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1.
Health Res Policy Syst ; 20(1): 68, 2022 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-35717233

RESUMO

BACKGROUND: COVID-19 has tested healthcare and research systems around the world, forcing the large-scale reorganization of hospitals, research infrastructure and resources. The United Kingdom has been singled out for the speed and scale of its research response. The efficiency of the United Kingdom's research mobilization was in large part predicated on the pre-existing embeddedness of the clinical research system within the National Health Service (NHS), a public, free-at-point-of-delivery healthcare system. In this paper we discuss the redeployment of the clinical research workforce to support the pandemic clinical services, detailing the process of organizing this redeployment, as well as the impacts redeployment has had on both staff and research delivery at one research-intensive acute NHS trust in London. METHODS: A social science case study of one large research-active NHS trust drawing on data from an online questionnaire; participant observation of key research planning meetings; semi-structured interviews with staff involved in research; and document analysis of emails and official national and trust communications. RESULTS: We found that at our case-study hospital trust, the research workforce was a resource that was effectively redeployed as part of the pandemic response. Research delivery workers were redeployed to clinical roles, to COVID-related research and to work maintaining the research system during the redeployment itself. Redeployed research workers faced some difficulties with technology and communication, but many had a positive experience and saw the redeployment as a significant and valuable moment in their career. CONCLUSIONS: This study explicates the role of the research delivery workforce for the United Kingdom's COVID response. Redeployed research workers facilitated the emergency response by delivering significant amounts of patient care. The public also benefited from having a well-developed research infrastructure in place that was able to flexibly respond to a novel virus. Many research workers feel that the NHS should provide more support for this distinctive workforce.


Assuntos
COVID-19 , Hospitais , Humanos , Pandemias , Medicina Estatal , Recursos Humanos
2.
Crit Care ; 24(1): 220, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32408883

RESUMO

BACKGROUND: The clinical effectiveness of neurally adjusted ventilatory assist (NAVA) has yet to be demonstrated, and preliminary studies are required. The study aim was to assess the feasibility of a randomized controlled trial (RCT) of NAVA versus pressure support ventilation (PSV) in critically ill adults at risk of prolonged mechanical ventilation (MV). METHODS: An open-label, parallel, feasibility RCT (n = 78) in four ICUs of one university-affiliated hospital. The primary outcome was mode adherence (percentage of time adherent to assigned mode), and protocol compliance (binary-≥ 65% mode adherence). Secondary exploratory outcomes included ventilator-free days (VFDs), sedation, and mortality. RESULTS: In the 72 participants who commenced weaning, median (95% CI) mode adherence was 83.1% (64.0-97.1%) and 100% (100-100%), and protocol compliance was 66.7% (50.3-80.0%) and 100% (89.0-100.0%) in the NAVA and PSV groups respectively. Secondary outcomes indicated more VFDs to D28 (median difference 3.0 days, 95% CI 0.0-11.0; p = 0.04) and fewer in-hospital deaths (relative risk 0.5, 95% CI 0.2-0.9; p = 0.032) for NAVA. Although overall sedation was similar, Richmond Agitation and Sedation Scale (RASS) scores were closer to zero in NAVA compared to PSV (p = 0.020). No significant differences were observed in duration of MV, ICU or hospital stay, or ICU, D28, and D90 mortality. CONCLUSIONS: This feasibility trial demonstrated good adherence to assigned ventilation mode and the ability to meet a priori protocol compliance criteria. Exploratory outcomes suggest some clinical benefit for NAVA compared to PSV. Clinical effectiveness trials of NAVA are potentially feasible and warranted. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01826890. Registered 9 April 2013.


Assuntos
Suporte Ventilatório Interativo/normas , Respiração Artificial/métodos , Fatores de Tempo , Adulto , Estudos de Viabilidade , Feminino , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Suporte Ventilatório Interativo/estatística & dados numéricos , Tempo de Internação/estatística & dados numéricos , Londres , Masculino , Pessoa de Meia-Idade , Respiração Artificial/estatística & dados numéricos
3.
Chromosome Res ; 21(6-7): 601-614, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24297756

RESUMO

The X-inactivation center is a hotbed of functional long noncoding RNAs in eutherian mammals. These RNAs are thought to help orchestrate the epigenetic transcriptional states of the two X-chromosomes in females as well as of the single X-chromosome in males. To balance X-linked gene expression between the sexes, females undergo transcriptional silencing of most genes on one of the two X-chromosomes in a process termed X-chromosome inactivation. While one X-chromosome is inactivated, the other X-chromosome remains active. Moreover, with a few notable exceptions, the originally established epigenetic transcriptional profiles of the two X-chromosomes is maintained as such through many rounds of cell division, essentially for the life of the organism. The stable and divergent transcriptional fates of the two X-chromosomes, despite residing in a shared nucleoplasm, make X-inactivation a paradigm of epigenetic transcriptional regulation. Originally proposed in 1961 by Mary Lyon, the X-inactivation hypothesis has been validated through much experimentation. In the last 25 years, the discovery and functional characterization has firmly established X-linked long noncoding RNAs as key players in choreographing X-chromosome inactivation.


Assuntos
Epigênese Genética/genética , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Animais , Mecanismo Genético de Compensação de Dose , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Cromossomo X/genética
4.
Proc Natl Acad Sci U S A ; 108(35): 14491-6, 2011 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-21849621

RESUMO

Stability and repair of DNA is of principal importance in cell survival. Heme oxygenase-1 (HO-1; Hmox1) is critical in maintaining cellular homeostasis, in large part through its ability to generate CO, but neither molecule has been studied in the setting of DNA damage. Naïve Hmox1(-/-) mice exhibit excessive tissue levels of γ-histone H2A, whereas administration of genotoxic stressors or irradiation in HO-1-deficient cells resulted in loss of ataxia-telangiectasia mutated/ataxia telangiectasia and Rad3-related protein and breast cancer 1, early onset induction with dysfunctional γ-H2AX foci and marked elevations in DNA damage. HO-1 induction or exposure to CO induced homologous recombination-mediated DNA repair through ataxia-telangiectasia mutated/ataxia telangiectasia and Rad3-related protein. In vivo, exposure of mice to CO followed by genotoxin (Adriamycin) or radiation-induced injury led to diminished tissue DNA damage and improved survival. We characterize a joint role for HO-1 and the gasotransmitter CO for appropriate DNA repair and provide a mechanism for their potent cytoprotective effects in various pathologies.


Assuntos
Monóxido de Carbono/farmacologia , Proteínas de Ciclo Celular/fisiologia , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Heme Oxigenase-1/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Quebras de DNA de Cadeia Dupla , Doxorrubicina/farmacologia , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos
5.
Proc Natl Acad Sci U S A ; 108(46): 18849-54, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22042868

RESUMO

The cellular response to an inflammatory stressor requires a proinflammatory cellular activation followed by a controlled resolution of the response to restore homeostasis. We hypothesized that biliverdin reductase (BVR) by binding biliverdin (BV) quells the cellular response to endotoxin-induced inflammation through phosphorylation of endothelial nitric oxide synthase (eNOS). The generated NO, in turn, nitrosylates BVR, leading to nuclear translocation where BVR binds to the Toll-like receptor-4 (TLR4) promoter at the Ap-1 sites to block transcription. We show in macrophages that BV-induced eNOS phosphorylation (Ser-1177) and NO production are mediated in part by Ca(2+)/calmodulin-dependent kinase kinase. Furthermore, we show that BVR is S-nitrosylated on one of three cysteines and that this posttranslational modification is required for BVR-mediated signaling. BV-induced nuclear translocation of BVR and inhibition of TLR4 expression is lost in macrophages derived from Enos(-/-) mice. In vivo in mice, BV provides protection from acute liver damage and is dependent on the availability of NO. Collectively, we elucidate a mechanism for BVR in regulating the inflammatory response to endotoxin that requires eNOS-derived NO and TLR4 signaling in macrophages.


Assuntos
Biliverdina/metabolismo , Núcleo Celular/metabolismo , Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Receptor 4 Toll-Like/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Endotoxinas/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Transcrição AP-1/metabolismo
6.
Nurse Res ; 32(1): 36-42, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38152019

RESUMO

BACKGROUND: Clinical research nurses and midwives (CRN/Ms) are highly specialised registered nurses. They combine their clinical nursing expertise with research knowledge and skills to aid in the delivery of rigorous, high-quality clinical research to improve health outcomes, the research participant's experience and treatment pathways ( Beer et al 2022 ). However, there is evidence that the transition into a CRN/M role is challenging for registered nurses. AIM: To discuss the development of a competency framework for CRN/Ms. DISCUSSION: The authors identified a gap in their organisation for standards that would support the development of CRN/Ms new to the role. The standards needed to be clear and accessible to use while encompassing the breadth of scope of CRN/Ms' practice. The authors used a systematic and inclusive process drawing on Benner's ( 1984 ) theory of competence development to develop a suitable framework. Stakeholders engaged in its development included research participants, inclusion agents and CRN/Ms. CONCLUSION: The project identified 15 elements that are core to the CRN/M role and the knowledge, skills and behaviours associated with it. IMPLICATIONS FOR PRACTICE: A large NHS trust has implemented the framework. It is also being shown to national and regional networks. Evaluation is under way.


Assuntos
Tocologia , Enfermeiras e Enfermeiros , Humanos , Gravidez , Feminino , Competência Clínica
7.
Nat Genet ; 2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39256583

RESUMO

X chromosome inactivation triggers a dramatic reprogramming of transcription and chromosome architecture. However, how the chromatin organization of inactive X chromosome is established de novo in vivo remains elusive. Here, we identified an Xist-separated megadomain structure (X-megadomains) on the inactive X chromosome in mouse extraembryonic lineages and extraembryonic endoderm (XEN) cell lines, and transiently in the embryonic lineages, before Dxz4-delineated megadomain formation at later stages in a strain-specific manner. X-megadomain boundary coincides with strong enhancer activities and cohesin binding in an Xist regulatory region required for proper Xist activation in early embryos. Xist regulatory region disruption or cohesin degradation impaired X-megadomains in extraembryonic endoderm cells and caused ectopic activation of regulatory elements and genes near Xist, indicating that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. These data reveal stepwise X chromosome folding and transcriptional regulation to achieve both essential gene activation and global silencing during the early stages of X chromosome inactivation.

8.
Nat Commun ; 15(1): 1721, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38409226

RESUMO

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.


Assuntos
Cromatina , Células-Tronco Embrionárias , Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , S-Adenosilmetionina/metabolismo
9.
Nurse Res ; 30(2): 12-17, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35545928

RESUMO

BACKGROUND: As clinical pressures evolved amid the COVID-19 pandemic, the importance of research activity came to the forefront of health and care service requirements. AIM: To illustrate through reflection the experiences of clinical research teams based in the UK during the pandemic. DISCUSSION: The article describes operational experiences in different settings and reflects on important themes and implications for future practice. The authors use a reflective model to share perspectives of leading research delivery roles in geographically and organisationally different settings. A patient's perspective was included from the outset of the reflective process. Delegates at an interactive masterclass conference in April 2021 also contributed their experiences. Seven themes characterise the research teams' response to the pandemic: prioritising, team-building, protection, limitation of autonomy, reduced bureaucracy, collaboration and transformation of process. Balance through compassionate leadership underpinned by ethically grounded decision-making was a theme throughout. CONCLUSION: Implicitly held, tacit knowledge progressed to explicit knowledge, formalising the research teams' responses to the pandemic partly into codified learning. The authors characterise the experience as an 'operational balancing act', whereby significant innovations were integrated into working practices and research delivery. IMPLICATIONS FOR PRACTICE: The pandemic demonstrated what research progress is possible when all resources are diverted to one novel virus. The value of research teams was elevated through treatment and vaccine trials and the contribution of those involved to patient care. This reinforces an invigorated commitment to resources as well as new acceptance of and belief in research as a core care activity across and throughout systems and organisations at all levels.


