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OBJECTIVE Timing of mobilization after chronic subdural hematoma (cSDH) surgery is highly heterogeneous among neurosurgical centers. Past studies have suggested that early mobilization may reduce medical complications without increasing recurrence, but evidence remains scarce. The purpose of this study was to compare an early mobilization protocol with a 48-hour bed rest practice, with a focus on the occurrence of medical complications. METHODS The GET-UP Trial is a prospective, randomized, unicentric, open-label study with an intention-to-treat primary analysis designed to evaluate the impact of an early mobilization protocol after burr hole craniostomy for cSDH on the occurrence of medical complications and functional outcomes. A total of 208 patients were recruited and randomly assigned to either an early mobilization group where they began head-of-bed elevation within the first 12 hours after surgery and proceeded to sedestation, orthostatism, and/or walking as rapidly as tolerated, or to a bed rest group where they remained recumbent with a head-of-bed angle inferior to 30° for 48 hours after surgery. The primary outcome was the occurrence of a medical complication (defined as either an infection, seizure, or thrombotic event) after surgery and until clinical discharge. Secondary outcomes included length of stay measured from randomization to clinical discharge, surgical hematoma recurrence at clinical discharge and 1 month after surgery, and Glasgow Outcome Scale–Extended (GOSE) assessment at clinical discharge and 1 month after surgery. RESULTS A total of 104 patients were randomly assigned to each group. No significant baseline clinical differences were observed before randomization. The primary outcome occurred in 36 (34.6%) patients included in the bed rest group and 20 (19.2%) in the early mobilization group (p = 0.012). At 1 month after surgery, a favorable functional outcome (defined as GOSE score ≥ 5) was observed in 75 (72.1%) patients in the bed rest group and 85 (81.7%) in the early mobilization group (p = 0.100). Surgical recurrence occurred in 5 (4.8%) patients in the bed rest group and 8 (7.7%) in the early mobilization group (p = 0.390). CONCLUSIONS The GET-UP Trial is the first randomized clinical trial to assess the impact of mobilization strategies on medical complications after burr hole craniostomy for cSDH. Early mobilization was associated with a reduction in medical complications without a significant effect on surgical recurrence, compared with a 48-hour bed rest protocol.

Early-life determinants are thought to be a major factor in the rapid increase of obesity. However, while maternal nutrition has been extensively studied, the effects of breastfeeding by the infant on the reprogramming of energy balance in childhood and throughout adulthood remain largely unknown. Here we show that delayed weaning in rat pups protects them against diet-induced obesity in adulthood, through enhanced brown adipose tissue thermogenesis and energy expenditure. In-depth metabolic phenotyping in this rat model as well as in transgenic mice reveals that the effects of prolonged suckling are mediated by increased hepatic fibroblast growth factor 21 (FGF21) production and tanycyte-controlled access to the hypothalamus in adulthood. Specifically, FGF21 activates GABA-containing neurons expressing dopamine receptor 2 in the lateral hypothalamic area and zona incerta. Prolonged breastfeeding thus constitutes a protective mechanism against obesity by affecting long-lasting physiological changes in liver-to-hypothalamus communication and hypothalamic metabolic regulation. This work was supported by grants from: L.M.S.: Instituto de Salud Carlos III and co-funded by FEDER (PI18/00998), FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (C.D.: BFU2017-87721; M.L.: RTI2018–101840-B-I00; R.N.: RTI2018-099413-B-I00 and RED2018-102379-T), Xunta de Galicia (M.L.: 2016-PG068; R.N.: 2016-PG057 and 2020-PG015), Fundación BBVA (R.N.: Fundación Atresmedia (to M.L. and R.N.), Fundación La Caixa (to M.L. and R.N.), Fundación Jesus Serra and European Foundation for the Study of Diabetes (to R.N.). The research leading to these results also received funding from the European Community’s H2020 Framework Programme under the following grant: ERC Synergy Grant-2019-WATCH- 810331 to M.S., V.P. and R.N. Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn). CIBERobn is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain, which is supported by FEDER funds. This work was supported by Inserm, Fondation pour la Recherche Médicale, ANR-EPITRACES (to E.V.). Research from the M.F. laboratory was supported by the Spanish Agencia Estatal de Investigación, co-funded by the FEDER Programme of the EU (BFU2016-80899-P and PID2019-105739GB-I00 grants; AEI/FEDER, UE). M.F. is recipient of a Ramón y Cajal award (RYC-2014-16779). C.F. is a recipient of a Sara Borrel award (ISCIII CD19/0078). O.A. is a recipient of the Miguel Servet grant (ISCIIII). Sí

