant for any aspect of the disorder, from disease progression to relapse, or even the appearance of therapy resistance. In the course of this work, three different human disorders were investigated. Our main objective was the study of Acute Myeloid Leukemia (AML), for which both experiments, ATAC-seq and ChIP-seq of the acetylation of lysine 27 of histone H3 (H3K27ac - a marker for active enhancers and super-enhancers) were performed. AML is a hematopoietic malignancy characterized by a broad genetic heterogeneity and the frequent occurrence of relapses, which compromises patient survival. In collaboration with other research groups, we additionally explored on the one hand, the mechanisms involved in the emergence of human breast cancer metastasis, and on the other hand, the response of the immune system in the context of severe autoimmune diseases using a mouse model commonly employed in the investigation of human multiple sclerosis. To deeply cover all relevant aspects of the AML disease concerning our main goal, I studied AML cell lines as in vitro model, an AML patient-derived xenograft (PDX) mouse model as in vivo model and, most importantly, paired patient-derived samples at the stages of primary diagnosis and relapse. The analysis of patient samples was of great interest to capture the nature of the disorder in its whole complexity, free of the limitations intrinsic to the two aforementioned models. The epigenetic landscape in terms of chromatin accessibility and enhancers was characterized based on 18 leukemic cell lines. They exhibited, as expected, a vast heterogeneity, setting the basis for an in vitro model to work with and validate hypothesis derived from the study of human samples. To explore the effects caused by the standard therapy over time in vivo we used PDX samples. Xenografted mice were subjected to several cycles of therapy. The derived samples were processed at three different time points to analyze the development of chromatin accessibility. Our experiments showed that at the beginning of the treatment chromatin changes barely occurred but as the therapy progressed, the effects accumulated, causing chromatin to significantly open up. The exploration and analysis performed on paired patient-derived samples clearly revealed a larger similarity with regard to the epigenetic landscape for the relapse stage among all patients analyzed. Especially apoptotic-related pathways, although primarily nonexistent in diagnosis, appeared to be commonly active in relapse. Nevertheless, due to the heterogeneity characterizing this disorder, no common pathways or genes could be identified for the whole patient cohort. Notably, for a patient subgroup, we could determine a bunch of relapse-associated relevant genes, some of which have been recently documented in the literature as playing an important role in the disease progression and even in therapy resistance, thus validating our approach.Im Rahmen der Erforschung menschlicher Erkrankungen, die Untersuchung der Zugänglichkeit des Chromatins mittels der Assay for Transposase Accessible Chromatin gefolgt von Sequenzierung (ATAC-seq) kombiniert mit der Untersuchung bestimmte Histonmodifikationen durch Chromatin Immunoprecipitation gefolgt von Sequenzierung (ChIP-seq) hat sich als robuster Ansatz erwiesen. Diese Strategie ermöglicht sowohl wesentliche transkriptionelle Regulationsschaltkreise als auch Gene oder genetische Pfade zu identifizieren. Diese weisen nicht unbedingt Mutationen auf, sind aber für manche Aspekte der Erkrankung relevant: von der Krankheitsprogression bis zum Rezidiv oder sogar dem Auftreten von Therapieresistenz. In dieser Arbeit wurden drei verschiedene Erkrankungen untersucht. Unser Hauptziel war dabei die Untersuchung der menschlichen Akuten Myeloischen Leukämie (AML) und dafür führte ich sowohl ATAC-seq als auch ChIP-seq für Acetylierung des Lysins 27 am Histon H3 (H3K27ac - ein Marker für aktive Enhancers und Super-enhancers) durch. AML ist eine hämatopoetische Malignität, die durch eine breite genetische Heterogenität und das häufige Auftreten von Rezidiven gekennzeichnet ist, wodurch das Überleben der Patienten beeinträchtigt wird. In Zusammenarbeit mit anderen Forschungsgruppen haben wir zwei zusätzliche menschliche Erkrankungen untersucht. Zum einen waren es die Mechanismen, die an der Entstehung von Brustkrebsmetastasen beteiligt sind. Zum anderen, die Reaktion des Immunsystems im Kontext schwerer Autoimmunerkrankungen. Dafür wurde ein Mausmodell angewendet, das häufig für die Erforschung menschlichen Multiplen Sklerose benutzt wird. Um alle relevanten Aspekte der AML umfassend abzudecken, untersuchte ich drei verschiedene Modelle: erstens, AML-Zelllinien als in-vitro-Modell; zweitens, ein von AML-Patienten abgeleitetes Xenograft (PDX)-Mausmodell als in vivo-Modell; und drittens, gepaarte Patientenproben in den Stadien der Erstdiagnose und des Rezidives. Die Analyse der Patientenproben war vom großen Interesse um die Natur der Störung in ihrer ganzen Komplexität zu erfassen, frei von den Einschränkungen, die in den zuvor genannten Modellen implizit enthalten sind. Die epigenetische Landschaft bezüglich der Chromatinzugänglichkeit und der Enhancer wurden an 18 Leukemie-Zelllinien charakterisiert. Diese Zelllinien wiesen erwartungsgemäß eine große Heterogenität auf. Das bildete die Grundlage für das in vitro-Modell, mit dem Hypothesen validiert werden können, die aus der Untersuchung menschlicher Proben abgeleitet worden sind. Mithilfe von PDX-Proben wurden die Auswirkungen der Standardtherapie über die Zeit in vivo untersucht. Dafür wurden xenotransplantierte Mäuse mehreren Therapiezyklen unterzogen. Zu bestimmten Zeitpunkten wurden die von Mäusen abgeleiteten Proben verarbeitet, um die Entwicklung der Chromatinzugänglichkeit zu analysieren. Die Ergebnisse der Experimente haben gezeigt, dass zu Beginn der Behandlung kaum Chromatinveränderungen auftraten, sich jedoch im Verlauf der Therapie die Wirkungen akkumulierten, wodurch sich das Chromatin deutlich öffnete. Die Analyse von gepaarten Patientenproben zeigte eine deutliche Ähnlichkeit in Bezug auf die epigenetische Landschaft für das Rezidivstadium bei allen analysierten Patienten. Insbesondere schienen apoptotisch bedingte Signalwege beim Rezidiv häufig aktiv zu sein, obwohl sie in der Diagnose nicht vorhanden waren. Jedoch aufgrund der Heterogenität, die diese Erkrankung charakterisiert, konnten für die gesamte Patientenkohorte keine gemeinsamen Signalwege oder Gene bestimmt werden. Nichtsdestotrotz konnten wir für eine Patientenuntergruppe eine Reihe von rezidivassoziierten relevanten Genen identifizieren, wobei einige von denen vor kurzem in der Literatur als wichtig für die Krankheitsprogression und auch für die Therapieresistenz dokumentiert wurden. Dadurch ist unser Ansatz grundsätzlich validiert

