DNA methylation memory: Understanding epigenetic reprogramming in vertebrates

DNA methylation is an epigenetic mark critical for vertebrate development and is associated with numerous cellular and organismal processes including X-chromosome inactivation, genomic imprinting, and regulation of gene expression. Importantly, DNA methylation patterns are faithfully inherited during cell division, providing an information memory module additional to the DNA code. This mark, along with other epigenetic modifications, plays an essential role in establishing and maintaining cell identity. DNA methylation dynamics has been studied in detail in eutherian mammals, where two major waves of demethylation, the first in the early embryo and the second during germline development, remove most marks. Erasure of epigenetic memory is associated with cell reprogramming, and in mammals, is inextricably linked to increased developmental potency. For divergent vertebrate models, this dynamic is largely untested and indirect evidence suggests epigenetic memory may be retained in the germline. Furthermore, the role of epigenetic memory and reprogramming in major cell fate transitions, such as sex determination and sex change, is underexplored despite being apparently driven by epigenetic mechanisms in at least some species. In order to understand how epigenetic memory is maintained, erased, and reprogrammed in divergent vertebrates, I have focused on two fish species. I have analysed the epigenome of the germline during gonad development in zebrafish (Danio rerio), and the transcriptome and methylome of bluehead wrasse (Thalassoma bifasciatum) during female-to-male sex change. Using a combination of techniques, including isolation of germline cells, whole genome bisulfite sequencing, and comparative epigenomics and transcriptomics, I explored epigenetic memory and reprogramming in these species. This thesis is presented as a collection of research and review papers, as well as a discussion synthesising my results

Whole-Exome Sequencing Enables Rapid Determination of Xeroderma Pigmentosum Molecular Etiology

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder haracterized by extreme sensitivity to actinic pigmentation changes in the skin and increased incidence of skin cancer. In some cases, patients are affected by neurological alterations. XP is caused by mutations in 8 distinct genes (XPA through XPG and XPV). The XP-V (variant) subtype of the disease results from mutations in a gene (XPV, also named POLH) which encodes for Polg, a member of the Y-DNA polymerase family. Although the presence and severity of skin and neurological dysfunctions differ between XP subtypes, there are overlapping clinical features among subtypes such that the sub-type cannot be deduced from the clinical features. In this study, in order to overcome this drawback, we undertook whole-exome sequencing in two XP sibs and their father. We identified a novel homozygous nonsense mutation (c.897T.G, p.Y299X) in POLH which causes the disease. Our results demonstrate that next generation sequencing is a powerful approach to rapid determination of XP genetic etiology

Leukocyte-specific DNA methylation biomarkers and their implication for pathological epigenetic analysis

Background: Distinct cell types can be identified by their DNA methylation patterns. Much research over the last decade has focused on DNA methylation changes in cancer or the use of cell-free circulating DNA in plasma to identify damaged tissue in cases of trauma or organ transplantation. However, there has been little research into the differential methylation patterns between leukocytes and other tissues and how they can be used as a detection tool for immune activity in a range of contexts. Results: We have identified several loci that are fully methylated in leukocytes but virtually devoid of methylation in a range of other mesoderm-, ectoderm-, and endoderm-derived tissues. We validated these biomarkers using amplicon- bisulphite-sequencing on saliva and in vitro mixing of peripheral blood mononuclear cells and intestinal organoid cells combined at a defined range of ratios. Interestingly, these methylation biomarkers have previously been identified as altered in various inflammatory diseases, including Alzheimer disease, inflammatory bowel disease, and psoriasis. We hypothesise this is due to leukocyte infiltration rather than being a feature of the diseased cells themselves. Moreover, we show a positive linear relationship between infiltrating leukocytes and DNA methylation levels at the HOXA3 locus in six cancer types, indicative of further immune cell infiltration. Conclusions: Our data emphasise the importance of considering cellular composition when undertaking DNA methylation analysis and demonstrate the feasibility of developing new diagnostic tests to detect inflammation and immune cell infiltration

Effects of exercise in addition to a family-based lifestyle intervention program on hepatic fat in children with overweight

OBJECTIVE Pediatric hepatic steatosis is highly prevalent and closely related to type 2 diabetes. This study aimed to determine whether the addition of supervised exercise to a family-based lifestyle and psycho-educational intervention results in greater reduction of percentage of hepatic fat (HF), adiposity, and cardiometabolic risk factors in children with overweight/obesity. RESEARCH DESIGN AND METHODS The study subjects of this nonrandomized, two-arm, parallel design clinical trial were 116 overweight/obese children (10.6 +/- 1.1 years of age, 53.4% girls) living in Vitoria-Gasteiz (Spain). For 22 weeks, they followed either a lifestyle and psycho-education program (control intervention [CInt], N = 57), consisting of two family-based education sessions/month, or the same plus supervised exercise (intensive intervention [II], N = 59) focused mainly on high-intensity aerobic workouts (3 sessions/week, 90 min/session). The primary outcome was the change in percentage of HF (as measured by MRI) between baseline and the end of the intervention period. Secondary outcomes included changes in BMI, fat mass index (FMI), abdominal fat (measured by DEXA), blood pressure, triglycerides, HDL, LDL, gamma-glutamyl transferase, glucose, and insulin concentrations. RESULTS A total of 102 children completed the trial (N = 53 and N = 49 in the CInt and II groups, respectively). Percentage of HF decreased only in the II group (-1.20 +/- 0.31% vs. 0.04 +/- 0.30%, II and CInt groups, respectively), regardless of baseline value and any change in adiposity (P < 0.01). BMI, FMI, abdominal fat (P <= 0.001), and insulin (P < 0.05) were reduced in both groups. CONCLUSIONS Multicomponent intervention programs that include exercise training may help to reduce adiposity, insulin resistance, and hepatic steatosis in overweight/obese children.This project was funded by the Spanish Ministry of Health 'Fondos de Investigation Sanitaria del Institute de Salud Carlos III' (PI13/01335), the Spanish Ministry of Industry and Competitiveness (DEP2016-78377-R), and by EU Fondos Estructurales de la Unión Europea (FEDER) funds ('Una manera de hacer Europa'). Support was also provided by grants from the Spanish Ministry of Education, Culture and Sports (FPU14/03329), the Education Department of the Government of the Basque Country (PRE_2016_1_0057, PRE_2017_2_0224, PRE_2018_2_0057), the University of Granada Plan Propio de Investigacion 2016-Excellence actions: Unit of Excellence on Exercise and Health (UCEES), and Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades (FEDER, ref. SOMM17/6107/UGR)

A sociodemographic, anthropometric and lifestyle-based prediction score for screening children with overweight and obesity for hepatic steatosis: the HEPAKID index