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment., SCOPUS: ar.j, info:eu-repo/semantics/published

[Background & Aims] Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to non-alcoholic fatty liver disease (NAFLD) remains unknown., [Methods] By performing proteomic analysis on livers from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples from patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the livers of mice., [Results] ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and non-alcoholic steatohepatitis) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoyltransferase 1a (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action., [Conclusions] Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis., This work has been supported by grants from FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (PA: RTI2018-095134-B-100; DS and LH: SAF2017-83813-C3-1-R; MLMC: RTC2019-007125-1; CD: BFU2017-87721; ML: RTI2018–101840-B-I00; GS; PID2019-104399RB-I00; RN: RTI2018-099413-B-I00 and RED2018-102379-T; MLMC: SAF2017-87301-R; TCD: RTI2018-096759-A-100), FEDER/Instituto de Salud Carlos III (AGR: PI19/00123), Xunta de Galicia (ML: 2016-PG068; RN: 2015-CP080 and 2016-PG057), Fundación BBVA (RN, GS and MLM), Proyectos Investigación en Salud (MLMC: DTS20/00138), Sistema Universitario Vasco (PA: IT971-16); Fundación Atresmedia (ML and RN), Fundación La Caixa (M.L., R.N. and M.C.), Gilead Sciences International Research Scholars Program in Liver Disease (MVR), Marató TV3 Foundation (DS: 201627), Government of Catalonia (DS: 2017SGR278) and European Foundation for the Study of Diabetes (RN and GS). This research also received funding from the European Community’s H2020 Framework Programme (ERC Synergy Grant-2019-WATCH- 810331, to RN, VP and MS). Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem). CIBERobn, CIBERehd and CIBERdem are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644).

Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation blastocyst and can be maintained indefinitely in vitro without losing their properties. Given their self-renewal and pluripotency, ESCs not only represent a key tool to study early embryonic development in a dish, but also an unlimited source of material for tissue replacement in regenerative medicine. Loss-of-function assays using RNA interference are a powerful tool to understand the roles of specific genes and are facilitated by lentiviral-mediated delivery of vector-encoded shRNAs which allows long-term silencing of single or multiple genes. Here, we describe the steps for rapid and cost-effective production and testing of lentiviral particles with vector-encoded shRNAs for gene silencing in ESCs. This protocol can be easily adapted for loss-of-function assays in other pluripotent cells or culture conditions of interest.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis

Extended pluripotent or expanded potential stem cells (EPSCs) possess superior developmental potential to embryonic stem cells (ESCs). However, the molecular underpinning of EPSC maintenance in vitro is not well defined. We comparatively studied transcriptome, chromatin accessibility, active histone modification marks, and relative proteomes of ESCs and the two well-established EPSC lines to probe the molecular foundation underlying EPSC developmental potential. Despite some overlapping transcriptomic and chromatin accessibility features, we defined sets of molecular signatures that distinguish EPSCs from ESCs in transcriptional and translational regulation as well as metabolic control. Interestingly, EPSCs show similar reliance on pluripotency factors Oct4, Sox2, and Nanog for self-renewal as ESCs. Our study provides a rich resource for dissecting the regulatory network that governs the developmental potency of EPSCs and exploring alternative strategies to capture totipotent stem cells in culture.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has proven to be a powerful system creating new opportunities to interrogate molecular mechanisms controlling cell fate determination. Under standard conditions, the generation of iPSCs upon overexpression of OCT4, SOX2, KLF4, and c-MYC (OSKM) is generally slow and inefficient due to the presence of barriers that confer resistance to cell fate changes. Hyperactivated endoplasmic reticulum (ER) stress has emerged as a major reprogramming barrier that impedes the initial mesenchymal-to-epithelial transition (MET) step to form iPSCs from mesenchymal somatic cells. Here, we describe several systems to detect ER stress in the context of OSKM reprogramming and chemical interventions to modulate this process for improving iPSC formation.

