Expression of microRNAs and isomiRs in the porcine endometrium: implications for gene regulation at the maternal-conceptus interface

Background: Embryo implantation is a complex, synchronized process that requires establishment of a reciprocal dialogue between a receptive endometrium and developing blastocysts. Recently, microRNAs (miRNAs),known to modulate gene expression through post-transcriptional mechanisms, were implicated in regulation of early pregnancy events including maternal recognition of pregnancy and implantation. To characterize complex transcriptomic changes, expression of miRNAs in pregnant and cyclic endometria collected on days 12, 16 and 20 was analyzed using Illumina deep sequencing and analyzed with bioinformatic pipeline. Moreover, expression profiles of ten genes related to miRNA synthesis and transport such as DROSHA, DGCR8, XPO5, DICER, TARBP2, TNRC6A, and AGO1-4 were determined. Results: Among genes involved in miRNA transport and synthesis DROSHA, XPO5, DICER1, TARBP, and AGO1 expression was affected by the reproductive status. Moreover, DICER1 and AGO2 proteins were localized in luminal and glandular epithelium with immunofluorescence staining. Several hundred mature, canonical and non-canonical miRNAs were found to be expressed in the endometrial samples. Detailed analysis revealed that miRNA length variants, isomiRs, accounted for the vast majority of defined sequences. Both miRNA and isomiR of miR-140-3p were shown to affect expression of putative targets in endometrial stromal cells in vitro. Computational analysis of putative target genes for miRNAs differentially expressed (DE) between pregnant and cyclic animals resulted in lists of biological processes and regulatory pathways indicating their role in cellular development, cell cycle, immunological response and organismal development. Among predicted target genes for DE miRNAs, vascular endothelial growth factor (VEGF),progesterone and estradiol receptors (PGR, ESR1) and leukemia inhibitory factor (LIF) were found. Conclusions: This research revealed a repertoire of pregnancy-related miRNAs in porcine endometrium during initial stages of conceptus implantation and during the estrous cycle, and sheds light on mechanisms regulating miRNA-mediated gene expression at the maternal-conceptus interface

Evaluation of two family-based intervention programs for children affected by rare disease and their families – research network (CARE-FAM-NET): study protocol for a rater-blinded, randomized, controlled, multicenter trial in a 2x2 factorial design

Background: Families of children with rare diseases (i.e., not more than 5 out of 10,000 people are affected) are often highly burdened with fears, insecurities and concerns regarding the affected child and its siblings. Although families caring for children with rare diseases are known to be at risk for mental disorders, the evaluation of special programs under high methodological standards has not been conducted so far. Moreover, the implementation of interventions for this group into regular care has not yet been accomplished in Germany. The efficacy and cost-effectiveness of a family-based intervention will be assessed. Methods/design: The study is a 2x2 factorial randomized controlled multicenter trial conducted at 17 study centers throughout Germany. Participants are families with children and adolescents affected by a rare disease aged 0 to 21 years. Families in the face-to-face intervention CARE-FAM, online intervention WEP-CARE or the combination of both will be treated over a period of roughly 6 months. Topics discussed in the interventions include coping, family relations, and social support. Families in the control condition will receive treatment as usual. The primary efficacy outcome is parental mental health, measured by the Structured Clinical Interview for DSM-IV (SCID-I) by blinded external raters. Further outcomes will be assessed from the parents’ as well as the children’s perspective. Participants are investigated at baseline, 6, 12 and 18 months after randomization. In addition to the assessment of various psychosocial outcomes, a comprehensive health-economic evaluation will be performed. Discussion: This paper describes the implementation and evaluation of two family-based intervention programs for Children Affected by Rare Disease and their Family’s Network (CARE-FAM-NET) in German standard care. A methodologically challenging study design is used to reflect the complexity of the actual medical care situation. This trial could be an important contribution to the improvement of care for this highly burdened group. Trial registration: German Clinical Trials Register: DRKS00015859 (registered 18 December 2018) and ClinicalTrials.gov: NCT04339465 (registered 8 April 2020). Protocol Version: 15 August 2020 (Version 6.1). Trial status: Recruitment started on 1 January 2019 and will be completed on 31 March 2021. © 2020, The Author(s)

The glioblastoma multiforme tumor site promotes the commitment of tumor-infiltrating lymphocytes to the TH17 lineage in humans

Although glioblastoma multiforme (GBM) is not an invariably cold tumor, checkpoint inhibition has largely failed in GBM. In order to investigate T cell-intrinsic properties that contribute to the resistance of GBM to endogenous or therapeutically enhanced adaptive immune responses, we sorted CD4(+) and CD8(+) T cells from the peripheral blood, normal-appearing brain tissue, and tumor bed of nine treatment-naive patients with GBM. Bulk RNA sequencing of highly pure T cell populations from these different compartments was used to obtain deep transcriptomes of tumor-infiltrating T cells (TILs). While the transcriptome of CD8(+) TILs suggested that they were partly locked in a dysfunctional state, CD4(+) TILs showed a robust commitment to the type 17 T helper cell (T(H)17) lineage, which was corroborated by flow cytometry in four additional GBM cases. Therefore, our study illustrates that the brain tumor environment in GBM might instruct T(H)17 commitment of infiltrating T helper cells. Whether these properties of CD4(+) TILs facilitate a tumor-promoting milieu and thus could be a target for adjuvant anti-T(H)17 cell interventions needs to be further investigated

Persistent Organic Pollutants in sediment and fish in the River Thames catchment (UK)

Some organic pollutants including polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and hexachlorobenzene (HCB) have been banned from production and use in the UK for > 30 years but due to their toxicity and persistence are still of concern. However, due to their hydrophobicity they are present at very low concentrations and are difficult to measure in water, and so other matrices need to be sampled in order to best assess contamination. This study measured concentrations of ΣICES 7 PCBs (PCB congeners 28, 52, 101, 118, 138, 153 and 180) and Σ6 PBDEs (PBDE congeners 28, 47, 99, 100, 153, 154) and HCB in both bed-sediments and wild roach (a common pelagic fish) in the Thames Basin. The highest sediment concentrations were detected in an urbanised tributary of the Thames, The Cut at Bracknell (HCB: 0.03–0.40 μg/kg dw; ICES 7 PCBs: 4.83–7.42 μg/kg dw; 6 BDEs: 5.82–23.10 μg/kg dw). When concentrations were expressed on a dry weight basis, the fish were much more contaminated than the sediments, but when sediment concentrations were normalised to organic carbon concentration they were comparable to the fish lipid normalised concentrations. Thus, despite the variability in the system, both sediments and wild fish can be considered suitable for representing the level of POPs contamination of the river system given sufficient sample numbers

Single-cell transcriptomics identifies potential cells of origin of MYC rhabdoid tumors

Rhabdoid tumors (RT) are rare and highly aggressive pediatric neoplasms. Their epigenetically-driven intertumoral heterogeneity is well described; however, the cellular origin of RT remains an enigma. Here, we establish and characterize different genetically engineered mouse models driven under the control of distinct promoters and being active in early progenitor cell types with diverse embryonic onsets. From all models only Sox2-positive progenitor cells give rise to murine RT. Using single-cell analyses, we identify distinct cells of origin for the SHH and MYC subgroups of RT, rooting in early stages of embryogenesis. Intra- and extracranial MYC tumors harbor common genetic programs and potentially originate from fetal primordial germ cells (PGCs). Using PGC specific Smarcb1 knockout mouse models we validate that MYC RT originate from these progenitor cells. We uncover an epigenetic imbalance in MYC tumors compared to PGCs being sustained by epigenetically-driven subpopulations. Importantly, treatments with the DNA demethylating agent decitabine successfully impair tumor growth in vitro and in vivo. In summary, our work sheds light on the origin of RT and supports the clinical relevance of DNA methyltransferase inhibitors against this disease

Diversity of fungi associated with petroglyph sites in the Negev Desert, Israel, and their potential role in bioweathering

The petroglyphs of the Negev Desert, Israel, are famous and valuable archaeological remains. Previous studies have investigated the microbial communities associated with petroglyphs and their potential role in stone deterioration; nevertheless, the role of fungi remains unclear. In this study, the fungal communities present on the stone and, as a comparison, in the surrounding environment (soil and air) at Negev petroglyph sites were analyzed by means of culture-dependent and -independent (metagenomic) techniques. The metagenomic results showed a high fungal biodiversity in the soil, and both approaches highlighted the prevalence of species producing melanized, large, thick-walled spores (mainly Alternaria spp.). From the air sampling, mostly Cladosporium spp. were retrieved. On the other hand, on the rock, the results seem to indicate a low presence of fungi, but with a rock-specialized mycobiota consisting of extremotolerant microcolonial fungi (MCF) (e.g., Vermiconidia and Coniosporium) and lichens (Flavoplaca). In addition, low proportions of cosmopolitan fungi were detected on the stone, but the comparison of the data clearly indicates that they are transients from the surrounding environment. The ability of the isolated strains to dissolve CaCO3 and therefore be a potential threat to the petroglyphs (limestone substrate) was tested, but only one strain resulted in positive acid production under laboratory conditions. Nevertheless, both lichens and MCF detected in this study are well-known stone deteriogens, which may have a significant impact on the petroglyph’s deterioration

Seasonal variations in the diagnosis of testicular germ cell tumors: a national cancer registry study in austria

SIMPLE SUMMARY: Seasonal variations in cancer diagnosis could already be demonstrated in prostate and breast cancer. The reasons for this observed seasonal pattern are still unclear. The health care system or other determinants such as the protective function of vitamin D3 in carcinogenesis could be assumed as one explanation. Testicular germ cell tumors are the most common developed malignancy among young men. The aim of our study was to investigate, for the first time, the seasonal variations in the clinical diagnosis of testicular germ cell tumors. We have been able to confirm that the frequency of monthly newly diagnosed cases of testicular cell tumors in Austria has a strong seasonality, with a significant reduction in the tumor incidence during the summer months and an increase during the winter months. ABSTRACT: We conducted a retrospective National Cancer Registry study in Austria to assess a possible seasonal variation in the clinical diagnosis of testicular germ cell tumors (TGCT). In total, 3615 testicular cancer diagnoses were identified during an 11-year period from 2008 to 2018. Rate ratios for the monthly number of TGCT diagnoses, as well as of seasons and half-years, were assessed using a quasi-Poisson model. We identified, for the first time, a statistically significant seasonal trend (p < 0.001) in the frequency of monthly newly diagnosed cases of TGCT. In detail, clear seasonal variations with a reduction in the tumor incidence during the summer months (Apr–Sep) and an increase during the winter months (Oct–Mar) were observed (p < 0.001). Focusing on seasonality, the incidence during the months of Oct–Dec (p = 0.008) and Jan–Mar (p < 0.001) was significantly higher compared to the months of Jul–Sep, respectively. Regarding histopathological features, there is a predominating incidence in the winter months compared to summer months, mainly concerning pure seminomas (p < 0.001), but not the non-seminoma or mixed TGCT groups. In conclusion, the incidence of TGCT diagnoses in Austria has a strong seasonal pattern, with the highest rate during the winter months. These findings may be explained by a delay of self-referral during the summer months. However, the hypothetical influence of vitamin D3 in testicular carcinogenesis underlying seasonal changes in TGCT diagnosis should be the focus of further research

ETMR-05: Single-cell transcriptomics of ETMR reveals developmental cellular programs and tumor-pericyte communications in the microenvironment [Abstract]

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are pediatric brain tumors bearing a grim prognosis, despite intensive multimodal therapeutic approaches. Insights into cellular heterogeneity and cellular communication of tumor cells with cells of the tumor microenvironment (TME), by applying single-cell (sc) techniques, potentially identify mechanisms of therapy resistance and target-directed treatment approaches. MATERIAL AND METHODS: To explore ETMR cell diversity, we used single-cell RNA sequencing (scRNA-seq) in human (n=2) and murine ETMR (transgenic mode; n=4) samples, spatial transcriptomics, 2D and 3D cultures (including co-cultures with TME cells), multiplex immunohistochemistry and drug screens. RESULTS: ETMR microenvironment is composed of tumor and non-tumor cell types. The ETMR malignant compartment harbour cells representing distinct transcriptional metaprograms, (NSC-like, NProg-like and Neuroblast-like), mirroring embryonic neurogenic cell states and fuelled by neurogenic pathways (WNT, SHH, Hippo). The ETMR TME is composed of oligodendrocyte and neuronal progenitor cells, neuroblasts, microglia, and pericytes. Tumor-specific ligand-receptor interaction analysis showed enrichment of intercellular communication between NProg-like ETMR cells and pericytes (PC). Functional network analyses reveal ETMR-PC interactions related to stem-cell signalling and extracellular matrix (ECM) organization, involving factors of the WNT, BMP, and CxCl12 networks. Results from ETMR-PC co-culture and spatial transcriptomics pointed to a pivotal role of pericytes in keeping ETMR in a germinal neurogenic state, enriched in stem-cell signalling. Drug screening considering cellular heterogeneity and cellular communication suggested novel therapeutic approaches. CONCLUSION: ETMR demonstrated diversity in the microenvironment, with enrichment of cell-cell communications with pericytes, supporting stem-cell signalling and interfering in the organization of the tumor extracellular matrix. Targeting ETMR-PC interactions might bring new opportunities for target-directed therapy

Influence of a high fat diet during adolescence on the reinforcing effects of cocaine and alcohol

RESUMEN La adolescencia es un período de especial vulnerabilidad en el desarrollo del ser humano, en el que se dan grandes cambios a nivel estructural y funcional (Spear, 2000; Chambers et al., 2003). En esta etapa de gran vulnerabilidad, los jóvenes muestran trastornos de la alimentación, consumo de sustancias, búsqueda de sensaciones y conductas de riesgo (Bava and Tapert, 2010). Actualmente, el consumo excesivo de comida especialmente rica en azúcares y grasas, se ha convertido en un serio problema para nuestra sociedad, habiéndose producido un incremento en las tasas de obesidad a nivel mundial que afecta particularmente a la población adolescente. La alimentación está regulada tanto por mecanismos homeostáticos como hedónicos. Un malfuncionamiento de cualquiera de estos sistemas puede provocar una alteración en la alimentación y producir obesidad (Kenny et al., 2011). El sistema de refuerzo, constituido esencialmente por el sistema dopaminérgico mesocorticolímbico, regula la motivación para buscar o consumir estímulos reforzantes como las drogas o la comida altamente palatable. Por lo tanto, el consumo de drogas o la alimentación por razones hedónicas activan las mismas vías del refuerzo. Inicialmente nuestro organismo se desarrolló en un contexto caracterizado por la escasez nutricional y conseguir alimentos era el principal objetivo a alcanzar en el día a día. Ya que nuestro cuerpo procesa los alimentos de forma rápida, mostramos una preferencia innata por las comidas con alto contenido calórico y energético. Este tipo de alimentos, al igual que la cocaína o el alcohol, producen liberación de dopamina en el sistema de recompensa cerebral. Hoy en día, en la sociedad de la abundancia y la variedad de alimentos, comemos no sólo por hambre, sino también por placer (Gold, 2011). Por lo tanto, comer por placer afecta a los mecanismos neurales relacionados con el refuerzo y promueve el mantenimiento de esta conducta (ver revisión Avena et al., 2008; Volkow et al., 2013). En resumen, tanto los reforzadores naturales como las drogas estimulan el circuito cerebral de recompensa, produciendo placer y euforia. El estado nutricional es un importante factor modulador en el desarrollo de la adicción, y algunos estudios han mostrado similitudes psicológicas y biológicas entre la ingesta de comida rápida y la adicción a las drogas, (Garber and Lustig, 2011; Avena et al., 2012). Por ejemplo, tanto la adicción a las drogas como la obesidad pueden ser definidas como trastornos en los que el valor de determinado refuerzo (droga o comida) está anormalmente incrementado en relación y a expensas de otros refuerzos. La adicción a drogas y los atracones de comida se caracterizan por una pérdida de control sobre la conducta consumatoria, la escalada, la dependencia y el ansia por consumir (craving), mostrando ambos trastornos una elevada comorbilidad (Swanson et al., 2011). Numerosos estudios han puesto de manifiesto que el consumo de drogas durante la adolescencia incrementa la probabilidad de desarrollar dependencia y problemas relacionados con estas sustancias en la edad adulta (Arteaga et al., 2010; Merline et al., 2004). Se ha propuesto igualmente una teoría de puerta de entrada también para la comida y el abuso de sustancias (Degenhardt y cols., 2008). Esta teoría postula que la ingesta compulsiva de comida puede facilitar el desarrollo de otros comportamientos desadaptativos, como es el consumo de drogas. Por el momento, los estudios preclínicos indican que el atracón de comida rica en azúcar provoca a una sensibilización conductual a la anfetamina (Avena y Hoebel, 2003) y aumenta la autoadministración de alcohol (Avena y cols., 2004). Se han realizado estudios utilizando comida rica en azúcar, sin embargo, los estudios con comida rica en grasa son escasos. Hasta la fecha todavía no se han evaluado las consecuencias de realizar atracones de grasa durante la adolescencia sobre el consumo de drogas como la cocaína y el alcohol. Tampoco conocemos los efectos de suspender el consumo de una dieta alta en grasas, sobre los efectos gratificantes de la cocaína o el alcohol. Así pues, el objetivo de la presente tesis doctoral ha sido evaluar cómo una dieta alta en grasa consumida durante la adolescencia, puede modificar los efectos reforzantes de la cocaína y el alcohol. Estudiamos dos tipos diferentes de consumo de la dieta alta en grasa, el acceso continuado a la dieta rica en grasa (los animales consumen la dieta durante todo el periodo de tiempo correspondiente, sin limitar su acceso) y el atracón, que es un acceso limitado e intermitente. Este último patrón se ha demostrado que induce un consumo de grasa en forma de atracones (Puhl y cols., 2011; Bocarsly y cols., 2011). En el protocolo de administración intermitente los ratones tienen acceso a la dieta rica en grasa solo durante 2 horas los lunes, miércoles y viernes con un acceso ilimitado a la dieta estándar. Como metodología principal para evaluar la respuesta a la cocaína y el alcohol utilizamos los paradigmas de Condicionamiento de la Preferencia de Lugar y el procedimiento de Autoadministración operante. El Condicionamiento de la Preferencia de lugar es el paradigma más utilizado para medir el efecto de las claves ambientadas asociadas con los efectos reforzantes de las drogas (Aguilar et al., 2013). Por otro lado, el modelo de autoadministración operante evalúa directamente la motivación del animal por conseguir la sustancia. Ambos modelos proporcionan una evaluación completa de los efectos reforzantes de las drogas, ya que nos permiten medir tanto las claves individuales como las externas. También hemos estudiado los efectos metabólicos que estos tipos de dieta grasa producían en los niveles plasmáticos de leptina, grelina y corticosterona. Igualmente, hemos estudiado mediante la reacción en cadena de la polimerasa (PCR) en tiempo real, la expresión de genes pertenecientes a los sistemas dopaminérgico, cannabinoide y opioide, debido a su implicación tanto en el refuerzo inducido por las drogas como por la comida. En cuanto a los resultados obtenidos, en el primer estudio hemos observado que, aunque no hay cambios en ninguna de las conductas estudiadas mientras se mantiene el consumo de grasa, el cese de esta dieta produce un incremento en el nivel de ansiedad, aumenta la respuesta aguda a la cocaína y produce condicionamiento de la preferencia de lugar con dosis subumbrales de cocaína. Todo esto se acompaña por cambios a nivel de expresión del gen para el receptor opioide mu, el cannabinoide CB1 y GHSR. Por otro lado, observamos el poder de la dieta rica en grasa como refuerzo alternativo, ya que cuando la administramos tras condicionar a los animales con una dosis efectiva, éstos tardan menos en extinguir la preferencia y no presentan reinstauración con dosis con las que el grupo control sí reinstaura la preferencia de lugar. En el segundo estudio hemos evaluado el efecto de realizar atracones de una dieta rica en grasa y hemos observado que provocan un aumento en la sensibilidad a dosis subumbrales de cocaína en el condicionamiento de la preferencia de lugar y se autoadministran más cocaína que el grupo con dieta estándar. Por otro lado, observamos también cambios en la expresión de los genes responsables de los receptores opioide mu, cannabinoide CB1 y de la grelina GHSR. En el tercer estudio realizamos el mismo procedimiento que en el estudio anterior, pero con el fin de evaluar si este tipo de administración de dieta rica en grasa también incrementaba la sensibilidad al alcohol. De nuevo encontramos que los animales que realizan atracones son más sensibles a dosis subumbrales de alcohol y se autoadministran más que el grupo con dieta estándar. Además, presentan cambios a nivel de expresión de genes tras la autoadministración, diferentes a los obtenidos con la simple administración de la grasa en estudios anteriores. En el cuarto estudio evaluamos si estos efectos sobre la ingesta de alcohol tenían un efecto a largo plazo, incluso habiendo cesado el consumo de grasa. Observamos cómo los animales que realizaron atracones durante la adolescencia y llevaban 15 días sin acceso a la grasa seguían presentando un incremento en la autoadministración de alcohol, pero ya no eran más sensibles a los efectos reforzantes de una dosis subumbral de alcohol en el condicionamiento de la preferencia de lugar. En el quinto estudio evaluamos el papel que tiene un estrés crónico, como es el aislamiento, en la modulación que produce la dieta rica en grasa en atracón sobre los efectos reforzantes de la cocaína. Observamos resultados opuestos a los obtenidos con animales agrupados, ya que los animales aislados con una dieta estándar fueron más sensibles a dosis subumbrales de cocaína que los que realizaron atracones de dieta rica en grasa. Además, los grupos que desarrollaron la preferencia (aislados dieta estándar y agrupados dieta atracón grasa) también presentaron niveles incrementados de leptina. En el sexto y último estudio trazamos un perfil conductual con todos los grupos que hemos utilizado en la presente tesis doctoral, observando que los animales que realizan atracones de grasa presentan pocas alteraciones conductuales. Únicamente exhiben un incremento en la actividad locomotora y conductas agresivas en la interacción social con un oponente. Sí que observamos déficits a nivel de aprendizaje espacial con los animales que comen comida rica en grasa de forma continuada y además de un comportamiento agresivo. En ambos tipos de dieta, el cese del consumo produce un incremento en el nivel de ansiedad. Los resultados obtenidos nos indican el consumo de una dieta alta en grasa de forma continuada produce efectos muy diferentes a los producidos por el consumo intermitente en atracón. Mientras que el consumo en forma de atracón no produce grandes efectos metabólicos, incrementa la sensibilidad a los efectos reforzantes del alcohol y la cocaína. Sin embargo, el consumo de grasa de forma continuada produce grandes cambios metabólicos, como hiperleptinemia y aumento de peso, pero no afecta la respuesta a la cocaína. Sin embargo, tras el cese en el consumo continuado de una dieta alta en grasa, aparece un incremento en la respuesta a los efectos condicionados reforzantes de la cocaína, confirmando así que el sistema de refuerzo ha quedado sensibilizado tras el consumo de grasa. Ambos tipos de dieta alta en grasa produce cambios en la expresión génica de los receptores opioide mu, cannabinoide CB1 y del receptor de grelina GHSR, indicando que ambos patrones de consumo de grasa modifican de forma diferente la función del sistema de refuerzo cerebral, produciendo consecuencias a largo plazo, incluso cuando la comida rica en grasa ya no está disponible. Nuestro trabajo demuestra que los hábitos nutricionales no solo producen alteraciones metabólicas en nuestro organismo o modifican nuestro peso corporal. También modifican nuestro SNC y alteran la forma en que respondemos a las drogas de abuso. Una de las principales aportaciones de este trabajo es la demostración de que la dieta grasa consumida de forma continuada, que induce grandes cambios metabólicos y obesidad, así como alteraciones conductuales, no parece modular la respuesta a las drogas hasta que esta deja de consumirse, momento en el que observamos una respuesta incrementada. Sin embargo, los atracones de grasa, que no alteran metabólicamente al sujeto, ni modifican su peso corporal o su conducta, sí que incrementan la respuesta a drogas como la cocaína o el etanol, incluso cuando ya ha dejado de consumirse. Por lo tanto, esperamos que los artículos que componen la presente Tesis Doctoral ayuden a incrementar el conocimiento y la conciencia sobre la importancia de los hábitos nutricionales en el abordaje de un problema multifactorial como es la adicción a las drogas.  ABSTRACT Adolescence is a highly vulnerable developmental period in which numerous structural and functional maturational changes occur (Spear, 2000; Chambers et al., 2003). In this early stage of vulnerability, adolescents exhibit eating disorders, substance abuse, novelty-seeking and risk-taking behaviors (Bava and Tapert, 2010). Regarding these environmental hazards, the rise in obesity rates worldwide has encouraged extensive research to improve understanding of this problem, in which the excessive intake of food, especially sugar-rich and high-fat food has become a serious problem for society. As we know, addiction is a chronic and multifactorial relapsing disorder resulting from an interaction of biological and environmental aspects, characterized by a loss of control in the use of the substance and relapse (Koob and Volkow, 2010). Developmental factors are important components of vulnerability, with strong evidence supporting that exposure to drugs of abuse during adolescence leads to a significantly higher likelihood of drug dependence and drug-related problems in adulthood. Feeding is regulated by homeostatic and hedonic mechanisms. Abnormalities in these functions can cause several feeding alterations and produce obesity (Kenny et al., 2011). The reward system, which is essentially constituted by the dopamine mesocorticolimbic system, regulates motivation to seek and take rewarding stimuli, such as drugs or highly palatable food. Therefore, drug abuse or hedonic eating activate the same reward pathways. Our body was initially developed in a context characterized by nutritional deficiencies and getting food was the daily main objective to achieve. It processes foods quickly and we have innate preference for high energy meals, especially those high in fat and sugar, because in a situation of scarcity those would be the energy stores that would help us to survive. This type of food, like drugs of abuse, produces a dopamine release in the brain reward system, which explains its pleasurable effects. Nowadays, there is such abundance and variety of food, that this survival-oriented evolutionary adaptation has lost its sense, since we eat not only for hunger, but also for pleasure (Gold, 2011). Thus, hedonic eating affects neural mechanisms connected with reward and maintains this behavior (For reviews: Avena et al., 2008; Volkow et al., 2013). In summary, both natural reinforcers and drugs stimulate the brain reward circuit, producing pleasure and euphoria. The nutritional status is an important factor in the development of addiction, since some studies indicate psychological and biological similarities between fast food intake and addiction to drugs, sharing common reward mechanisms (Garber and Lustig, 2011; Avena et al., 2012). For example, both drug addiction and obesity can be defined as disorders in which the value of the type of reinforcement (drug or food, respectively) is abnormally increased in relationship to other reinforcements (Avena et al., 2012; Volkow et al., 2013). Drug addiction and binge eating are characterized by a loss of control over consummatory behaviors, exhibiting escalation, dependence and craving when the reward is not available, and both present a high comorbidity (Swanson et al., 2011). Several studies report that drug use during adolescence often predicts an increased likelihood of continued use into adulthood (Arteaga et al., 2010; Merline et al., 2004). Based on these relationships, a Theory of Gateway has also been proposed for eating disorders and substance abuse (Degenhardt et al., 2008), in which it is postulated that eating disorders, such as binge eating, can lead to the development of another desadaptive behavior, such as drug abuse. At the moment, preclinical studies indicate, for example, that intake of certain types of sugar leads to a sensitization to amphetamine (Avena and Hoebel, 2003) and to an increase in alcohol self-administration (Avena et al., 2004). Studies with sugar are abundant, unlike studies with high-fat food and drug vulnerability. For example, there are no studies reporting the effects of bingeing on fat during adolescence and vulnerability to drug abuse, like cocaine or alcohol, nor are there studies examining the effects of interrupting fat consumption on the subsequent vulnerability to drugs like cocaine or alcohol. Thus, the objective of the present work was to evaluate how a high-fat diet exposure during adolescence modulates the reinforcing effects of cocaine and alcohol. We studied two different types of fat diet consumption: continuous access (animals have ad libitum access to high-fat food without a limit) and the intermittent and limited access. The latter has been shown to induce a binge eating pattern (Puhl and cols., 2011; Bocarsly et al., 2011), and our protocol is based on that used by Corwin et al. (1998). Here, mice had access to the high-fat diet only for 2 hours every Monday, Wednesday and Friday, with ad libitum access to the standard diet. The main methodology employed to assess the vulnerability to cocaine and alcohol rewarding effects was the Conditioned Place Preference paradigm and the operant Self-Administration procedure. The conditioned place preference is the most commonly used test to evaluate the environmental cues conditioned to the rewarding effects of the drug (Aguilar et al., 2013), while the self-administration procedure evaluates directly the animals’ motivation to obtain the substance of abuse. Both models provide a complete scenario to evaluate vulnerability to drug abuse, since they allow us to measure both the external and the individual cues. We have also studied some metabolic effects on circulating leptin, ghrelin and corticosterone levels and examined changes in gene expression with real time polymerase chain reaction (rt-PCR) related to dopaminergic, opioid and endocannabinoid systems, because of their involvement in the rewarding properties of drugs and food. Regarding our results, in the first study we observed no effects on the behavioral response to drugs while fat is available. However, following fat discontinuation, mice exhibited increased anxiety, augmented locomotor response to cocaine, and developed preference for subthreshold doses of cocaine. In addition, there were changes in MOr, CB1 and GHSR gene expression. On the other hand, we observed how high-fat diets act as an alternative reinforcer, as its administration after conditioned place preference reduces the number of sessions required to extinguish the preference and decreased sensitivity to drug priming-induced reinstatement. In the second study, we observed that animals that binged on fat were more sensitive to the reinforcing effects of a subthreshold dose of cocaine in the conditioned place preference and enhanced self-administration. We also observed changes in MOr, CB1 and GHSR gene expression. In the third study, we employed the same procedure as in the former study, but with the aim of evaluating if the increased sensitivity also arises with alcohol. Animals in the high-fat binge group presented more sensitivity to the rewarding effects of subthreshold doses of ethanol and greater ethanol consumption with a higher motivation to obtain the drug. In addition, they presented changes in gene expression after the self-administration procedure which are different from those found with the fat administration alone. In the fourth study, we evaluated if these effects of bingeing on fat and alcohol intake would have a long term consequences, even when fat consumption is interrupted. Our results showed how after 15 days from the last binge session, animals still presented a greater ethanol consumption, but they were not sensitive to subthreshold doses in the conditioned place preference anymore. The fifth study aimed to evaluate the role of chronic stress, such as isolation, on the modulatory effects of fat on the rewarding properties of cocaine. We observed opposite effects as those found in grouped animals, as isolated mice with a standard diet access were more sensitive to subthreshold doses of cocaine than those fed with a fat binge eating pattern. In addition, the groups that developed preference (isolated with standard diet and grouped with high-fat binge) were those who presented increased circulating leptin levels. In the sixth and last study, we drew a behavioral profile with the groups used in the present doctoral thesis, and found a few alterations in animals that binge on fat, such as hyperlocomotion and aggressive behaviors. On the other hand, we observed marked spatial learning deficits in animals eating fat continuously as well as increased attack behaviors with conspecifics. In both diet patterns, discontinuation of fat led to an increase in anxiety levels. Our results show that bingeing on fat is quite different from eating fat continuously. While the former does not produce great metabolic effects, it produces significant changes in the sensitivity to drugs such as alcohol and cocaine. On the other hand, continuously eating fat produces big metabolic changes like hyperleptinemia and increased bodyweight and but has no effect regarding vulnerability to drug use. However, when access to fat is interrupted (withdrawal period), the increased response to the rewarding effects of drugs arises, confirming that the reward system has become sensitized. Both fat consumption patterns produce several changes in gene expression of mu opioid receptor, of cannabinoid receptor CB1 and of ghrelin receptor GHSR, indicating that both patterns of fat consumption changed the reward system function in different manners, having long-lasting effects, even when fat is no longer available. Our work shows that the nutritional habits not only produce metabolic alterations in our organism or modify our body weight, they also modify our CNS and change the way w