Background: Hepatic steatosis (HS) is currently the most prevalent hepatic disease in paediatric population and a major risk factor for type 2 diabetes and cardiovascular diseases. The proper identification of children with HS is therefore of great public health interest. Objective: To develop a new prediction score using anthropometric, sociodemographic and lifestyle factors to identify children with HS (the HEPAKID index). Previously published biochemical paediatric screening tools were validated in the same cohort. Methods: A total of 115 pre-adolescent children aged 8 to 12 years with overweight/obesity, recruited at hospital paediatric units were enrolled in this cross-sectional study. HS (≥5.5% hepatic fat) was assessed by magnetic resonance imaging (MRI). Anthropometric, sociodemographic and lifestyle variables were collected by validated tests/questionnaires. Results: Forty-one children had MRI-diagnosed HS (35.6%, 49% girls). These children had (P <.01) a higher waist-height ratio, a lower cardiorespiratory fitness, a younger gestational age, and consumed more sugar-sweetened beverages than their HS-free peers. Children with HS were more likely to belong to an ethnic minority (P <.01) and to spend longer viewing screens than recommended (P <.05). The addition of these variables to the multivariate logistic regression model afforded a HEPAKID index with high discriminatory capacity (area under the receiver-operating characteristic curve: 0.808, 95% CI 0.715-0.901), and score of ≥25.0 was associated with high sensitivity (82%, 95% CI 68%-96%). Biochemical biomarker-based paediatric tools for identifying HS showed only moderate discriminatory capacity and low sensitivity (5%-41%) in this cohort. Conclusions: The HEPAKID index is the first simple, non-invasive, sensitive, inexpensive and easy-to-perform screening that can identify children with overweight or obesity who have HS.EFIGRO project was funded by the Spanish Ministry of Health ‘Fondos de Investigación Sanitaria del Instituto de Salud Carlos III’ (PI13/01335), the Spanish Ministry of Industry and Competitiveness (DEP2016‐78377‐R), by EU Fondos Estructurales de la Unión Europea (FEDER) funds (‘Una manera de hacer Europa’), and partially funded by the University of Granada Plan Propio de Investigación 2016 Excellence actions: Unit of Excellence on Exercise and Health (UCEENS, https://uceens.ugr.es ), and the Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades (ERDF: ref. SOMM17/6107/UGR). M.O. is supported by a grant from the Spanish Ministry of Economy and Competitiveness, grant number BES‐2017‐080770. L.A. is supported by the Education Department of the Government of the Basque Country (PRE_2016_1_0057, PRE_2017_2_0224, PRE_2018_2_0057, PRE_2019_2_0004)

A short exposure to a semi-natural habitat alleviates the honey bee hive microbial imbalance caused by agricultural stress

Honeybee health and the species' gut microbiota are interconnected. Also noteworthy are the multiple niches present within hives, each with distinct microbiotas and all coexisting, which we termed "apibiome". External stressors (e.g. anthropization) can compromise microbial balance and bee resilience. We hypothesised that (1) the bacterial communities of hives located in areas with different degrees of anthropization differ in composition, and (2) due to interactions between the multiple microbiomes within the apibiome, changes in the community of a niche would impact the bacteria present in other hive sections. We characterised the bacterial consortia of different niches (bee gut, bee bread, hive entrance and internal hive air) of 43 hives from 3 different environments (agricultural, semi-natural and natural) through 16S rRNA amplicon sequencing. Agricultural samples presented lower community evenness, depletion of beneficial bacteria, and increased recruitment of stress related pathways (predicted via PICRUSt2). The taxonomic and functional composition of gut and hive entrance followed an environmental gradient. Arsenophonus emerged as a possible indicator of anthropization, gradually decreasing in abundance from agriculture to the natural environment in multiple niches. Importantly, after 16 days of exposure to a semi-natural landscape hives showed intermediate profiles, suggesting alleviation of microbial dysbiosis through reduction of anthropization.This work was funded by the Dept. of Economic Development and Competitiveness of the Basque Government (Gobierno Vasco/Eusko Jaurlaritza), R&D&I grants for the agricultural, food and fishing sectors of the Basque Autonomous Community (37-2017-00044), and the Research Group IT1233-19 of the Basque University System. JG was supported by the Department of Agriculture, Fisheries and Food of the Basque Government (Gobierno Vasco/Eusko Jaurlaritza) through a subsidy programme of training aid and support. These funding bodies provided the financial support to the research, but did not participate in the design of the study, analysis and interpretation of data, and writing of the manuscript

Adquisición de señales analógicas de instrumentación con LOGO! Soft V8.3 mediante generador de señales y el sensor PT100