Effect of Different Technological Factors on the Gelation of a Low-Lectin Bean Protein Isolate

Gelling ability of a bean protein isolate (BPI) obtained from a naturally low-lectin variety (Phaseolus vulgaris var. Almonga) was analysed. For that purpose differences on gels processing: concentration (14% and 17%), salt addition (0 and 2%), and pH (6.5 –lot A- and 7 –lot B), were studied to obtain suitable colour, mechanical and viscoelastic properties for making appropriate meat and seafood analogues. Gelation at pH 7 at both 14 and 17% BPI concentrations, produced less rigid, more flexible, time-stable and cohesive gel networks. Colour of the resulting gels was white enough to be considered as an adequate base for making plant-based analogues. The content of total galactoside, inositol phosphates and trypsin inhibitors (bioactive compounds) present in one serving (100 g) of these BPI gels were up to 0.80 mg/g, 8.06 mg/g and 239 TIUs, respectively

A mixture of four dietary fibres ameliorates adiposity and improves metabolic profile and intestinal health in cafeteria-fed obese rats: an integrative multi-omics approach

Aims: Atherosclerosis is the main pathological process contributing to cardiovascular disease, with diet being the most important factor involved. Although the lipidome of atheromatous plaque has been studied previously, the use of comparative lipidomics and metabolomics in plasma in early atherogenesis could lead to the discovery of plasma biomarkers that allow not only disease prediction but also measurement of disease progression. Methods and results: High-throughput techniques, such as liquid chromatography/mass spectrometry, allowed us to compare the circulating and aortic lipidome and plasma metabolome in order to look for new molecular targets involved in atherogenesis. To achieve this objective, we chose the hamster (Mesocricetus auratus) as the best small animal model for diet-induced early atherosclerosis, because its lipoprotein metabolism is similar to that of humans. The results revealed the existence of several, previously unreported, changes in lipid and amino-acid metabolism, the peroxisome proliferator-activated receptor γ pathway, and oxidative and endoplasmic reticulum stress, also involving cell senescence. Furthermore, as a proof of concept in the modelling of dietary influences in atherogenesis, we have measured the effect of a potential anti-atherogenic polyphenol extract on the reported pathways. Our results support a previously unknown role for taurocholic acid as a potential plasma biomarker of early atheromatous plaque formation. Conclusion: The use of comparative liquid chromatography/mass spectrometry-based lipidomics and metabolomics allows the discovery of novel pathways in atherogenesis, as well as new potential plasma biomarkers, which could allow us to predict disease in its early stages and measure its progression

Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential in vivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells in vivo

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Labour and social security law in Spain in 2015

El informe ha sido elaborado por la Sección Juvenil de la Asociación Española de Derecho del Trabajo y Seguridad SocialEste Informe deja constancia de los cambios normativos más relevantes y de las tendencias judiciales más paradigmáticas del ordenamiento laboral en 2015. En él se observa el imparable dinamismo del Derecho del Trabajo y de la Seguridad Social en España. El documento, consciente de tal mutabilidad, recoge una minuciosa selección de cuestiones esenciales, a juicio de las personas que abordan cada una de las materias, de las que son especialistas; los autores y las autoras, que forman parte de la Sección Juvenil de la Asociación Española de Derecho del Trabajo y de la Seguridad Social, se adscriben a los grupos temáticos por afinidad con sus principales líneas de investigación y su labor docente universitaria. En síntesis, en el Informe “El Derecho del Trabajo y de la Seguridad Social en España en 2015” se puede encontrar información muy útil para los profesionales del iuslaboralismo en materia de derechos fundamentales inespecíficos, contratación laboral y empleo, vicisitudes del contrato de trabajo, derechos colectivos, igualdad y corresponsabilidad, Seguridad Social o prevención de riesgos laborales.This report has as aim leaving a record of the most relevant normative changes and the most paradigmatic judicial trends in Labour Law in 2015. One can easily observe the unstoppable dynamismof Labour and Social Security Law in Spain. The document, conscious of that mutability, collects a thorough selection of key issues, according to the judgement of the authors, all of them specialists and all of them members of the Young Scholars’ Section of the Spanish Association for Labour and Social Security Law. They are part of thematic groups, linked to their main research lines and their teaching task. Summing up, in this report “Labour and Social Security Law in Spain in 2015”, one can easily find useful information for labour lawyers in subjects such as unspecific fundamental rights, work contracts and employment, issues of the labour relationship, collective rights, equality and co-responsibility, Social Security or occupational risk prevention

El Derecho del Trabajo y de la Seguridad Social en España en 2015. Sección Juvenil de la Asociación Española de Derecho del Trabajo y de la Seguridad Social.

Este Informe deja constancia de los cambios normativos más relevantes y de las tendencias judiciales más paradigmáticas del ordenamiento laboral en 2015. En él se observa el imparable dinamismo del Derecho del Trabajo y de la Seguridad Social en España. El documento, consciente de tal mutabilidad, recoge una minuciosa selección de cuestiones esenciales, a juicio de las personas que abordan cada una de las materias, de las que son especialistas; los autores y las autoras, que forman parte de la Sección Juvenil de la Asociación Española de Derecho del Trabajo y de la Seguridad Social, se adscriben a los grupos temáticos por afinidad con sus principales líneas de investigación y su labor docente universitaria. En síntesis, en el Informe “El Derecho del Trabajo y de la Seguridad Social en España en 2015” se puede encontrar información muy útil para los profesionales del iuslaboralismo en materia de derechos fundamentales inespecíficos, contratación laboral y empleo, vicisitudes del contrato de trabajo, derechos colectivos, igualdad y corresponsabilidad, Seguridad Social o prevención de riesgos laborales. This report has as aim leaving a record of the most relevant normative changes and the most paradigmatic judicial trends in Labour Law in 2015. One can easily observe the unstoppable dynamismof Labour and Social Security Law in Spain. The document, conscious of that mutability, collects a thorough selection of key issues, according to the judgement of the authors, all of them specialists and all of them members of the Young Scholars’ Section of the Spanish Association for Labour and Social Security Law. They are part of thematic groups, linked to their main research lines and their teaching task. Summing up, in this report “Labour and Social Security Law in Spain in 2015”, one can easily find useful information for labour lawyers in subjects such as unspecific fundamental rights, work contracts and employment, issues of the labour relationship, collective rights, equality and co-responsibility, Social Security or occupational risk prevention

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI