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Forkhead box (FOX)O proteins are transcription factors (TFs) with four members in mammals designated FOXO1, FOXO3, FOXO4, and FOXO6. FOXO TFs play a pivotal role in the cellular adaptation to diverse stress conditions. FOXO proteins act as context-dependent tumor suppressors and their dysregulation has been implicated in several age-related diseases. FOXO3 has been established as a major gene for human longevity. Accordingly, FOXO proteins have emerged as potential targets for the therapeutic development of drugs and geroprotectors. In this review, we provide an overview of the most recent advances in our understanding of FOXO regulation and function in various pathological conditions. We discuss strategies targeting FOXOs directly or by the modulation of upstream regulators, shedding light on the most promising intervention points. We also reveal the most relevant clinical indications and discuss the potential, trends, and challenges of modulating FOXO activity for therapeutic purposes., This work was supported by the Spanish Ministry of Science, Innovation, and Universities through Grant RTI2018-094629-B-I00 to W.L. This work was supported by the Kuakini Medical Center, the US National Institutes of Health [contract N01-AG-4-2149, grants 5U01AG019349-05, 5R01AG027060 (Kuakini Hawaii Lifespan Study) (B.J.W.), 5R01AG038707 (Kuakini Hawaii Healthspan Study), and 1P20GM125526-01A1 (Kuakini Center of Biomedical Research Excellence for Clinical and Translational Research on Aging) (B.J.W.)], contract N01-HC-05102 from the National Heart, Lung, and Blood Institute (B.J.W.), P01CA214274 from the National Cancer Institute (J.P.), MCL-RI from the Leukemia and Lymphoma Society (J.P.), and R56AG048284 from the National Institute of Aging (J.P.). J.B. thanks the Fundação para a Ciência e Tecnologia (FCT) and the Comissão de Coordenação e Desenvolvimento Regional do Algarve (CCDR Algarve) for supporting the work with the project ALG-01-0145-FEDER-28044 ‘VITAL’, PTDC/BTM-TEC/28044/2017, and the project ALG-01-0145-FEDER-072586 ‘ALFA Score’ #072586. This review is supported by STRATAGEM COST Action, CA17104.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of death due to cancer in the world. Therefore, the identification of novel druggable targets is urgently needed. Tribbles proteins belong to a pseudokinase family, previously recognized in CRC as oncogenes and potential therapeutic targets. Here, we analyzed the expression of TRIB1, TRIB2, and TRIB3 simultaneously in 33 data sets from CRC based on available GEO profiles. We show that all three Tribbles genes are overrepresented in CRC cell lines and primary tumors, though depending on specific features of the CRC samples. Higher expression of TRIB2 in the tumor microenvironment and TRIB3 overexpression in an early stage of CRC development, unveil a potential and unexplored role for these proteins in the context of CRC. Differential Tribbles expression was also explored in diverse cellular experimental conditions where either genetic or pharmacological approaches were used, providing novel hints for future research. This comprehensive bioinformatic analysis provides new insights into Tribbles gene expression and transcript regulation in CRC. info:eu-repo/semantics/publishedVersion

BACKGROUND AND AIMS Peritoneal protein loss has been for many years seen as a detrimental consequence of peritoneal dialysis (PD). Many studies correlated this leakage with mortality, malnutrition and inflammation. More recent knowledge has brought overhydration to this equation. This study aims to review classic and recent factors associated with peritoneal protein clearance and its consequences on overall mortality. METHOD Prevalent and incident PD patients were included. Interleukin 6 (IL-6; dialysate and serum) was obtained during a 3.86% glucose peritoneal equilibration test. Hydration and nutritional status was assessed by bioimpedance. Linear and Cox regression were performed. RESULTS A total of 78 patients were included (54 incident, 24 prevalent), the mean age was 54.6 ± 18.1 years, 65% were males, and the mean follow-up was 33.9 ± 29.3 months. The mean Charlson comorbidity index (CCI) was 4.4 ± 2.4. The residual GFR was 6.7 ± 3.6 mL/min/1.73 m2 and the D/P creatinine 0.65 ± 0.12. The IL-6 concentration in the dialysate was 24.9 ± 30.8 pg/mL and in serum was 37.3 ± 5.1 pg/mL. The phase angle was 5.4 ± 1.1°, overhydration 0.88 ± 0.94 L and peritoneal protein clearance 85.6 ± 54.7 mL/24 h. Overall, eight patients died. Linear univariable analysis showed positive associations between peritoneal protein clearance and (i) small solute transport, as measured by D/P creatinine, (ii) body composition, as measured by phase angle and overhydration and (iii) CCI. The peritoneal protein clearance also correlated positively with dialysate IL-6, but not with serum IL-6. By linear multivariable analysis, using the backward method, a significant positive association between peritoneal protein and overhydration (P In the exploratory survival analysis, no relationship was found between mortality and peritoneal protein clearance. A univariable positive association was shown with serum IL-6 concentration, overhydration and CCI. A higher phase angle was associated with lower mortality. No relationship with dialysate IL-6 or D/P creatinine was found. In this early-stage PD population, with globally preserved residual kidney function, an effect of this variable on mortality was not evident. Cox regression, conditional backward method, showed CCI (OR: 1.896, IC: 1.235–2.913, P = 0.003), overhydration (OR: 10.034, IC: 1.426–70.587, P = 0.021) and peritoneal protein clearance (OR: 0.576, IC: 0.339–0.978, P = 0.041) were predictors for mortality. CONCLUSION A better nutrition status and overhydration are the major determinants of peritoneal protein clearance. The survival analysis showed that mortality is higher in overhydrated patients, with higher CCI, but not with higher peritoneal protein clearance. Inflammation, local or systemic, as assessed by IL-6 concentration, did not reveal to be such a strong prognosis predictor as overhydration in these PD patients.

BACKGROUND AND AIMS FGF-21 is a hepatokine that stimulates glucose uptake through GLUT-1. The presence of this glucose transporter in peritoneal fibroblasts is driven by peritoneal glucose exposure and is related to impaired ultrafiltration, predisposing to overhydration. The study was designed to determine FGF-21 levels in peritoneal effluent and serum, and its possible association with overhydration. METHOD Prevalent and incident peritoneal dialysis (PD) patients without diabetes were included. FGF-21 (dialysate and serum) was obtained during a 3.86% glucose peritoneal equilibration test. Peritoneal glucose load was calculated. The presence or absence of local peritoneal production was assessed by comparing the dialysate to plasma (D/P) ratio of FGF-21 (MW 32 kD) with that of albumin (MW 67 kD). Hydration status was assessed by bioimpedance. Spearman's correlations were performed. RESULTS A total of 58 patients (38 incident, 20 prevalent) were included, mean age 54 years, 67% males, PD duration from 1 to 75 months, mean 10.4. Residual GFR was 6.4 ± 3.5 mL/min/1.73 m2 and D/P creatinine 0.65 ± 0.12. Peritoneal glucose load was 110.5 g/day (range 40.8–226.9). Dialysate FGF-21 was 42.5 ± 47.0 pg/mL and serum FGF-21 943.9 ± 1164.8 pg/mL, giving a D/P ratio of 0.07. D/P albumin was 0.009, indicating that dialysate FGF-21 concentrations can be explained by transport from the circulation, without local peritoneal production. Serum FGF-21 was positively correlated with dialysate FGF-21, extracellular water excess and total peritoneal glucose load (all P CONCLUSION FGF-21 in peritoneal effluent is likely not locally produced, but caused by transport from the circulation. The associations between plasma FGF-21 levels with PD duration, peritoneal glucose load and overhydration, all indicate an effect of peritoneal glucose absorption from the peritoneal cavity into the interstitium with an insulin-independent storage into interstitial adipocytes, where it may stimulate the expression of GLUT-1.

Congenital hypothyroidism (CH) is the most common congenital endocrine disorder in neonates and children with a global incidence of 1 in 3,000–4,000. Adequate thyroid hormone levels in the bloodstream are essential for the normal growth and development of the nervous system in children. Consequently, CH is associated with irreversible central nervous system and mental health problems, as well as poor growth of untreated children with CH. Girls are more likely to be affected than boys (female to male ratios are 2:1) [1–3]. Early diagnosis methods for CH are still necessary to establish more efficient treatments of CH, which is an important public health issue worldwide, including developed countries [4]. CH can be classified either as primary or as central hypothyroidism, and it is an autosomal recessive disorder. But in a significant percentage of patients, CH is sporadic and non-hereditary and caused by de novo developmental defects in the thyroid gland (primary congenital hypothyroidism). Hereditary forms of CH with decreased thyroid hormone synthesis are either due to a defective thyroid gland development (thyroid dysgenesis) present in 80% of patients or to a failure in thyroid hormone synthesis (thyroid dyshormonogenesis) present in 20% of the patients [5, 6]. Thyroid dysgenesis (TD; OMIM 218700), which refers to the most common cause of congenital hypothyroidism and abnormal structural malformations in the thyroid, is subcategorized into: (i) thyroid ectopy, an abnormally located and small thyroid gland; (ii) thyroid athyreosis, complete lack of the thyroid gland in imaging studies; and (iii) hypoplasia, a smaller gland of thyroid tissue but in a normal position. Dyshormonogenesis (OMIM 274400–274900) refers to the failure of thyroid hormone synthesis by a structurally normal thyroid gland [7]. In recent years, the overall incidence of CH has increased, including the transient form of CH in children, which is a consequence of thyroid hormone deficiency due to low thyroxine and elevated thyrotropin expression. Temporary CH may show symptoms of mild dyshormonogenesis during the first months of life due to insufficient production of thyroxine (T4) [8, 9]. Transient CH usually resolves itself in the first few months of infancy because of an increase in thyroxine production, but some of these children may also be treated for CH in infancy with levothyroxine (a manufactured form of the thyroid hormone thyroxine). However, after treatment endogenous hormonal levels are normalized, and by the age of 3, these children no longer need medication. info:eu-repo/semantics/publishedVersion

Induced pluripotent stem cells (iPSC) technology has propelled the field of stem cells biology, providing new cells to explore the molecular mechanisms of pluripotency, cancer biology and aging. A major advantage of human iPSC, compared to the pluripotent embryonic stem cells, is that they can be generated from virtually any embryonic or adult somatic cell type without destruction of human blastocysts. In addition, iPSC can be generated from somatic cells harvested from normal individuals or patients, and used as a cellular tool to unravel mechanisms of human development and to model diseases in a manner not possible before. Besides these fundamental aspects of human biology and physiology that are revealed using iPSC or iPSC-derived cells, these cells hold an immense potential for cell-based therapies, and for the discovery of new or personalized pharmacological treatments for many disorders. Here, we review some of the current challenges and concerns about iPSC technology. We introduce the potential held by iPSC for research and development of novel health-related applications. We briefly present the efforts made by the scientific and clinical communities to create the necessary guidelines and regulations to achieve the highest quality standards in the procedures for iPSC generation, characterization and long-term preservation. Finally, we present some of the audacious and pioneer clinical trials in progress with iPSC-derived cells.

Left Ventricular Noncompaction Cardiomyopathy (LVNC) is characterized by abnormal number and prominence of trabeculations of the left ventricle of the heart. Although LVNC has been associated with mutations in several genes encoding for transcriptional regulators, ion channels, sarcomeric and mitochondrial proteins, approximately 60% of LVNC patients do not present these genetic alterations. Here, we describe an induced pluripotent stem cell (hiPSC) line (UALGi002-A) originated from a LVNC female patient (LVNC-hiPSC) who does not present any previously known mutations associated to LVNC. The LVNC-hiPSC exhibited full pluripotency and differentiation potential and retained a normal karyotype after reprogramming. Moreover, the LVNC-hiPSC differentiated into contracting cardiomyocytes. This cellular model will be useful to study the molecular, genetic and functional aspects of LVNC in vitro. ALG-01-0145-FEDER-28044 PPBI-POCI-01-0145-FEDER-022122 info:eu-repo/semantics/acceptedVersion

Left Ventricular Noncompaction Cardiomyopathy (LVNC) is characterized by excessive trabeculation of the left ventricle. To date, mutations in more than 40 genes have been associated with LVNC, however the exact mechanisms underlying the disease remain unknown. Here, we describe an induced pluripotent stem cell (iPSC) line (UALGi001-A) from a LVNC patient (LVNC-iPSC) that does not present mutations in the genes most commonly associated with the disease (van Waning et al., 2019). The LVNC-iPSC exhibited full pluripotency and differentiation potential, and retained a normal karyotype after reprogramming. This in vitro cellular model will be useful to study the molecular, genetic and functional aspects of LVNC. ALG-01-0145-FEDER-28044, PTDC/BTM-TEC/28044/2017, PPBI-POCI-01-0145FEDER-022122 info:eu-repo/semantics/publishedVersion

This study aimed to evaluate the modulatory effect of dietary taurine supplementation on protein turnover of meagre juveniles fed semi-purified plant-based diets. For that purpose, fish (3.2 +/- 0.2 g) were fed with five isoproteic (61.2%), isolipidic (16.6%) and isoenergetic (21.9 kJ g(-1)) experimental diets supplemented with 0.5% (Tau0.5), 1% (Tau1), 1.5% (Tau1.5) and 2% (Tau2) taurine or not (Tau0) for 38 days. the resulting growth parameters, protein expression levels and the activity of several proteolytic markers involved in the two major degradation pathways in vertebrates (autophagy-lysosomal and ubiquitin-proteasome) were assessed in the liver, intestine and white muscle of meagre. Increasing dietary taurine concentrations promoted fish growth, feed utilization and whole-body protein, lipid and energy contents. Dietary taurine seemed to modulate the activity of cathepsins D and B in white muscle and cathepsin L in the liver. Although, no significant effects were observed on proteasome activity, 1% dietary taurine supplementation decreased the expression of the Psmb4 proteasome subunit in the liver of fish when compared to lower taurine concentrations. No taurine effects were detected on the proteolytic markers analysed in the intestine of meagre. Hsp70 expression levels were also not affected by any of the experimental dietary taurine concentrations. Overall, experimental diet Tau2 promoted the highest growth rate which can be explained by a significant decrease in protein degradation in the liver and white muscle preferentially via the autophagy-lysosomal pathway (cathepsins) over the ubiquitin-proteasome route. Diversiaqua project [Mar2020 16-02-01-FMP-0066] Diversiaqua II project [Mar2020-P02M01-0656P] Fundacao para a Ciencia e a Tecnologia FCT Portuguese Foundation for Science and Technology [SFRH/BPD/117504/2016] info:eu-repo/semantics/publishedVersion

Transcriptomic data have become a fundamental resource for stem cell (SC) biologists as well as for a wider research audience studying SC-related processes such as aging, embryonic development and prevalent diseases including cancer, diabetes and neurodegenerative diseases. Access and analysis of the growing amount of freely available transcriptomics datasets for SCs, however, are not trivial tasks. Here, we present StemMapper, a manually curated gene expression database and comprehensive resource for SC research, built on integrated data for different lineages of human and mouse SCs. It is based on careful selection, standardized processing and stringent quality control of relevant transcriptomics datasets to minimize artefacts, and includes currently over 960 transcriptomes covering a broad range of SC types. Each of the integrated datasets was individually inspected andmanually curated. StemMapper's user-friendly interface enables fast querying, comparison, and interactive visualization of quality-controlled SC gene expression data in a comprehensive manner. A proof-of-principle analysis discovering novel putative astrocyte/neural SC lineage markers exemplifies the utility of the integrated data resource. We believe that StemMapper can open the way for new insights and advances in SC research by greatly simplifying the access and analysis of SC transcriptomic data. Portuguese Fundacao para a Ciencia e Tecnologia (FCT) [SFRH/BPD/96890/2013, PTDC/BEX-BID/5410/2014, IF/00881/2013, UID/BIM/04773/2013, UID/Multi/04326/2013]; Programa Doutoral ProRegeM-Mecanismos de Doenca e Medicina Regenerativa [PD/00117/2012]; Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, Devon, UK info:eu-repo/semantics/publishedVersion

CITED2 (CBP/p300-interacting transactivator with Glu/Asp-rich C-terminal domain, 2) is a ubiquitously expressed protein exhibiting a high affinity for the CH1 domain of the transcriptional co-activators CBP/p300, for which it competes with hypoxia-inducible factors (HIFs). CITED2 is particularly efficient in the inhibition of HIF-1α-dependent transcription in different contexts, ranging from organ development and metabolic homeostasis to tissue regeneration and immunity, being also potentially involved in various other physiological processes. In addition, CITED2 plays an important role in inhibiting HIF in some diseases, including kidney and heart diseases and type 2-diabetes. In the particular case of cancer, CITED2 either functions by promoting or suppressing cancer development depending on the context and type of tumors. For instance, CITED2 overexpression promotes breast and prostate cancers, as well as acute myeloid leukemia, while its expression is downregulated to sustain colorectal cancer and hepatocellular carcinoma. In addition, the role of CITED2 in the maintenance of cancer stem cells reveals its potential as a target in non-small cell lung carcinoma and acute myeloid leukemia, for example. But besides the wide body of evidence linking both CITED2 and HIF signaling to carcinogenesis, little data is available regarding CITED2 role as a negative regulator of HIF-1α specifically in cancer. Therefore, comprehensive studies exploring further the interactions of these two important mediators in cancer-specific models are sorely needed and this can potentially lead to the development of novel targeted therapies. info:eu-repo/semantics/publishedVersion