Improved 6-year overall survival in AT/RT - results of the registry study Rhabdoid 2007

Atypical teratoid rhabdoid tumors (AT/RT) are characterized by mutations and subsequent inactivation of SMARCB1 (INI1, hSNF5), a predilection for very young children and an unfavorable outcome. The European Registry for rhabdoid tumors (EU-RHAB) was established to generate a common European database and to establish a standardized treatment regimen as the basis for phase I/II trials. Thus, genetic analyses, neuropathologic and radiologic diagnoses, and a consensus treatment regimen were prospectively evaluated. From 2005 to 2009, 31 patients with AT/RT from four countries were recruited into the registry study Rhabdoid 2007 and treated with systemic and intraventricular chemotherapy. Eight patients received high-dose chemotherapy, 23 radiotherapy, and 17 maintenance therapy. Reference evaluations were performed in 64% (genetic analyses, FISH, MLPA, sequencing) up to 97% (neuropathology, INI1 stain). Germ-line mutations (GLM) were detected in 6/21 patients. Prolonged overall survival was associated with age above 3years, radiotherapy and achievement of a complete remission. 6-year overall and event-free survival rates were 46% (+/- 0.10) and 45% (+/- 0.09), respectively. Serious adverse events and one treatment-related death due to insufficiency of a ventriculo peritoneal shunt (VP-shunt) and consecutive herniation were noted. Acquisition of standardized data including reference diagnosis and a standard treatment schedule improved data quality along with a survival benefit. Treatment was feasible with significant but manageable toxicity. Although our analysis is biased due to heterogeneous adherence to therapy, EU-RHAB provides the best available basis for phase I/II clinical trials