Applied Hepatic Bioengineering: Modeling the Human Liver Using Organoid and Liver-on-a-Chip Technologies.

The liver is the most important metabolic hub of endo and xenobiotic compounds. Pre-clinical studies using rodents to evaluate the toxicity of new drugs and cosmetics may produce inconclusive results for predicting clinical outcomes in humans, moreover being banned in the European Union. Human liver modeling using primary hepatocytes presents low reproducibility due to batch-to-batch variability, while iPSC-derived hepatocytes in monolayer cultures (2D) show reduced cellular functionality. Here we review the current status of the two most robust in vitro approaches in improving hepatocyte phenotype and metabolism while mimicking the hepatic physiological microenvironment: organoids and liver-on-chip. Both technologies are reviewed in design and manufacturing techniques, following cellular composition and functionality. Furthermore, drug screening and liver diseases modeling efficiencies are summarized. Finally, organoid and liver-on-chip technologies are compared regarding advantages and limitations, aiming to guide the selection of appropriate models for translational research and the development of such technologies.

Soluble factors of mesenchimal stem cells (FS-MSC) as a potential tool to reduce inflammation in donor's lungs after hypovolemic shock. / Fatores solúveis de células-tronco mesenquimais (FS-CTM) como uma ferramenta potencial para reduzir a inflamação nos pulmões de doadores após choque hipovolêmico.

OBJECTIVE: The shortage of viable lungs is still a major obstacle for transplantation. Trauma victims who represent potential lung donors commonly present hypovolemic shock leading to pulmonary inflammation and deterioration and rejection after transplantation. Seeking to improve lung graft, new approaches to donor treatment have been tested. This study focuses on treatment with mesenchymal stem cells (MSCs) or soluble factors produced by MSCs (FS-MSC) using a rat model for lung donors after hemorrhagic shock. METHODS: Forty-eight rats were divided into four groups: Sham (n=12), animals without induction of hypovolemic shock; Shock (n=12), animals submitted to hypovolemic shock (mean arterial pressure 40 mmHg); MSC (n=12), animals submitted to hypovolemic shock and treated with MSCs, and FS (n=12), animals submitted to hypovolemic shock and treated with FS-MSC. The animals were subjected to a 50-minute hypovolemic shock (40 mmHg) procedure. The treated animals were monitored for 115 minutes. We performed histopathology of lung tissue and quantification of inflammatory markers (TNF-α, IL-1ß, IL-6, IL-10, iCAM and vCAM) in lung tissue and peripheral blood leukocytes (PBLs). RESULTS: Hemorrhagic shock resulted in higher PBLs and neutrophil infiltrate in the lungs. FS animals had lower neutrophil density comparing with Shock and MSC animals (p<0.001). No differences in the cytokine levels in lung tissue were observed between the groups. CONCLUSIONS: The lungs of rats submitted to hemorrhagic shock and treated with FS-MSC showed reduced inflammation indicated in a decrease in lung neutrophil infiltrate.

OBJETIVO: A escassez de pulmões viáveis ainda é um grande obstáculo para o transplante. As vítimas de trauma, que constituem potenciais doadores de pulmão, comumente apresentam choque hipovolêmico que acarreta inflamação e deterioração pulmonar e rejeição após o transplante. Buscando melhorar o enxerto pulmonar, testaram-se novas abordagens ao tratamento do doador. Este estudo foca o tratamento com células-tronco mesenquimais (CTMs) ou fatores solúveis produzidos pelas CTMs (FS-CTMs), usando um modelo com ratos para doadores de pulmão após choque hemorrágico. MÉTODOS: Quarenta e oito ratos foram divididos em quatro grupos: Controle (n=12), animais sem indução de choque hipovolêmico; Choque (n=12), animais submetidos a choque hipovolêmico (pressão arterial média de 40 mmHg); CTM (n=12), animais submetidos a choque hipovolêmico e tratados com CTMs; e FS (n=12), animais submetidos a choque hipovolêmico e tratados com FS-CTMs. Os animais foram submetidos a um procedimento de choque hipovolêmico (40 mmHg) com 50 minutos de duração. Os animais tratados foram monitorados por 115 minutos. Realizamos análise histopatológica do tecido pulmonar e quantificação dos marcadores inflamatórios (TNF-α, IL-1ß, IL-6, IL-10, iCAM e vCAM) no tecido pulmonar e leucócitos no sangue periférico (LSPs). RESULTADOS: O choque hemorrágico resultou em taxas mais altas de LSPs e infiltrado de neutrófilos nos pulmões. Os animais do grupo FS apresentaram menor densidade de neutrófilos em comparação com os animais dos grupos Choque e CTM (p<0,001). Não foram observadas diferenças entre os grupos quanto aos níveis de citocinas no tecido pulmonar. CONCLUSÃO: Os pulmões dos ratos submetidos a choque hemorrágico e tratados com FS-CTM apresentaram inflamação reduzida indicada por uma diminuição do infiltrado de neutrófilos nos pulmões.