Assuntos
COVID-19 , Pandemias , Humanos
10.
Nat Commun ; 13(1): 2602, 2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35545632

RESUMO

XX female and XY male therian mammals equalize X-linked gene expression through the mitotically-stable transcriptional inactivation of one of the two X chromosomes in female somatic cells. Here, we describe an essential function of the X-linked homolog of an ancestral X-Y gene pair, Kdm5c-Kdm5d, in the expression of Xist lncRNA, which is required for stable X-inactivation. Ablation of Kdm5c function in females results in a significant reduction in Xist RNA expression. Kdm5c encodes a demethylase that enhances Xist expression by converting histone H3K4me2/3 modifications into H3K4me1. Ectopic expression of mouse and human KDM5C, but not the Y-linked homolog KDM5D, induces Xist in male mouse embryonic stem cells (mESCs). Similarly, marsupial (opossum) Kdm5c but not Kdm5d also upregulates Xist in male mESCs, despite marsupials lacking Xist, suggesting that the KDM5C function that activates Xist in eutherians is strongly conserved and predates the divergence of eutherian and metatherian mammals. In support, prototherian (platypus) Kdm5c also induces Xist in male mESCs. Together, our data suggest that eutherian mammals co-opted the ancestral demethylase KDM5C during sex chromosome evolution to upregulate Xist for the female-specific induction of X-inactivation.


Assuntos
Marsupiais , Ornitorrinco , RNA Longo não Codificante , Animais , Feminino , Genes Ligados ao Cromossomo X , Histona Desmetilases , Masculino , Mamíferos/genética , Marsupiais/genética , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromossomo X/genética , Cromossomo X/metabolismo , Inativação do Cromossomo X/genética
11.
Methods Mol Biol ; 2372: 123-144, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34417748

RESUMO

Long non-coding RNAs (lncRNAs) have been postulated to function in a number of DNA-based processes, most notably transcription. The detection of lncRNAs in situ can offer insights into their function. Fluorescence in situ hybridization (FISH) enables the detection of specific nucleic acid sequences, including lncRNAs, within individual cells. Current RNA FISH techniques can inform both the localization and expression level of RNA transcripts. Together with advances in microscopy, these in situ techniques now allow for visualization and quantification of even lowly expressed or unstable lncRNAs. When combined with detection of associated proteins and chromatin modifications by immunofluorescence, RNA FISH can lend essential insights into lncRNA function. Here, we describe an integrated set of protocols to detect, individually or in combination, specific RNAs, DNAs, proteins, and histone modifications in single cells at high sensitivity using conventional fluorescence microscopy.


Assuntos
RNA Longo não Codificante/genética , DNA , Imunofluorescência , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Proteínas
12.
Nat Commun ; 11(1): 961, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098950

RESUMO

India is located at a critical geographic crossroads for understanding the dispersal of Homo sapiens out of Africa and into Asia and Oceania. Here we report evidence for long-term human occupation, spanning the last ~80 thousand years, at the site of Dhaba in the Middle Son River Valley of Central India. An unchanging stone tool industry is found at Dhaba spanning the Toba eruption of ~74 ka (i.e., the Youngest Toba Tuff, YTT) bracketed between ages of 79.6 ± 3.2 and 65.2 ± 3.1 ka, with the introduction of microlithic technology ~48 ka. The lithic industry from Dhaba strongly resembles stone tool assemblages from the African Middle Stone Age (MSA) and Arabia, and the earliest artefacts from Australia, suggesting that it is likely the product of Homo sapiens as they dispersed eastward out of Africa.

13.
Elife ; 82019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30938678

RESUMO

Imprinted X-inactivation silences genes exclusively on the paternally-inherited X-chromosome and is a paradigm of transgenerational epigenetic inheritance in mammals. Here, we test the role of maternal vs. zygotic Polycomb repressive complex 2 (PRC2) protein EED in orchestrating imprinted X-inactivation in mouse embryos. In maternal-null (Eedm-/-) but not zygotic-null (Eed-/-) early embryos, the maternal X-chromosome ectopically induced Xist and underwent inactivation. Eedm-/- females subsequently stochastically silenced Xist from one of the two X-chromosomes and displayed random X-inactivation. This effect was exacerbated in embryos lacking both maternal and zygotic EED (Eedmz-/-), suggesting that zygotic EED can also contribute to the onset of imprinted X-inactivation. Xist expression dynamics in Eedm-/- embryos resemble that of early human embryos, which lack oocyte-derived maternal PRC2 and only undergo random X-inactivation. Thus, expression of PRC2 in the oocyte and transmission of the gene products to the embryo may dictate the occurrence of imprinted X-inactivation in mammals.