Ten-eleven translocation-2 (TET2) is a crucial driver of cell fate outcomes in a myriad of biological processes, including embryonic development and tissue homeostasis. TET2 catalyzes the demethylation of 5-methylcytosine on DNA, affecting transcriptional regulation. New exciting research has provided evidence for TET2 catalytic activity in post-transcriptional regulation through RNA hydroxymethylation. Here we review the current understanding of TET2 functions on both DNA and RNA, and the influence of these chemical modifications in normal development and pluripotency contexts, highlighting TET2 versatility in influencing genome regulation and cellular phenotypes.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has proven to be a powerful system creating new opportunities to interrogate molecular mechanisms controlling cell fate determination. Under standard conditions, the generation of iPSCs upon overexpression of OCT4, SOX2, KLF4, and c-MYC (OSKM) is generally slow and inefficient due to the presence of barriers that confer resistance to cell fate changes. Hyperactivated endoplasmic reticulum (ER) stress has emerged as a major reprogramming barrier that impedes the initial mesenchymal-to-epithelial transition (MET) step to form iPSCs from mesenchymal somatic cells. Here, we describe several systems to detect ER stress in the context of OSKM reprogramming and chemical interventions to modulate this process for improving iPSC formation.

Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation blastocyst and can be maintained indefinitely in vitro without losing their properties. Given their self-renewal and pluripotency, ESCs not only represent a key tool to study early embryonic development in a dish, but also an unlimited source of material for tissue replacement in regenerative medicine. Loss-of-function assays using RNA interference are a powerful tool to understand the roles of specific genes and are facilitated by lentiviral-mediated delivery of vector-encoded shRNAs which allows long-term silencing of single or multiple genes. Here, we describe the steps for rapid and cost-effective production and testing of lentiviral particles with vector-encoded shRNAs for gene silencing in ESCs. This protocol can be easily adapted for loss-of-function assays in other pluripotent cells or culture conditions of interest.

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Although pluripotent cells lack circadian oscillations, this study reveals a novel function of the master circadian regulator BMAL1 in metabolism and early differentiation of embryonic stem cells., BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMAL1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-of-function of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination.

Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos, although the molecular mechanism underlying such an expanded 2CLC potency remains elusive. We examine global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We find that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with activation of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by DUX and post-transcriptionally represses ZMYM2 and its partner LSD1, and ZMYM2 recruits LSD1/HDAC corepressor complex to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the previously unappreciated DUX-miR-344-Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.

Background and Aims G protein-coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l-alpha-lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown. Approach and Results We measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up-regulated in patients with NASH. LPI induced adenosine monophosphate-activated protein kinase activation of acetyl-coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing beta-oxidation. The inhibition of GPR55 and ACC alpha blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high-fat diet and in mice fed a methionine-choline-deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC. Conclusions The LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC. Supported by grants from the Fondo Europeo de Desarrollo Regional (FEDER)/Ministerio de Ciencia, Innovacion y Universidades (MCIU)/Agencia Estatal de Investigacion (AEI) (C.D.: BFU2017-87721; M.L.: RTI2018-101840-B-I00; R.N.: BFU2015-70664R; A.G.-R.: PI16/00823; C.G.-M.: PI17/00535), Xunta de Galicia (M.L.: 2015-CP079 and 2016-PG068; R.N.: 2015-CP080 and 2016-PG057), Fundacion Banco Bilbao Vizcaya Argentaria (BBVA; to R.N.), Fundacion Atresmedia (M.L. and R.N.), European Foundation for the Study of Diabetes (R. N.), and Fundacion Francisco Cobos (A.G.-R.). MCIU/AEI/FEDER, European Union, (RTI2018-095134-B-100 to P.A.) provided aid to support the research groups of Sistema Universitario Vasco (IT971-16 to P. A). MCIU provided SAF2017-87301-R and RTI2018-096759-1-100, which were integrated into the Plan Estatal de Investigacion Cientifica y Tecnica e Innovacion and were cofinanced with FEDER (to M.L.M.-C. and T.C. D. respectively), and La Caixa Foundation Program and 2018 Fundacion BBVA Grants for Scientific Research Teams (to M.L.M.-C.). The research leading to these results has also received funding from the European Community's H2020 Framework Programme under the following grant: European Research Council Synergy Grant 2019-WATCH-810331 to R.N. Centro de Investigacion Biomedica en Red (CIBER) de Fisiopatologia de la Obesidad y Nutricion and CIBER de Enfermedades Hepaticas y Digestivas are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain, which is supported by FEDER funds, Gilead Sciences International Research Scholars Program in Liver Disease (to MVR), PI16/01548 (to MM) and the Red de Trastornos Adictivos-RTA (RD16/0017/0023). This article was partially supported by grants from the Fondo Nacional de Desarrollo Cientifico y Tecnologico grants 1191145 (to M.A.), 1200227 to JPA and 1191183 (to F. B.) and by the Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT, AFB170005, CARE Chile UC, Basal Centre for Excellence in Science and Technology; to M.A.). We thank MINECO for the Severo Ochoa Excellence Accreditation provided to the Center for Cooperative Research in Biosciences (SEV-2016-0644).