Pre-coating decellularized liver with HepG2-conditioned medium improves hepatic recellularization.

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

Fatores solúveis de células-tronco mesenquimais (FS-CTM) como uma ferramenta potencial para reduzir a inflamação nos pulmões de doadores após choque hipovolêmico / Soluble factors of mesenchimal stem cells (FS-MSC) as a potential tool to reduce inflammation in donor's lungs after hypovolemic shock

RESUMO Objetivo A escassez de pulmões viáveis ainda é um grande obstáculo para o transplante. As vítimas de trauma, que constituem potenciais doadores de pulmão, comumente apresentam choque hipovolêmico que acarreta inflamação e deterioração pulmonar e rejeição após o transplante. Buscando melhorar o enxerto pulmonar, testaram-se novas abordagens ao tratamento do doador. Este estudo foca o tratamento com células-tronco mesenquimais (CTMs) ou fatores solúveis produzidos pelas CTMs (FS-CTMs), usando um modelo com ratos para doadores de pulmão após choque hemorrágico. Métodos Quarenta e oito ratos foram divididos em quatro grupos: Controle (n=12), animais sem indução de choque hipovolêmico; Choque (n=12), animais submetidos a choque hipovolêmico (pressão arterial média de 40 mmHg); CTM (n=12), animais submetidos a choque hipovolêmico e tratados com CTMs; e FS (n=12), animais submetidos a choque hipovolêmico e tratados com FS-CTMs. Os animais foram submetidos a um procedimento de choque hipovolêmico (40 mmHg) com 50 minutos de duração. Os animais tratados foram monitorados por 115 minutos. Realizamos análise histopatológica do tecido pulmonar e quantificação dos marcadores inflamatórios (TNF-α, IL-1β, IL-6, IL-10, iCAM e vCAM) no tecido pulmonar e leucócitos no sangue periférico (LSPs). Resultados O choque hemorrágico resultou em taxas mais altas de LSPs e infiltrado de neutrófilos nos pulmões. Os animais do grupo FS apresentaram menor densidade de neutrófilos em comparação com os animais dos grupos Choque e CTM (p<0,001). Não foram observadas diferenças entre os grupos quanto aos níveis de citocinas no tecido pulmonar. Conclusão Os pulmões dos ratos submetidos a choque hemorrágico e tratados com FS-CTM apresentaram inflamação reduzida indicada por uma diminuição do infiltrado de neutrófilos nos pulmões.

ABSTRACT Objective The shortage of viable lungs is still a major obstacle for transplantation. Trauma victims who represent potential lung donors commonly present hypovolemic shock leading to pulmonary inflammation and deterioration and rejection after transplantation. Seeking to improve lung graft, new approaches to donor treatment have been tested. This study focuses on treatment with mesenchymal stem cells (MSCs) or soluble factors produced by MSCs (FS-MSC) using a rat model for lung donors after hemorrhagic shock. Methods Forty-eight rats were divided into four groups: Sham (n=12), animals without induction of hypovolemic shock; Shock (n=12), animals submitted to hypovolemic shock (mean arterial pressure 40 mmHg); MSC (n=12), animals submitted to hypovolemic shock and treated with MSCs, and FS (n=12), animals submitted to hypovolemic shock and treated with FS-MSC. The animals were subjected to a 50-minute hypovolemic shock (40 mmHg) procedure. The treated animals were monitored for 115 minutes. We performed histopathology of lung tissue and quantification of inflammatory markers (TNF-α, IL-1β, IL-6, IL-10, iCAM and vCAM) in lung tissue and peripheral blood leukocytes (PBLs). Results Hemorrhagic shock resulted in higher PBLs and neutrophil infiltrate in the lungs. FS animals had lower neutrophil density comparing with Shock and MSC animals (p<0.001). No differences in the cytokine levels in lung tissue were observed between the groups. Conclusions The lungs of rats submitted to hemorrhagic shock and treated with FS-MSC showed reduced inflammation indicated in a decrease in lung neutrophil infiltrate.

Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells.