In the industry in general it is required to control physical variables such as temperature and pressure to produce products, for this reason, specialists are required in the field of Instrumentation; Consequently, the final price of the manufactured product increases. The objective of this work was the acquisition of physical variables in LOGO! Soft V8.3 using the signal generator and PT100 as part of the overall research project. Through the signal generator, we replace a current and voltage transducer to simulate the damaged signals that are sent to the Programmable Logic Controller. This investigation is fragmented into three phases, the first stage studies the generation of current and voltage signals based on ISA standards, the second stage is the physical connections of the signal generator with Logo Soft V8.3, and the last stage programming was carried out in LOGO! Soft Comfort V8.3 of the SIEMENS brand to use the selected signals. This article allows us to have a tool for instrumentation and data acquisition issues that will later be sent through IoT 2040 to the cloud.En la industria en general se requiere controlar variables físicas como temperatura y presión para elaborar productos, por ello se requiere especialistas en el tema de Instrumentación; consecuencia de esto se incrementa el precio final del producto elaborado. El objetivo de este trabajo es la adquisición de las variables físicas en LOGO! Soft V8.3 mediante el generador de señales y PT100 como parte del proyecto general de investigación. Mediante el generador de señales reemplazamos a un transductor de corriente y voltaje para simular las señales analógicas que son enviadas al Controlador Lógico Programable. Esta investigación se fragmentó en tres fases, la primera etapa se estudia sobre la generación de señales analógicas de corriente y voltaje basado en normas ISA, la segunda etapa es las conexiones físicas del generador de señales con el Logo Soft V8.3, y la última etapa se realizó la programación en LOGO! Soft Comfort V8.3 de la marca SIEMENS para utilizar las señales analógicas. Este artículo, nos permite tener una herramienta para temas de instrumentación y adquisición de datos que posteriormente será enviado por medio del IoT 2040 a la nube

Real-world efficacy and safety of eribulin in advanced and pretreated HER2-negative breast cancer in a Spanish comprehensive cancer center

Background Eribulin improves survival in pre-treated HER2-negative advanced breast cancer (ABC). However, limited data exist on co-morbidities and central nervous system (CNS) efficacy. The purpose of this study was to review eribulin's efficacy and safety in everyday clinical practice with special focus on age, body mass index (BMI) and central nervous system (CNS) activity. Methods An observational study was conducted in a series of HER2-negative ABC patients treated from January'14-December'17 outside a clinical trial. Objective Response Rate (ORR), Progression Free Survival (PFS), Overall Survival (OS), and association of clinical and pathological variables with outcome were evaluated. Results Ninety-five women were treated with at least one cycle of eribulin. Median age was 57 (33-83), and 18% were obese. Median number of prior chemotherapies for ABC was 3 (2-5) and 76% of patients had visceral metastases, including 21% with CNS involvement. Most tumors were estrogen receptor-positive (79%). ORR and stable disease (SD) at 6 months were 26.2 and 37.5%, respectively. Remarkably, relevant CNS efficacy was observed with eribulin: 20% of patients obtained partial response and 25% SD. Treatment was generally well tolerated and manageable, with 29% grade 3 and 10.9% grade 4 toxicities. Median PFS and OS were 4.1 months (CI95% 3.2-4.9) and 11.1 months (CI95% 9.5-14.7), respectively. Triple-negative disease, > 2organs involved and being younger than 70 years old were independent prognosis factors for worse OS in multivariate analysis. Most patients (75%) progressed in pre-existing metastases sites. Conclusion In everyday clinical practice, eribulin's efficacy seems similar to pivotal trials. CNS-efficacy was observed. TNBC, > 2 organs involved and being younger than 70 years old were independent prognosis factors for worse OS. Remarkably, less incidence of grade 4-toxicity compared to previous studies was found

The effect of a multicomponent intervention on steatosis is partially mediated by the reduction of intermuscular abdominal adipose tissue in children with overweight or obesity: the EFIGRO Project