Topological-associated domains (TADs) are thought to be relatively stable across cell types, although some TAD reorganization has been observed during cellular differentiation. However, little is known about the mechanisms through which TAD reorganization affects cell fate or how master transcription factors affect TAD structures during cell fate transitions. Here, we show extensive TAD reorganization during somatic cell reprogramming, which is correlated with gene transcription and changes in cellular identity. Manipulating TAD reorganization promotes reprogramming, and the dynamics of concentrated chromatin loops in OCT4 phase separated condensates contribute to TAD reorganization. Disrupting OCT4 phase separation attenuates TAD reorganization and reprogramming, which can be rescued by fusing an intrinsically disordered region (IDR) to OCT4. We developed an approach termed TAD reorganization-based multiomics analysis (TADMAN), which identified reprogramming regulators. Together, these findings elucidate a role and mechanism of TAD reorganization, regulated by OCT4 phase separation, in cellular reprogramming.

AMPK is a cellular gauge that is activated under conditions of low energy, increasing energy production and reducing energy waste. Current evidence links hypothalamic AMPK with the central regulation of energy balance. However, it is unclear whether targeting hypothalamic AMPK has beneficial effects in obesity. Here, we show that genetic inhibition of AMPK in the ventromedial nucleus of the hypothalamus (VMH) protects against high-fat diet (HFD)–induced obesity by increasing brown adipose tissue (BAT) thermogenesis and subsequently energy expenditure. Notably, this effect depends upon the AMPKα1 isoform in steroidogenic factor 1 (SF1) neurons of the VMH, since mice bearing selective ablation of AMPKα1 in SF1 neurons display resistance to diet-induced obesity, increased BAT thermogenesis, browning of white adipose tissue, and improved glucose and lipid homeostasis. Overall, our findings point to hypothalamic AMPK in specific neuronal populations as a potential druggable target for the treatment of obesity and associated metabolic disorders.

Summary Yin Yang 1 (YY1) regulates early embryogenesis and adult tissue formation. However, the role of YY1 in stem cell regulation remains unclear. YY1 has a Polycomb group (PcG) protein-dependent role in mammalian cells. The PcG-independent functions of YY1 are also reported, although their underlying mechanism is still undefined. This paper reports the role of YY1 and BAF complex in the OCT4-mediated pluripotency network in mouse embryonic stem cells (mESCs). The interaction between YY1 and BAF complex promotes mESC proliferation and pluripotency. Knockdown of Yy1 or Smarca4, the core component of the BAF complex, downregulates pluripotency markers and upregulates several differentiation markers. Moreover, YY1 enriches at both promoter and super-enhancer regions to stimulate transcription. Thus, this study elucidates the role of YY1 in regulating pluripotency through its interaction with OCT4 and the BAF complex and the role of BAF complex in integrating YY1 into the core pluripotency network., Graphical Abstract, Highlights • YY1 is integrated into the core pluripotency network through the BAF complex • YY1 and the BAF complex promote ESC proliferation • YY1 activates gene expression through the BAF complex to maintain pluripotency • YY1 is enriched at the ESC-specific super-enhancers to promote gene expression, In this article, Junjun Ding and colleagues show the role of YY1 in regulating embryonic stem cells through its interaction with OCT4 and the BAF complex to activate transcription, promote ESC proliferation, and maintain pluripotency, and the role of BAF complex in integrating YY1 into the core pluripotency network.

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editing induces aberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity during cell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

International audience; Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the CNS to regulate energy balance.

International audience; Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.

RNA chemical modifications are intricately linked to transcriptome structural and functional diversity. Whereas somatic cell reprogramming involves a profound rewiring of epigenetic and transcriptomic landscapes, how RNA modifications contribute to overriding somatic cell identity is incompletely understood. Here we demonstrate that loss of Adenosine-to-Inosine (A-to-I) editing of RNA by ADAR1 hampers mesenchymal-to-epithelial transition (MET) and impedes induced pluripotent stem cell (iPSC) formation. Absence of RNA editing leads to aberrant innate immune response (IIR) expression programs, which unleash endoplasmic reticulum (ER) stress and hinder epithelial fate acquisition. Using chemical and genetic approaches, we show a dual role for A-to-I editing in preventing MDA5-dependent activation of ER stress and ensuring proper double strand RNA (dsRNA) compartmentalization for PERK activation. Thus, our study establishes a critical role for A-to-I RNA modification in cell fate transitions during reprogramming, which delineates a novel regulatory layer underlying MET control for efficient reprogramming.

Epigenetic and epitranscriptomic networks have important functions in maintaining the pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However, the mechanisms integrating the actions of these distinct networks are only partially understood. Here we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates the transcription of key pluripotency genes, and modulates N6-methyladenosine (m(6)A) deposition on their transcripts by sequestering the enzyme m(6)A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m(6)A RNA levels, and enhances m(6)A modification of the Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3 associated, and is enriched at promoters of m(6)A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m(6)A RNA modification to ensure ESC identity.

Summary: RNA-binding proteins are key regulators of cell identity and function, which underscores the need for unbiased and versatile protocols to identify and characterize novel protein-RNA interactions. Here, we describe a simple and cost-effective in vitro RNA immunoprecipitation (iv-RIP) method to assess the direct or indirect RNA-binding ability of any protein of interest. The versatility of this method relies on the adaptability of the immunoprecipitation conditions and the choice of the RNA, which exponentially broadens the range of potential applications.For complete details on the use and execution of this protocol, please refer to Guallar et al. (2020).