Neural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Differential gene expression elicited by ZIKV infection in trophoblasts from congenital Zika syndrome discordant twins.

Zika virus (ZIKV) causes congenital Zika syndrome (CZS), which is characterized by fetal demise, microcephaly and other abnormalities. ZIKV in the pregnant woman circulation must cross the placental barrier that includes fetal endothelial cells and trophoblasts, in order to reach the fetus. CZS occurs in ~1-40% of cases of pregnant women infected by ZIKV, suggesting that mothers' infection by ZIKV during pregnancy is not deterministic for CZS phenotype in the fetus. Therefore, other susceptibility factors might be involved, including the host genetic background. We have previously shown that in three pairs of dizygotic twins discordant for CZS, neural progenitor cells (NPCs) from the CZS-affected twins presented differential in vitro ZIKV susceptibility compared with NPCs from the non-affected. Here, we analyzed human-induced-pluripotent-stem-cell-derived (hiPSC-derived) trophoblasts from these twins and compared by RNA-Seq the trophoblasts from CZS-affected and non-affected twins. Following in vitro exposure to a Brazilian ZIKV strain (ZIKVBR), trophoblasts from CZS-affected twins were significantly more susceptible to ZIKVBR infection when compared with trophoblasts from the non-affected. Transcriptome profiling revealed no differences in gene expression levels of ZIKV candidate attachment factors, IFN receptors and IFN in the trophoblasts, either before or after ZIKVBR infection. Most importantly, ZIKVBR infection caused, only in the trophoblasts from CZS-affected twins, the downregulation of genes related to extracellular matrix organization and to leukocyte activation, which are important for trophoblast adhesion and immune response activation. In addition, only trophoblasts from non-affected twins secreted significantly increased amounts of chemokines RANTES/CCL5 and IP10 after infection with ZIKVBR. Overall, our results showed that trophoblasts from non-affected twins have the ability to more efficiently activate genes that are known to play important roles in cell adhesion and in triggering the immune response to ZIKV infection in the placenta, and this may contribute to predict protection from ZIKV dissemination into fetuses' tissues.

Safety, Tumor Reduction, and Clinical Impact of Zika Virus Injection in Dogs with Advanced-Stage Brain Tumors.

Malignant brain tumors are among the most aggressive cancers with poor prognosis and no effective treatment. Recently, we reported the oncolytic potential of Zika virus infecting and destroying the human central nervous system (CNS) tumors in vitro and in immunodeficient mice model. However, translating this approach to humans requires pre-clinical trials in another immunocompetent animal model. Here, we analyzed the safety of Brazilian Zika virus (ZIKVBR) intrathecal injections in three dogs bearing spontaneous CNS tumors aiming an anti-tumoral therapy. We further assessed some aspects of the innate immune and inflammatory response that triggers the anti-tumoral response observed during the ZIKVBR administration in vivo and in vitro. For the first time, we showed that there were no negative clinical side effects following ZIKVBR CNS injections in dogs, confirming the safety of the procedure. Furthermore, the intrathecal ZIKVBR injections reduced tumor size in immunocompetent dogs bearing spontaneous intracranial tumors, improved their neurological clinical symptoms significantly, and extended their survival by inducing the destruction specifically of tumor cells, sparing normal neurons, and activating an immune response. These results open new perspectives for upcoming virotherapy using ZIKV to destroy and induce an anti-tumoral immune response in CNS tumors for which there are currently no effective treatments.

Safety, Tumor Reduction, and Clinical Impact of Zika Virus Injection in Dogs with Advanced-Stage Brain Tumors

Malignant brain tumors are among the most aggressive cancers with poor prognosis and no effective treatment. Recently, we reported the oncolytic potential of Zika virus infecting and destroying the human central nervous system (CNS) tumors in vitro and in immunodeficient mice model. However, translating this approach to humans requires pre-clinical trials in another immunocompetent animal model. Here, we analyzed the safety of Brazilian Zika virus (ZIKVBR) intrathecal injections in three dogs bearing spontaneous CNS tumors aiming an anti-tumoral therapy. We further assessed some aspects of the innate immune and inflammatory response that triggers the anti-tumoral response observed during the ZIKVBR administration in vivo and in vitro. For the first time, we showed that there were no negative clinical side effects following ZIKVBR CNS injections in dogs, confirming the safety of the procedure. Furthermore, the intrathecal ZIKVBR injections reduced tumor size in immunocompetent dogs bearing spontaneous intracranial tumors, improved their neurological clinical symptoms significantly, and extended their survival by inducing the destruction specifically of tumor cells, sparing normal neurons, and activating an immune response. These results open new perspectives for upcoming virotherapy using ZIKV to destroy and induce an anti-tumoral immune response in CNS tumors for which there are currently no effective treatments.

