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1.
Dev Cell ; 59(4): 465-481.e6, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38237590

RESUMO

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.


Assuntos
Epigênese Genética , Células-Tronco Pluripotentes , Animais , Camundongos , Metilação de DNA/genética , Cromatina/metabolismo , DNA/metabolismo , Diferenciação Celular/genética , Camadas Germinativas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
2.
bioRxiv ; 2023 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-36993548

RESUMO

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naïive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

3.
Global Spine J ; 12(4): 646-653, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-33131331

RESUMO

STUDY DESIGN: Retrospective cohort study. OBJECTIVES: The OF classification is a new classification for osteoporotic vertebral fractures. The aim of this study was to clarify the relationship between preoperative OF subgroups and the postoperative outcome after kyphoplasty in patients with such fractures. METHODS: Patients who underwent kyphoplasty of a single osteoporotic vertebral fracture were included and divided into groups according to the OF subgroups. Pre- and postoperative plain radiographs were analyzed in regard to the restoration of vertebral body height and local kyphotic angle (LKA). Additionally, clinical data including pre- and postoperative Visual Analogue Scale pain scores was documented. The clinical and radiological results were compared pre- and postoperatively within groups and between groups. RESULTS: A total of 156 patients from OF subgroups 2 to 4 were included (OF 2: n = 58; OF 3: n = 36; OF 4: n = 62). Patients from all groups experienced significant pain relief postoperatively (P < .001). Patients with OF 2 fractures showed a repositioning of the vertebral body height in the anterior and middle portions (both P < .001), but no significant improvement in LKA. For OF 3 and 4 fractures, there was a significant restoration of vertebral body height (P < .001 for both) and a significant improvement of LKA (P < .001 for both). The highest average restoration was noted in the OF 4 group. CONCLUSIONS: A higher OF subgroup is related to a higher radiological benefit from kyphoplasty. This confirms that the OF classification is an appropriate tool for the preoperative assessment of osteoporotic fractures.

4.
Eur Spine J ; 29(10): 2560-2567, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32556626

RESUMO

PURPOSE: To clarify the relationship between operative timing and the early post-operative radiological and clinical outcome after kyphoplasty. METHODS: We conducted a retrospective cohort study including patients who underwent kyphoplasty of a single vertebra. Patients were divided into three groups (acute [< 2 weeks], subacute [2-6 weeks] or chronic [6-51 weeks]) based on the interval between fracture and surgery. The relative vertebral body height (VBH) and local kyphotic angle (LKA) of the fractured vertebra (measured on plain radiographs) as well as pain and use of analgesics were compared pre- and post-operatively (day 2) and between the groups. RESULTS: A total of 230 patients (100 with acute, 91 with subacute and 39 with chronic fractures) with fractures from T4 to L5 were included. In all groups, there was a significant post-operative improvement in the anterior (8.9-12.9%) and middle (10.7-13.4%) VBH (all groups: p < 0.001), LKA (acute: 3.8°, p < 0.001; subacute: 4.3°, p < 0.001; chronic: 1.7°, p = 0.046) and pain. The use of analgesics significantly decreased post-operatively in the acute and subacute groups, but did not significantly change in the chronic group. Patients from acute (p = 0.042) and subacute (p = 0.027) groups showed significantly better post-operative correction of the LKA than the chronic group. CONCLUSION: Kyphoplasty is effective for vertebral height restoration as well as pain relief for both acute, subacute and chronic fractures. However, the achievable correction of the fracture-related local kyphosis decreases significantly after 6 weeks. Therefore, we recommend making a final decision about conservative vs. operative treatment within 6 weeks to ensure better height restoration in surgically treated patients.


Assuntos
Fraturas por Compressão , Cifoplastia , Fraturas por Osteoporose , Fraturas da Coluna Vertebral , Seguimentos , Humanos , Medição da Dor , Estudos Retrospectivos , Fraturas da Coluna Vertebral/diagnóstico por imagem , Fraturas da Coluna Vertebral/cirurgia , Resultado do Tratamento
5.
Blood Purif ; 49(1-2): 55-62, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31527371

RESUMO

BACKGROUND AND AIMS: Acute and acute on chronic liver failure are life-threatening conditions, and bridging to transplantation is complicated by a paucity of suitable organs for children. While different modalities of extracorporeal liver support exist, their use in children is complicated by a large extracorporeal volume, and data on their use in children is limited. The aim of this analysis was to investigate the efficacy and safety of single-pass albumin dialysis (SPAD) in children with liver failure. METHODS: Retrospective medical chart review of pediatric patients with liver failure treated with SPAD. The decrease in hepatic encephalopathy (HE) and the serum levels of bilirubin and ammonia were measured to determine efficacy. Adverse events were documented to assess safety. RESULTS: Nineteen pediatric patients with a median age of 25.5 months and a median body weight of 11.9 kg were treated with SPAD between January 2011 and March 2018. Total bilirubin (p < 0.001) and ammonia (p = 0.02) significantly decreased after treatment with SPAD. As clinical outcome parameter, HE significantly improved (p = 0.001). Twelve patients were bridged successfully to liver transplantation. In all patients, 71 SPAD sessions were run. Clotting in the dialysis circuit was observed in 49% of all sessions. Heparin and citrate were used for anticoagulation and were significantly superior to dialysis without any anticoagulation (p= 0.03). Transfusion of packed blood cells (57%) and catecholamine therapy (49%) were frequently necessary. CONCLUSIONS: Treatment with SPAD was effective in detoxification, as measured by significant improvement of HE and clearance from surrogate laboratory parameters.


Assuntos
Anticoagulantes/administração & dosagem , Ácido Cítrico/administração & dosagem , Heparina/administração & dosagem , Encefalopatia Hepática/terapia , Falência Hepática/terapia , Albumina Sérica Humana , Adolescente , Criança , Pré-Escolar , Feminino , Encefalopatia Hepática/sangue , Humanos , Lactente , Falência Hepática/sangue , Transplante de Fígado , Masculino , Estudos Retrospectivos
6.
Nat Commun ; 9(1): 5304, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30546012

RESUMO

Wnt/Wg-signalling is critical signalling in all metazoans. Recent studies suggest that IFT-A proteins and Kinesin-2 modulate canonical Wnt/Wg-signalling independently of their ciliary role. Whether they function together in Wnt-signalling and their mechanistic role in the pathway remained unresolved. Here we demonstrate that Kinesin-2 and IFT-A proteins act as a complex during Drosophila Wg-signalling, affecting pathway activity in the same manner, interacting genetically and physically, and co-localizing with ß-catenin, the mediator of Wnt/Wg-signalling on microtubules. Following pathway activation, Kinesin-2/IFT-A mutant cells exhibit high cytoplasmic ß-catenin levels, yet fail to activate Wg-targets. In mutant tissues in both, Drosophila and mouse/MEFs, nuclear localization of ß-catenin is markedly reduced. We demonstrate a conserved, motor-domain dependent function of the Kinesin-2/IFT-A complex in promoting nuclear translocation of ß-catenin. We show that this is mediated by protecting ß-catenin from a conserved cytoplasmic retention process, thus identifying a mechanism for Kinesin-2/IFT-A in Wnt-signalling that is independent of their ciliary role.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Cinesinas/metabolismo , Asas de Animais/embriologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Proteínas de Transporte/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Cinesinas/genética , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Via de Sinalização Wnt/fisiologia
7.
Elife ; 62017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29168693

RESUMO

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.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/embriologia , Proteína Nodal/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Deleção de Genes , Camundongos , Células-Tronco Pluripotentes/fisiologia , Fatores de Transcrição/genética
8.
Dev Dyn ; 245(5): 547-57, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26845388