OBJECTIVE: In adults, there is evidence that improvement of metabolic-associated fatty liver disease (MAFLD) depends on the reduction of myosteatosis. In children, in whom the prevalence of MAFLD is alarming, this muscle-liver crosstalk has not been tested. Therefore, we aimed to explore whether the effects of a multicomponent intervention on hepatic fat is mediated by changes in intermuscular abdominal adipose tissue (IMAAT) in children with overweight/obesity. RESEARCH DESIGN AND METHODS: A total of 116 children with overweight/obesity were allocated to a 22-week family-based lifestyle and psychoeducational intervention (control group, n = 57) or the same intervention plus supervised exercise (exercise group, n = 59). Hepatic fat percentage and IMAAT were acquired by MRI at baseline and at the end of the intervention. RESULTS: Changes in IMAAT explained 20.7% of the improvements in hepatic steatosis (P < 0.05). Only children who meaningfully reduced their IMAAT (i.e., responders) had improved hepatic steatosis at the end of the intervention (within-group analysis: responders -20% [P = 0.005] vs. nonresponders -1.5% [P = 0.803]). Between-group analysis showed greater reductions in favor of IMAAT responders compared with nonresponders (18.3% vs. 0.6%, P = 0.018), regardless of overall abdominal fat loss. CONCLUSIONS: The reduction of IMAAT plays a relevant role in the improvement of hepatic steatosis after a multicomponent intervention in children with overweight/obesity. Indeed, only children who achieved a meaningful reduction in IMAAT at the end of the intervention had a reduced percentage of hepatic fat independent of abdominal fat loss. Our findings suggest that abdominal muscle fat infiltration could be a therapeutic target for the treatment of MAFLD in childhood.This project was funded by the Spanish Ministry of Health's Fondos de Investigación Sanitaria del Instituto de Salud Carlos III (PI13/01335), the Spanish Ministry of the Economy Industry and Competitiveness (DEP2016-78377-R), and by European Regional Development Funds (ERDF): Una Manera de Hacer Europa. Support was also provided by the Regional Government of Navarra's Department of Economic Development (0011-1365-2019-000152 & 0011-1365-2020-000243), co-funded by European Regional Development Funds (ERDF 2014-2020 for Navarra). CC-S is supported by the Spanish Ministry of Science and Innovation (FJC2018-037925-I). MM is supported by Junta de Andalucía and European Union (SNGJ Ref-8025). MO is supported by the Spanish Ministry of Economy and Competitiveness (BES-2017-080770). This study was supported by the University of Granada Plan Propio de Investigación 2021 -Excellence actions: Unit of Excellence on Exercise, Nutrition and Health (UCEENS)- and the Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades, European Regional Development Funds (ref. SOMM17/6107/UGR)

Victimización secundaria y reparación integral en la atención a mujeres víctimas de violencia en Quito

La violencia contra la mujer constituye un fenómeno de gran preocupación en Ecuador, que ocurre en un contexto social de inequidad de género y que, permanentemente, se expresa y válida en comportamientos y actitudes que refuerzan el poder masculino sobre lo femenino. Cuando una mujer denuncia violencia, además, se ve expuesta a una naturalización institucional sobre sus causas, que plantean una serie de limitaciones en el acceso a la justicia y en la reparación integral, entendiéndose dichos obstáculos como victimización secundaria. El objetivo de esta investigación consiste en identificar la percepción de las dinámicas de victimización secundaria y reparación integral en funcionarios públicos que brindan atención a mujeres víctimas de violencia en Quito. El método utilizado está basado en la teoría fundamentada que permite desarrollar de manera más amplia los conceptos y categorías en estudio. Se realizaron entrevistas semiestructuradas a ocho operadores de justicia y cuatro psicólogas de distintas instituciones judiciales y de salud. Los resultados evidencian que los participantes reconocen que estas dinámicas de victimización secundaria están presentes en la atención a las mujeres, que se expresa en la extensión de procesos judiciales, la desarticulación entre instituciones, el desfase en la reparación y la escasa formación en los funcionarios en enfoques de protección constituye un maltrato adicional a las mujeres, que posibilita una distancia de la justicia ante la vulneración de sus derechos. Se discuten las implicaciones de estos hallazgos y del requerimiento hacia el Estado en el abordaje de la violencia y las reales posibilidades que las mujeres tienen para acceder a una reparación que abarque todos los impactos de las situaciones de violencia