Differential gene expression elicited by ZIKV infection in trophoblasts from congenital Zika syndrome discordant twins

Zika virus (ZIKV) causes congenital Zika syndrome (CZS), which is characterized by fetal demise, microcephaly and other abnormalities. ZIKV in the pregnant woman circulation must cross the placental barrier that includes fetal endothelial cells and trophoblasts, in order to reach the fetus. CZS occurs in ~1–40% of cases of pregnant women infected by ZIKV, suggesting that mothers’ infection by ZIKV during pregnancy is not deterministic for CZS phenotype in the fetus. Therefore, other susceptibility factors might be involved, including the host genetic background. We have previously shown that in three pairs of dizygotic twins discordant for CZS, neural progenitor cells (NPCs) from the CZS-affected twins presented differential in vitro ZIKV susceptibility compared with NPCs from the non-affected. Here, we analyzed human-induced-pluripotent-stem-cell-derived (hiPSC-derived) trophoblasts from these twins and compared by RNA-Seq the trophoblasts from CZS-affected and non-affected twins. Following in vitro exposure to a Brazilian ZIKV strain (ZIKVBR), trophoblasts from CZS-affected twins were significantly more susceptible to ZIKVBR infection when compared with trophoblasts from the non-affected. Transcriptome profiling revealed no differences in gene expression levels of ZIKV candidate attachment factors, IFN receptors and IFN in the trophoblasts, either before or after ZIKVBR infection. Most importantly, ZIKVBR infection caused, only in the trophoblasts from CZS-affected twins, the downregulation of genes related to extracellular matrix organization and to leukocyte activation, which are important for trophoblast adhesion and immune response activation. In addition, only trophoblasts from non-affected twins secreted significantly increased amounts of chemokines RANTES/CCL5 and IP10 after infection with ZIKVBR. Overall, our results showed that trophoblasts from non-affected twins have the ability to more efficiently activate genes that are known to play important roles in cell adhesion and in triggering the immune response to ZIKV infection in the placenta, and this may contribute to predict protection from ZIKV dissemination into fetuses’ tissues.

Safety, tumor reduction, and clinical impact of Zika Virus injection in dogs with advanced-stage brain tumors

Malignant brain tumors are among the most aggressive cancers with poor prognosis and no effective treatment. Recently, we reported the oncolytic potential of Zika virus infecting and destroying the human central nervous system (CNS) tumors in vitro and in immunodeficient mice model. However, translating this approach to humans requires pre-clinical trials in another immunocompetent animal model. Here, we analyzed the safety of Brazilian Zika virus (ZIKVBR) intrathecal injections in three dogs bearing spontaneous CNS tumors aiming an anti-tumoral therapy. We further assessed some aspects of the innate immune and inflammatory response that triggers the anti-tumoral response observed during the ZIKVBR administration in vivo and in vitro. For the first time, we showed that there were no negative clinical side effects following ZIKVBR CNS injections in dogs, confirming the safety of the procedure. Furthermore, the intrathecal ZIKVBR injections reduced tumor size in immunocompetent dogs bearing spontaneous intracranial tumors, improved their neurological clinical symptoms significantly, and extended their survival by inducing the destruction specifically of tumor cells, sparing normal neurons, and activating an immune response. These results open new perspectives for upcoming virotherapy using ZIKV to destroy and induce an anti-tumoral immune response in CNS tumors for which there are currently no effective treatments.

3D bioprinting of liver spheroids derived from human induced pluripotent stem cells sustain liver function and viability in vitro.

The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver's epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-ß.

BACKGROUND: Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPS-derived cells could represent great challenges for a translational perspective. METHODS: Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day 12. RESULTS: We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-ß and Wnt signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-ß/Wnt signaling activity. CONCLUSION: Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.

TET Upregulation Leads to 5-Hydroxymethylation Enrichment in Hepatoblastoma.