O trabalho foi desenvolvido no DGH do INSA em colaboração com participantes do DGH do Porto e de Lisboa ao abrigo do projeto PTDC/BIM-MEC/4762/2014. Fabry Disease (FD) is a multisystemic X-linked disorder that belongs to the group of lysosomal storage disorders (LSDs). Causal mutations on alpha-galactosidase A (α-Gal A) commonly lead to abnormal protein and consequently to FD. Since it is an X-linked disease, males are primarily affected. This work describes the generation of induced Pluripotent Stem Cells (iPSCs) from skin fibroblasts from a FD patient, using non-integrative episomal vectors. Differentiation of iPSCs can be applied to generate a variety of cell types with high degree of genetic complexity that would otherwise be difficult to obtain. Portuguese Foundation of Science and Technology (FCT) project PTDC/BIM-MEC/4762/2014 (PI-O.A.) and UIDB/00211/2020. R.S. was the recipient of an FCT Grant from project PTDC/BIM-MEC/4762/2014 (MCTES). info:eu-repo/semantics/publishedVersion

The CITED family of transcriptional modulators plays multiple roles in the development of vertebrates. These proteins are characterized by the conservation of a carboxy-terminal domain which defines this family and has a high affinity for the transcriptional co-activators CBP/p300. In humans, mutations in some CITED genes or deregulation of their expression are associated with developmental anomalies. In particular, CITED2 dysfunction has been associated with congenital heart disease. Although most studies have reported the critical function of CITED proteins during development, there is increasing evidence supporting an important role for these proteins in physiological functions of adult organisms and pathologies such as cardiomyopathies and cancer. In addition, recent studies have pointed out the involvement of CITED proteins in embryonic and adult stem cells self-renewal, and cell fate decisions. Here, we present an overview of the CITED transcriptional modulators, their protein interactors and gene networks, with an emphasis on their importance in pluripotent stem cells and cardiogenesis.

Mutations and inadequate methylation profiles of CITED2 are associated with human congenital heart disease (CHD). In mouse, Cited2 is necessary for embryogenesis, particularly for heart development, and its depletion in embryonic stem cells (ESC) impairs cardiac differentiation. We have now determined that Cited2 depletion in ESC affects the expression of transcription factors and cardiopoietic genes involved in early mesoderm and cardiac specification. Interestingly, the supplementation of the secretome prepared from ESC overexpressing CITED2, during the onset of differentiation, rescued the cardiogenic defects of Cited2-depleted ESC. In addition, we demonstrate that the proteins WNT5A and WNT11 held the potential for rescue. We also validated the zebrafish as a model to investigate cited2 function during development. Indeed, the microinjection of morpholinos targeting cited2 transcripts caused developmental defects recapitulating those of mice knockout models, including the increased propensity for cardiac defects and severe death rate. Importantly, the co-injection of anti-cited2 morpholinos with either CITED2 or WNT5A and WNT11 recombinant proteins corrected the developmental defects of Cited2-morphants. This study argues that defects caused by the dysfunction of Cited2 at early stages of development, including heart anomalies, may be remediable by supplementation of exogenous molecules, offering the opportunity to develop novel therapeutic strategies aiming to prevent CHD. Agência financiadora: Fundação para a Ciência e a Tecnologia (FCT) Comissão de Coordenação e Desenvolvimento Regional do Algarve (CCDR Algarve) ALG-01-0145-FEDER-28044; DFG 568/17-2 Algarve Biomedical Center (ABC) Municipio de Loulé info:eu-repo/semantics/publishedVersion

Recent advances in oncology have evidenced the existence of a sub-population of cells, the cancer stem cells (CSC), which self-renew and are able to originate differentiated tumour cells responsible for the overall organization and heterogeneity of the tumour tissue. CSC have been identified and isolated from a wide range of human tumours, including solid tumours, and are thought to play a central role in the tumour initiation, progression and metastatic ability, as well as resistance to conventional cancer treatment and relapse of the tumours. Studies of animal models and human cancers, as well as advances in the understanding of the biological processes regulating normal tissue stem cells, have provided further knowledge into CSC biology. Here, we present the specific characteristics of CSC and the functional similarities shared with normal tissue stem cells, including the signalling pathways and microenvironment. We will also address recent advances that have revealed the complexity of these cells and present new prospects in the treatment of cancer by a combined use of standard therapies with agents specifically targeting CSC.