RESUMO

The notochord is a structure common to all chordates, and the feature that the phylum Chordata has been named after. It is a rod-like mesodermal structure that runs the anterior-posterior length of the embryo, adjacent to the ventral neural tube. The notochord plays a critical role in embryonic tissue patterning, for example the dorsal-ventral patterning of the neural tube. The cells that will come to form the notochord are specified at gastrulation. Axial mesodermal cells arising at the anterior primitive streak migrate anteriorly as the precursors of the notochord and populate the notochordal plate. Yet, even though a lot of interest has centered on investigating the functional and structural roles of the notochord, we still have a very rudimentary understanding of notochord morphogenesis. The events driving the formation of the notochord are rapid, taking place over the period of approximately a day in mice. In this commentary, we provide an overview of our current understanding of mouse notochord morphogenesis, from the initial specification of axial mesendodermal cells at the primitive streak, the emergence of these cells at the midline on the surface of the embryo, to their submergence and organization of the stereotypically positioned notochord. We will also discuss some key open questions. Developmental Dynamics 245:547-557, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Morfogênese , Notocorda/embriologia , Animais , Mesoderma/citologia , Camundongos , Linha Primitiva
9.
Dev Cell ; 34(6): 705-18, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26364750

RESUMO

The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, we conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. Our results show no effect on Hedgehog signaling. In contrast, our screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting ß-catenin/Armadillo activity.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Cílios/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Flagelos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
10.
Nat Commun ; 6: 6751, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25849195

RESUMO

A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo.


Assuntos
Fator 1 de Ribosilação do ADP/genética , Complexo 1 de Proteínas Adaptadoras/genética , Polaridade Celular/genética , Proteínas de Drosophila/genética , Drosophila/embriologia , Asas de Animais/embriologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Desgrenhadas , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fosfoproteínas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
11.
EMBO Rep ; 14(8): 718-25, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23797875

RESUMO

Wnt/ß-catenin signalling is central to development and its regulation is essential in preventing cancer. Using phosphorylation of Dishevelled as readout of pathway activation, we identified Drosophila Wnk kinase as a new regulator of canonical Wnt/ß-catenin signalling. WNK kinases are known for regulating ion co-transporters associated with hypertension disorders. We demonstrate that wnk loss-of-function phenotypes resemble canonical Wnt pathway mutants, while Wnk overexpression causes gain-of-function canonical Wnt-signalling phenotypes. Importantly, knockdown of human WNK1 and WNK2 also results in decreased Wnt signalling in mammalian cell culture, suggesting that Wnk kinases have a conserved function in ensuring peak levels of canonical Wnt signalling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Desgrenhadas , Proteínas de Drosophila , Drosophila melanogaster , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lentivirus/genética , Antígenos de Histocompatibilidade Menor , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK , beta Catenina/metabolismo
12.
Am J Physiol Gastrointest Liver Physiol ; 294(6): G1335-43, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18388184

RESUMO

During the early postnatal period lymphocytes migrate into the mouse small intestine. Migrating infiltrative lymphocytes have the potential to affect the epithelial cells via secreted cytokines. Such cross talk can result in the elicitation of an epithelial transcriptional response. Knowledge about such physiological cross talk between the immune system and the epithelium in the postnatal small intestinal mucosa is lacking. We have investigated the transcriptome changes occurring in the postnatal mouse small intestine using DNA microarray technology, immunocytochemistry, and quantitative real-time RT-PCR analysis. The DNA microarray data were analyzed bioinformatically by using a combination of projections to latent structures analysis and functional annotation analysis. The results show that infiltrating lymphocytes appear in the mouse small intestine in the late postweaning period and give rise to distinct changes in the epithelial transcriptome. Of particular interest is the expression of three genes encoding a mucin (Muc4), a mucinlike protein (16000D21Rik), and ATP citrate lyase (Acly). All three genes were shown to be expressed by the epithelium and to be upregulated in response to lymphocytic migration into the small intestinal mucosa.


Assuntos
Comunicação Celular/fisiologia , Células Epiteliais/fisiologia , Intestino Delgado/metabolismo , Linfócitos/citologia , Proteoma/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Animais , Animais Recém-Nascidos , Movimento Celular/fisiologia , Células Cultivadas , Células Epiteliais/citologia , Linfócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL
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