Hepatoblastoma is an embryonal liver tumor carrying few genetic alterations. We previously disclosed in hepatoblastomas a genome-wide methylation dysfunction, characterized by hypermethylation at specific CpG islands, in addition to a low-level hypomethylation pattern in non-repetitive intergenic sequences, in comparison to non-tumoral liver tissues, shedding light into a crucial role for epigenetic dysregulation in this type of cancer. To explore the underlying mechanisms possibly related to aberrant epigenetic modifications, we evaluated the expression profile of a set of genes engaged in the epigenetic machinery related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L, UHRF1, TET1, TET2, and TET3), as well as the 5-hydroxymethylcytosine (5hmC) global level. We observed in hepatoblastomas a general disrupted expression of these genes from the epigenetic machinery, mainly UHRF1, TET1, and TET2 upregulation, in association with an enrichment of 5hmC content. Our findings support a model of active demethylation by TETs in hepatoblastoma, probably during early stages of liver development, which in combination with UHRF1 overexpression would lead to DNA hypomethylation and an increase in overall 5hmC content. Furthermore, our data suggest that decreased 5hmC content might be associated with poor survival rate, highlighting a pivotal role of epigenetics in hepatoblastoma development and progression.

Publisher Correction: Discordant congenital Zika syndrome twins show differential in vitro viral susceptibility of neural progenitor cells.

The original PDF version of this Article contained errors in the spelling of Luiz Carlos Caires-Júnior, Uirá Souto Melo, Bruno Henrique Silva Araujo, Alessandra Soares-Schanoski, Murilo Sena Amaral, Kayque Alves Telles-Silva, Vanessa van der Linden, Helio van der Linden, João Ricardo Mendes de Oliveira, Nivia Maria Rodrigues Arrais, Joanna Goes Castro Meira, Ana Jovina Barreto Bispo, Esper Abrão Cavalheiro, and Robert Andreata-Santos, which were incorrectly given as Luiz Carlos de Caires Jr., UiráSouto Melo, Bruno Silva Henrique Araujo, Alessandra Soares Schanoski, MuriloSena Amaral, Kayque Telles Alves Silva, Vanessa Van der Linden, Helio Van der Linden, João Mendes Ricardo de Oliveira, Nivia Rodrigues Maria Arrais, Joanna Castro Goes Meira, Ana JovinaBarreto Bispo, EsperAbrão Cavalheiro, and Robert Andreata Santos. Furthermore, in both the PDF and HTML versions of the Article, the top panel of Fig. 3e was incorrectly labeled '10608-1' and should have been '10608-4', and financial support from CAPES and DECIT-MS was inadvertently omitted from the Acknowledgements section. These errors have now been corrected in both the PDF and HTML versions of the Article.

Discordant congenital Zika syndrome twins show differential in vitro viral susceptibility of neural progenitor cells.

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

Discordant congenital Zika syndrome twins show differential in vitro viral susceptibility of neural progenitor cells

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

Discordant congenital Zika syndrome twins show differential in vitro viral susceptibility of neural progenitor cells

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

Altered behavior of adult obese rats by monosodium l-glutamate neonatal treatment is related to hypercorticosteronemia and activation of hypothalamic ERK1 and ERK2.

OBJECTIVES: Obesity is a metabolic and hormonal disorder with serious social and psychological impacts. There is a close relationship among obesity, neuroendocrine homeostasis and behavioral patterns. However, few data are available in the literature regarding this subject. This study assessed behavior and memory of adult obese rats by monosodium l-glutamate (MSG) neonatal treatment or highly palatable dietary treatment. METHODS: MSG obesity was induced by subcutaneous injections of MSG (4 mg/g) during the first 5 days of life (Ob-MSG); control group (C-MSG), received saline solution equimolar. Both groups were fed with commercial chow. To induce dietary obesity, 21-day-old rats were assigned to two experimental diets: highly palatable diet (Ob-Diet) and control diet (C-Diet) composed of commercial chow. Ninety-day-old animals were submitted to behavioral assessment by the open-field test and short- and long-term memory by the object recognition test. Biometric variables were obtained, the Lee index was calculated and mass of retroperitoneal and perigonadal fat pads was measured. Furthermore, an altered behavioral profile was investigated by quantification of plasmatic corticosterone, expression, and activity of hypothalamic extracellular signal-regulated kinase protein (ERK) 1 and 2. RESULTS: Increased Lee index and fat pads were observed in Ob-MSG and Ob-Diet groups. Ob-MSG presented a higher level of anxiety and impaired long-term memory compared to C-MSG, while there was no difference between Ob-Diet and C-Diet. The Ob-MSG group presented a higher level of plasmatic corticosterone and increased phosphorylation of hypothalamic ERK1 and 2. DISCUSSION: Both treatments induced obesity but only Ob-MSG showed altered behavioral parameters, which is related to increased concentration of corticosterone and hypothalamic ERK1 and 2 activation.