Assuntos
Camundongos/embriologia , Complexo Repressor Polycomb 2/metabolismo , Inativação do Cromossomo X , Animais , Camundongos Knockout , Complexo Repressor Polycomb 2/deficiência , RNA Longo não Codificante/metabolismo
15.
Methods Mol Biol ; 1861: 177-203, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30218368

RESUMO

X-chromosome inactivation is a dosage compensation mechanism that equalizes X-linked gene expression between male and female mammals through the transcriptional silencing of most genes on one of the two X-chromosomes in females. With a few key exceptions, once the X-chromosome is inactivated replicated copies of that X-chromosome are maintained as inactive in all descendant cells. X-inactivation is therefore a paradigm of epigenetic inheritance. Imprinted X-inactivation is a specialized form of X-inactivation that results in the silencing of the paternally derived X-chromosome. Due to its parent-of-origin-specific pattern of inactivation, imprinted X-inactivation is a model of mitotic as well as meiotic, i.e., transgenerational, epigenetic inheritance. All cells of the early mouse embryo undergo imprinted X-inactivation, a pattern that is subsequently maintained in extraembryonic cell types in vivo and in vitro. Here, we describe both high- and low-throughput approaches to interrogate imprinted X-inactivation in the mouse embryo as well in cultured extraembryonic stem cells.


Assuntos
Alelos , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica/métodos , Hibridização in Situ Fluorescente/métodos , Inativação do Cromossomo X , Animais , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Células-Tronco Embrionárias , Epigenômica/métodos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA/métodos
16.
Genome Biol ; 18(1): 82, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28468635

RESUMO

BACKGROUND: Polycomb repressive complex 2 (PRC2) catalyzes histone H3K27me3, which marks many transcriptionally silent genes throughout the mammalian genome. Although H3K27me3 is associated with silenced gene expression broadly, it remains unclear why some but not other PRC2 target genes require PRC2 and H3K27me3 for silencing. RESULTS: Here we define the transcriptional and chromatin features that predict which PRC2 target genes require PRC2/H3K27me3 for silencing by interrogating imprinted mouse X-chromosome inactivation. H3K27me3 is enriched at promoters of silenced genes across the inactive X chromosome. To abrogate PRC2 function, we delete the core PRC2 protein EED in F1 hybrid trophoblast stem cells (TSCs), which undergo imprinted inactivation of the paternally inherited X chromosome. Eed -/- TSCs lack H3K27me3 and Xist lncRNA enrichment on the inactive X chromosome. Despite the absence of H3K27me3 and Xist RNA, only a subset of the inactivated X-linked genes is derepressed in Eed -/- TSCs. Unexpectedly, in wild-type (WT) TSCs these genes are transcribed and are enriched for active chromatin hallmarks on the inactive-X, including RNA PolII, H3K27ac, and H3K36me3, but not the bivalent mark H3K4me2. By contrast, PRC2 targets that remain repressed in Eed -/- TSCs are depleted for active chromatin characteristics in WT TSCs. CONCLUSIONS: A comparative analysis of transcriptional and chromatin features of inactive X-linked genes in WT and Eed -/- TSCs suggests that PRC2 acts as a brake to prevent induction of transcribed genes on the inactive X chromosome, a mode of PRC2 function that may apply broadly.


Assuntos
Impressão Genômica , Complexo Repressor Polycomb 2/metabolismo , Cromossomo X/genética , Animais , Cromatina/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Inativação Gênica , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Complexo Repressor Polycomb 2/genética , Trofoblastos/citologia , Inativação do Cromossomo X
17.
J Cell Biol ; 216(12): 3981-3990, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29021220

RESUMO

Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.