Mestrado em Engenharia Mecânica Este projeto curricular consiste num estudo acerca do efeito da geometria da aresta de corte na superfície maquinada e do seu respetivo desgaste, através da realização de ensaios de fresagem a alta velocidade numa operação de acabamento a seco no aço AISI D2, em dois estados endurecidos, 55 e 61 HRc. Foram utilizadas três ferramentas de corte de metal duro revestidas por PVD, duas delas por AlTiN e outra por TiAlN + TiN. A diferença entre as ferramentas de corte estava na sua geometria. Raio de ponta, zona da aresta de corte a 90 (face alisadora) e preparação da aresta negativa (chanfro) foram as características a serem estudadas. Foram realizados ensaios de corte longos e curtos. Os primeiros visaram avaliar o desgaste da ferramenta (VB) de modo a obter a curva de desgaste, numa operação onde as ferramentas maquinam em superfícies planas com diferentes durezas. Os ensaios curtos permitiram avaliar a rugosidade da superf ície maquinada onde a ferramenta maquinou em dois tipos de superfície, nomeadamente plana vertical e 3D. Efetuou-se um estudo e levantamento geométrico da ferramenta de corte já existente, que por sua vez, foi modelada com recurso ao software CAD, So- lidWorks. Posteriormente, no âmbito do projeto curricular, desenvolveu-se uma nova ferramenta de corte que pudesse conjugar todas as características das ferramentas de corte utilizadas, nomeadamente o raio de ponta, a face alisadora e chanfro. Concluiu-se que, para maquinagens em superfície plana vertical, a ferramenta com a geometria com menor raio de ponta e face alisadora apresentou maior tempo de maquinagem e menor valor de rugosidade, mesmo com o aumento da dureza do material. No entanto, para maquinagens em superfícies 3D, a referida ferramenta não é aconselhável. Ferramentas de corte com raios de ponta elevados, com faces alisadoras e chanfros representaram ferramentas capazes de maquinar superfícies 3D e, por sua vez, obter valores de rugosidade média aritmética abaixo do valor proposto. Contudo, para maquinagens em superfícies planas, estas apresentaram os menores tempos de maquinagem até a obtenção do V Bmax e maiores valores de rugosidade na superfície maquinada. This curricular project consists on a study about the effect of the cutting edge's geometry in the machined surface and its respective wear, through the completion of milling tests with a high speed machine in a dry nishing operation on AISI D2 steel, in two hardened states, 55 and 61 HRc. Three hard metal cutting tools, coated with PVD, two of them with AlTiN and another with TiAlN +TiN. The difference between the cutting tools were in their geometry. Radius edge, cutting egde zone at 90 (face straightener) and the negative preparation edge (chamfer) were the characteristics to be studied. Long and short cut tests were conducted. The first aimed to evaluate the tool wear (VB) in order to obtain the wear curve, in an operation where the tools are used on at surfaces with different hardness values. The short test allowed to evaluate the roughness of the machined surface on two types of surface, vertical at and 3D. It was performed a geometric survey of a cutting tool available in the market which, in turn, was modeled using a CAD software,SolidWorks. Later, in the context of the curricular project, a new cutting tool was developed, in a way that could bring together all the characteristics of the cutting tools used, mainly the radius, the face straightener and chamfer. It was concluded that, while machining vertical at surfaces, a tool geometry with smaller radius of edge and face straightener, there was an increased machining time and the lowest value of surface roughness, even with the increase of the material hardness. This tool allowed, for the same conditions, to obtain a better surface finish with good dimensional accuracy. However, for 3D machining, this tool is not advisable. Tools with high edge radius, face straightener and chamfer are tools capable of 3D surface machining and get values of arithmetic surface roughness below the expected. However, format surface machining, this tool obtained smaller machining time until the achievement of the V Bmax and higher values of surface roughness in the machined surface.

SummarySuper-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination.

Pluripotency is defined by a cell's potential to differentiate into any somatic cell type. How pluripotency is transited during embryo implantation, followed by cell lineage specification and establishment of the basic body plan, is poorly understood. Here we report the transcription factor Zfp281 functions in the exit from naive pluripotency occurring coincident with pre-to-post-implantation mouse embryonic development. By characterizing Zfp281 mutant phenotypes and identifying Zfp281 gene targets and protein partners in developing embryos and cultured pluripotent stem cells, we establish critical roles for Zfp281 in activating components of the Nodal signaling pathway and lineage-specific genes. Mechanistically, Zfp281 cooperates with histone acetylation and methylation complexes at target gene enhancers and promoters to exert transcriptional activation and repression, as well as epigenetic control of epiblast maturation leading up to anterior-posterior axis specification. Our study provides a comprehensive molecular model for understanding pluripotent state progressions in vivo during mammalian embryonic development.

In mammals, all somatic development originates from lineage segregation in early embryos. However, the dynamics of transcriptomes and epigenomes acting in concert with initial cell fate commitment remains poorly characterized. Here we report a comprehensive investigation of transcriptomes and base-resolution methylomes for early lineages in peri- and postimplantation mouse embryos. We found allele-specific and lineage-specific de novo methylation at CG and CH sites that led to differential methylation between embryonic and extraembryonic lineages at promoters of lineage regulators, gene bodies, and DNA-methylation valleys. By using Hi-C experiments to define chromatin architecture across the same developmental period, we demonstrated that both global demethylation and remethylation in early development correlate with chromatin compartments. Dynamic local methylation was evident during gastrulation, which enabled the identification of putative regulatory elements. Finally, we found that de novo methylation patterning does not strictly require implantation. These data reveal dynamic transcriptomes, DNA methylomes, and 3D chromatin landscapes during the earliest stages of mammalian lineage specification.

Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.

This paper addresses the need to use the knowledge about the human perceived quality, adding machine learning models to the objective quality estimation. A new technique is proposed based on the division of images into several cells where the mean of the SSIM metric is computed. A sliding window over a grid of cells that divide the image will define a set of image descriptors that are aggregated using a bag of words. This model is able to improve the typical values provided by SSIM and defines a new path for the application of machine learning to image quality evaluation.