CITED2 is a ubiquitously expressed nuclear protein exhibiting a high affinity for the cysteine-histidine-rich domain 1 (CH1) of the transcriptional co-activators CBP/p300. CITED2 is particularly efficient in the inhibition of the hypoxia-inducible factor-1α (HIF-1α) dependent transcription by competing with it for the interaction with the CH1 domain. Here we report a direct and specific interaction between CITED2 and the F-box and leucine rich repeat protein 5 (FBXL5), a substrate adaptor protein which is part of E3 ubiquitin ligase complexes mediating protein degradation by the proteasome. We demonstrated that depletion of FBXL5 by RNA interference led to an increase of CITED2 protein levels. Conversely, overexpression of FBXL5 caused the decrease of CITED2 protein levels in a proteasome-dependent manner, and impaired the interaction between CITED2 and the CH1 domain of p300 in living cells. In undifferentiated mouse embryonic stem cells, the overexpression of FBXL5 also reduced Cited2 protein levels. Finally, we evidenced that FBXL5 overexpression and the consequent degradation of CITED2 enabled the transcriptional activity of the N-terminal transactivation domain of HIF-1α. Collectively, our results highlighted a novel molecular interaction between CITED2 and FBXL5, which might regulate the steady state CITED2 protein levels and contribute to the modulation of gene expression by HIF-1α.

The CBFβ gene encodes a transcription factor that, in combination with CBFα (also called Runx, runt-related transcription factor) regulates expression of several target genes. CBFβ interacts with all Runx family members, such as RUNX2, a regulator of bone-related gene transcription that contains a conserved DNA-binding domain. CBFβ stimulates DNA binding of the Runt domain, and is essential for most of the known functions of RUNX2. A comparative analysis of the zebrafish cbfβ gene and protein, and of its orthologous identified homologous proteins in different species indicates a highly conserved function. We cloned eleven zebrafish cbfβ gene transcripts, one resulting in the known Cbfβ protein (with 187 aa), and three additional variants resulting from skipping exon 5a (resulting in a protein with 174 aa) or exon 5b (resulting in a protein with 201 aa), both observed for the first time in zebrafish, and a completely novel isoform containing both exon 5a and 5b (resulting in a protein with 188 aa). Functional analysis of these isoforms provides insight into their role in regulating gene transcription. From the other variants two are premature termination Cbfβ forms, while the others show in-frame exon-skipping causing changes in the Cbfβ domain that may affect its function.

The somatic cell count (SCC) of refrigerated raw milk reflects the occurrence of mastitis in the herd and the management of rural property. Therefore, this study aimed to evaluate the SCC of the refrigerated raw milk and its determinant factors in dairy properties of Viçosa, State of Minas Gerais. Monthly, milk samples were taken from 44 cooling tanks during 2012. A structured questionnaire was applied to evaluate the production and management characteristics of the herds. There was a significant variation (p < 0.05) in the mean SCC of the milk, which was lower from June to August. Higher mean SCC and percentages of samples above the limit set by the legislation were observed in months of higher rainfall and ambient temperature (p < 0.05). Mechanical milking, lower milk yield, productivity, inadequate milking procedures, equipment hygiene, and water quality were all factors that influenced (p < 0.05) the SCC of raw refrigerated milk. Milker training is required for production of low SCC milk.

Neurogenic niches constitute a powerful endogenous source of new neurons that can be used for brain repair strategies. Neuronal differentiation of these cells can be regulated by molecules such as retinoic acid (RA) or by mild levels of reactive oxygen species (ROS) that are also known to upregulate RA receptor alpha (RAR alpha) levels. Data showed that neural stem cells from the subventricular zone (SVZ) exposed to blue light (405 nm laser) transiently induced NADPH oxidase-dependent ROS, resulting in beta-catenin activation and neuronal differentiation, and increased RAR alpha levels. Additionally, the same blue light stimulation was capable of triggering the release of RA from light-responsive nanoparticles (LR-NP). The synergy between blue light and LR-NP led to amplified neurogenesis both in vitro and in vivo, while offering a temporal and spatial control of RA release. In conclusion, this combinatory treatment offers great advantages to potentiate neuronal differentiation, and provides an innovative and efficient application for brain regenerative therapies. Statement of Significance Controlling the differentiation of stem cells would support the development of promising brain regenerative therapies. Blue light transiently increased reactive oxygen species, resulting in neuronal differentiation and increased retinoic acid receptor (RAR alpha) levels. Additionally, the same blue light stimulation was capable of triggering the release of RA from light-responsive nanoparticles (LR-NP). The synergy between blue light and LR-NP led to amplified neurogenesis, while offering a temporal and spatial control of RA release. In this sense, our approach relying on the modulation of endogenous stem cells for the generation of new neurons may support the development of novel clinical therapies. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. FEDER [FCOMP-01-0124-FEDER-041099, POCI-COMPETE2020] COMPETE [BD/79526/2011, Centro-07-ST24-FEDER-002008] L'Oreal-UNESCO Portugal for Women in Science Professor Francisco Pulido Valente Foundation, EC [ERC 307384] Operational Programme Competitiveness and Internationalization in Axis-I [007491] FCT [Project UID/Multi/00709]