Assuntos
Citocinese , Camadas Germinativas/ultraestrutura , Morfogênese/genética , Células-Tronco Pluripotentes/ultraestrutura , Actinas/genética , Actinas/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Calnexina/genética , Calnexina/metabolismo , Linhagem Celular , Polaridade Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Interfase , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Mitose , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Análise de Célula Única , Imagem com Lapso de Tempo
18.
Methods Mol Biol ; 1402: 147-164, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26721489

RESUMO

Fluorescence in situ hybridization (FISH) enables the detection of specific nucleic acid sequences within single cells. For example, RNA FISH provides information on both the expression level and localization of RNA transcripts and, when combined with detection of associated proteins and chromatin modifications, can lend essential insights into long noncoding RNA (lncRNA) function. Epigenetic effects have been postulated for many lncRNAs, but shown for only a few. Advances in in situ techniques and microscopy, however, now allow for visualization of lncRNAs that are expressed at very low levels or are not very stable. FISH-based detections of RNA and DNA coupled with immunological staining of proteins/histone modifications offer the possibility to connect lncRNAs to epigenetic effects. Here, we describe an integrated set of protocols to detect, individually or in combination, specific RNAs, DNAs, proteins, and histone modifications in single cells at a high level of sensitivity using conventional fluorescence microscopy.


Assuntos
Cromatina/química , DNA/análise , Hibridização in Situ Fluorescente/métodos , Microscopia de Fluorescência/métodos , RNA Longo não Codificante/análise , Animais , Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/química , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Feminino , Imunofluorescência/métodos , Humanos , Camundongos
19.
Nat Commun ; 6: 8564, 2015 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-26477563

RESUMO

The transcriptional imbalance due to the difference in the number of X chromosomes between male and female mammals is remedied through X-chromosome inactivation, the epigenetic transcriptional silencing of one of the two X chromosomes in females. The X-linked Xist long non-coding RNA functions as an X inactivation master regulator; Xist is selectively upregulated from the prospective inactive X chromosome and is required in cis for X inactivation. Here we discover an Xist antisense long non-coding RNA, XistAR (Xist Activating RNA), which is encoded within exon 1 of the mouse Xist gene and is transcribed only from the inactive X chromosome. Selective truncation of XistAR, while sparing the overlapping Xist RNA, leads to a deficiency in Xist RNA expression in cis during the initiation of X inactivation. Thus, the Xist gene carries within its coding sequence an antisense RNA that drives Xist expression.


Assuntos
RNA Antissenso/metabolismo , RNA Longo não Codificante , Inativação do Cromossomo X , Animais , Linhagem Celular , Mapeamento Cromossômico , Feminino , Hibridização in Situ Fluorescente , Masculino , Camundongos Endogâmicos C57BL
20.
Nat Commun ; 5: 4209, 2014 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-24979243

RESUMO

Imprinted X-inactivation is a paradigm of mammalian transgenerational epigenetic regulation resulting in silencing of genes on the paternally inherited X-chromosome. The preprogrammed fate of the X-chromosomes is thought to be controlled in cis by the parent-of-origin-specific expression of two opposing long non-coding RNAs, Tsix and Xist, in mice. Exclusive expression of Tsix from the maternal-X has implicated it as the instrument through which the maternal germline prevents inactivation of the maternal-X in the offspring. Here, we show that Tsix is dispensable for inhibiting Xist and X-inactivation in the early embryo and in cultured stem cells of extra-embryonic lineages. Tsix is instead required to prevent Xist expression as trophectodermal progenitor cells differentiate. Despite induction of wild-type Xist RNA and accumulation of histone H3-K27me3, many Tsix-mutant X-chromosomes fail to undergo ectopic X-inactivation. We propose a novel model of lncRNA function in imprinted X-inactivation that may also apply to other genomically imprinted loci.


Assuntos
Diferenciação Celular , Impressão Genômica , Camundongos/genética , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X , Cromossomo X/genética , Animais , Feminino , Masculino , Camundongos/embriologia , Camundongos/metabolismo , RNA Longo não Codificante/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo
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