Cited2 plays crucial roles in mouse embryonic stem cells self-renewal, the initiation of the somatic reprogramming process into induced pluripotent stem cells (iPSC) and the suppression of cell senescence. Here, we investigated the potential of CITED2 expression in combination with the Oct4, Sox2, Klf4 and c-Myc factors for reprogramming of primary mouse embryonic fibroblasts (MEF) at passage 2 and 4. The ectopic CITED2 expression in primary MEF prior to the onset of the reprogramming process, generated iPSC with less variability in the expression of endogenous pluripotency-related genes. In contrast, part of the MEF reprogrammed without ectopic expression of CITED2 at passage 4 originated partially reprogrammed iPSC or pre-iPSC. However, the overexpression of CITED2 in the pre-iPSC was insufficient to complete the reprogramming process into iPSC. These results indicated that ectopic CITED2 expression at the onset of the reprogramming process in combination with the reprogramming factors promotes a complete and homogeneous conversion of somatic cells into iPSC. FCT - Fundacao para a Ciencia e a Tecnologia [PTDC/BIM-MEC/4762/2014] Merck Sharp & Dhome Foundation - Portugal

Conducting polymer electrodes based on poly (3, 4 ethylenedioxythiophene): polystyrene sulfonate were used to record electrophysiological signals from autonomous cardiac contractile cells present in embryoid bodies. Signal detection was carried out by measuring the displacement current across the polymer/electrolyte double-layer capacitance, and compared with voltage detection. While for relatively low capacitance electrodes, the voltage amplification provides higher signal quality, and for high capacitive electrodes, the displacement current method exhibits a higher signal-to-noise ratio. It is proposed that the displacement current method combined with high capacitive polymer-based electrodes is adequate to measure clusters of cells and whole organs. Our approach has a great potential in fundamental studies of drug discovery and safety pharmacology. Portuguese Foundation for Science and Technology through the Implantable Organic Devices for Advanced Therapies Project [PTDC/EEI-AUT/5442/2014] Instituto de Telecomunicacoes [UID/Multi/04326/2013] info:eu-repo/semantics/publishedVersion

CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be insufficient.

Many aspects of physiology and behavior follow a circadian rhythm. Brain and muscle Arnt-like protein-1 (BMAL1) is a key component of the mammalian molecular clock, which controls circadian oscillations. In the rat, the gene encodingBmal1is located within hypertension susceptibility loci. We analyzed the SNP distribution pattern in a congenic interval associated with hypertension in the spontaneously hypertensive rat (SHR), and we show thatBmal1maps close to a region genetically divergent between SHR and its normotensive (Wistar–Kyoto) counterpart.Bmal1sequencing in rat strains identified 19 polymorphisms, including an SHR promoter variant that significantly affects Gata-4 activation of transcription in transient transfection experiments. A genetic association study designed to test the relevance of these findings in 1,304 individuals from 424 families primarily selected for type 2 diabetes showed that twoBMAL1haplotypes are associated with type 2 diabetes and hypertension. This comparative genetics finding translated from mouse and rat models to human provides evidence of a causative role ofBmal1variants in pathological components of the metabolic syndrome.

Summary The transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC., Graphical Abstract, Highlights • Overexpression of CITED2 in ESC promotes cardiogenesis upon differentiation • Cited2 depletion reduces ESC ability to generate cardiac cells • Cited2 expression is enriched in cardiac progenitors • CITED2 and ISL1 cooperate to promote ESC differentiation toward cardiomyocytes, In this article, Bragança and colleagues show that the transcriptional regulator Cited2 is essential for cardiac commitment of mouse embryonic stem cells (ESC), and its depletion in ESC impairs the expression of early mesoderm markers and cardiogenic transcription factors. In addition, they demonstrate that CITED2 and ISL1 proteins interact and cooperate to promote ESC differentiation toward cardiomyocytes.

Herein, we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors, decreased stemness, and increased proneurogenic gene expression. Importantly, this work reports for the first time a nanoparticle formulation able to modulate in vivo the subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases.

Part 16: Flexible and Transparent Oxide Electronics; International audience; This work presents an experimental characterization of electrochemically gated graphene field-effect transistors (EGFETs) to measure extracellular cell signals. The performance of the EGFETs was evaluated using cardiomyocytes cells. Extracellular signals with a peak value of 0.4 pico-amperes (pA) embedded in a noise level of 0.1 pA were recorded. Signals in current mode were compared with signals recorded as a voltage. Signals below 28 µV of magnitude can be detected in a noise floor of 7 µV with a signal-to-noise ratio of 4.

Conducting polymer electrodes based on poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) are used to record extracellular signals from autonomous cardiac contractile cells and glioma cell cultures. The performance of these conducting polymer electrodes is compared with Au electrodes. A small-signal impedance analysis shows that in the presence of an electrolyte, both Au and polymer electrodes establish high capacitive double-layers. However, the polymer/electrolyte interfacial resistance is 3 orders of magnitude lower than the resistance of the metal/electrolyte interface. The polymer low interfacial resistance minimizes the intrinsic thermal noise and increases the system sensitivity. However, when measurements are carried out in current mode a low interfacial resistance partially acts as a short circuit of the interfacial capacitance, this affects the signal shape.

In vertebrates, stem cells play a fundamental role in embryogenesis and lifetime homeostasis of adult tissues. The transcriptional regulator Cited2 is essential for many mouse developmental and morphological processes, and mutations in human CITED2 gene have repeatedly been associated with congenital heart defects. An increasing number of studies have now described the importance of Cited2 in self-renewal and cell fate decision of embryonic stem cells (ESC) and some adult stem cells. Interestingly, the loss of stem cell properties due to aging or extensive regeneration of some adult stem cells has been associated with a decrease of Cited2 expression, while an aberrant increase in CITED2 expression might play a critical role in malignant oncologic processes. Here, we review recent advances unravelling the complexity of Cited2 function as a regulator of the maintenance and cell fate decision of ESC and adult stem cells, and present its potential role in pathological conditions by modulating stem cells properties.

Malformations of the septum, outflow tract and aortic arch are the most common congenital cardiovascular defects and occur in mice lacking Cited2, a transcriptional coactivator of TFAP2. Here we show that Cited2(-/-) mice also develop laterality defects, including right isomerism, abnormal cardiac looping and hyposplenia, which are suppressed on a mixed genetic background. Cited2(-/-) mice lack expression of the Nodal target genes Pitx2c, Nodal and Ebaf in the left lateral plate mesoderm, where they are required for establishing laterality and cardiovascular development. CITED2 and TFAP2 were detected at the Pitx2c promoter in embryonic hearts, and they activate Pitx2c transcription in transient transfection assays. We propose that an abnormal Nodal-Pitx2c pathway represents a unifying mechanism for the cardiovascular malformations observed in Cited2(-/-) mice, and that such malformations may be the sole manifestation of a laterality defect.

Cited2 (CBP/p300 interacting transactivator with ED-rich tail 2) is required for embryonic development, coactivation of transcription factor AP-2, and inhibition of hypoxia-inducible factor 1 transactivation. Cited2 is induced by multiple growth factors and cytokines and oncogenically transforms cells. Here, we show that the proliferation of Cited2(-/-) mouse embryonic fibroblasts ceases prematurely. This is associated with a reduction in growth fraction, senescent cellular morphology, and increased expression of the cell proliferation inhibitors p16(INK4a), p19(ARF), and p15(INK4b). Deletion of INK4a/ARF (encoding p16(INK4a) and p19(ARF)) completely rescued the defective proliferation of Cited2(-/-) fibroblasts. However, the deletion of INK4a/ARF did not rescue the embryonic malformations observed in Cited2(-/-) mice, indicating that INK4a/ARF-independent pathways are likely to be involved here. We found that Cited2(-/-) fibroblasts had reduced expression of the polycomb-group genes Bmi1 and Mel18, which function as INK4a/ARF and Hox repressors. Complementation with CITED2-expressing retrovirus enhanced proliferation, induced Bmi1/Mel18 expression, and decreased INK4a/ARF expression. Bmi1- and Mel18-expressing retroviruses enhanced the proliferation of Cited2(-/-) fibroblasts, indicating that they function downstream of Cited2. Our results provide genetic evidence that Cited2 controls the expression of INK4a/ARF and fibroblast proliferation, at least in part via the polycomb-group genes Bmi1 and Mel18.