RESUMO
Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.
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Blastômeros/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Blastocisto/citologia , Blastômeros/metabolismo , Linhagem da Célula , Células Cultivadas , Quimera , Embrião de Mamíferos/citologia , Endoderma/citologia , Epigênese Genética , Epigenômica , Feminino , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Placenta/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Gravidez , Análise de Célula Única , Transcriptoma , Trofoblastos/citologiaRESUMO
MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Saúde da População , Software , Animais , Estudos de Associação Genética , Humanos , Camundongos , FenótipoRESUMO
The rapid expansion of flow cytometry applications has outpaced the functionality of traditional manual analysis tools used to interpret flow cytometry data. Scientists are faced with the daunting prospect of manually identifying interesting cell populations in 50-dimensional datasets, equalling the complexity previously only reached in mass cytometry. Data can no longer be analyzed or interpreted fully by manual approaches. While automated gating has been the focus of intense efforts, there are many significant additional steps to the analytical pipeline (e.g., cleaning the raw files, event outlier detection, extracting immunophenotypes). We review the components of a customized automated analysis pipeline that can be generally applied to large scale flow cytometry data. We demonstrate these methodologies on data collected by the International Mouse Phenotyping Consortium (IMPC).
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Biologia Computacional , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Algoritmos , Animais , Citometria de Fluxo/estatística & dados numéricos , Humanos , Imunofenotipagem/estatística & dados numéricos , Camundongos , SoftwareRESUMO
We present a simple and quick system for accurately scoring the developmental progress of mouse embryos harvested on embryonic day 14 (E14.5). Based solely on the external appearance of the maturing forelimb, we provide a convenient way to distinguish six developmental sub-stages. Using a variety of objective morphometric data obtained from the commonly used C57BL/6N mouse strain, we show that these stages correlate precisely with the growth of the entire embryo and its organs. Applying the new staging system to phenotype analyses of E14.5 embryos of 58 embryonic lethal null mutant lines from the DMDD research programme (https://dmdd.org.uk) and its pilot, we show that homozygous mutant embryos are frequently delayed in development. To demonstrate the importance of our staging system for correct phenotype interpretation, we describe stage-specific changes of the palate, heart and gut, and provide examples in which correct diagnosis of malformations relies on correct staging.
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Desenvolvimento Embrionário/fisiologia , Fenótipo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/fisiologia , Especificidade da EspécieRESUMO
Accurate identification of abnormalities in the mouse embryo depends not only on comparisons with appropriate, developmental stage-matched controls, but also on an appreciation of the range of anatomical variation that can be expected during normal development. Here we present a morphological, topological and metric analysis of the heart and arteries of mouse embryos harvested on embryonic day (E)14.5, based on digital volume data of whole embryos analysed by high-resolution episcopic microscopy (HREM). By comparing data from 206 genetically normal embryos, we have analysed the range and frequency of normal anatomical variations in the heart and major arteries across Theiler stages S21-S23. Using this, we have identified abnormalities in these structures among 298 embryos from mutant mouse lines carrying embryonic lethal gene mutations produced for the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme. We present examples of both commonly occurring abnormal phenotypes and novel pathologies that most likely alter haemodynamics in these genetically altered mouse embryos. Our findings offer a reference baseline for identifying accurately abnormalities of the heart and arteries in embryos that have largely completed organogenesis.
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Artérias/patologia , Embrião de Mamíferos/patologia , Coração/embriologia , Mutação , Miocárdio/patologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1(tm1a/tm1a) and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1(tm1a/tm1a) HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.
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Diferenciação Celular/genética , Endopeptidases/genética , Hematopoese/genética , Linfócitos/metabolismo , Animais , Contagem de Células Sanguíneas , Western Blotting , Endopeptidases/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Genótipo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Linfócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Turning genetic discoveries identified in genome-wide association (GWA) studies into biological mechanisms is an important challenge in human genetics. Many GWA signals map outside exons, suggesting that the associated variants may lie within regulatory regions. We applied the formaldehyde-assisted isolation of regulatory elements (FAIRE) method in a megakaryocytic and an erythroblastoid cell line to map active regulatory elements at known loci associated with hematological quantitative traits, coronary artery disease, and myocardial infarction. We showed that the two cell types exhibit distinct patterns of open chromatin and that cell-specific open chromatin can guide the finding of functional variants. We identified an open chromatin region at chromosome 7q22.3 in megakaryocytes but not erythroblasts, which harbors the common non-coding sequence variant rs342293 known to be associated with platelet volume and function. Resequencing of this open chromatin region in 643 individuals provided strong evidence that rs342293 is the only putative causative variant in this region. We demonstrated that the C- and G-alleles differentially bind the transcription factor EVI1 affecting PIK3CG gene expression in platelets and macrophages. A protein-protein interaction network including up- and down-regulated genes in Pik3cg knockout mice indicated that PIK3CG is associated with gene pathways with an established role in platelet membrane biogenesis and thrombus formation. Thus, rs342293 is the functional common variant at this locus; to the best of our knowledge this is the first such variant to be elucidated among the known platelet quantitative trait loci (QTLs). Our data suggested a molecular mechanism by which a non-coding GWA index SNP modulates platelet phenotype.
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Cromatina/genética , Estudo de Associação Genômica Ampla , Animais , Plaquetas/metabolismo , Cromossomos Humanos Par 7/genética , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Proteínas de Ligação a DNA/metabolismo , Eritroblastos/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Macrófagos/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Fenótipo , Proto-Oncogenes , Locos de Características Quantitativas , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
Two large-scale phenotyping efforts, the European Mouse Disease Clinic (EUMODIC) and the Wellcome Trust Sanger Institute Mouse Genetics Project (SANGER-MGP), started during the late 2000s with the aim to deliver a comprehensive assessment of phenotypes or to screen for robust indicators of diseases in mouse mutants. They both took advantage of available mouse mutant lines but predominantly of the embryonic stem (ES) cells resources derived from the European Conditional Mouse Mutagenesis programme (EUCOMM) and the Knockout Mouse Project (KOMP) to produce and study 799 mouse models that were systematically analysed with a comprehensive set of physiological and behavioural paradigms. They captured more than 400 variables and an additional panel of metadata describing the conditions of the tests. All the data are now available through EuroPhenome database (www.europhenome.org) and the WTSI mouse portal (http://www.sanger.ac.uk/mouseportal/), and the corresponding mouse lines are available through the European Mouse Mutant Archive (EMMA), the International Knockout Mouse Consortium (IKMC), or the Knockout Mouse Project (KOMP) Repository. Overall conclusions from both studies converged, with at least one phenotype scored in at least 80% of the mutant lines. In addition, 57% of the lines were viable, 13% subviable, 30% embryonic lethal, and 7% displayed fertility impairments. These efforts provide an important underpinning for a future global programme that will undertake the complete functional annotation of the mammalian genome in the mouse model.
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Genoma , Camundongos/genética , Animais , Europa (Continente) , Células Germinativas , Mutação , FenótipoRESUMO
The broad aim of biomedical science in the postgenomic era is to link genomic and phenotype information to allow deeper understanding of the processes leading from genomic changes to altered phenotype and disease. The EuroPhenome project (http://www.EuroPhenome.org) is a comprehensive resource for raw and annotated high-throughput phenotyping data arising from projects such as EUMODIC. EUMODIC is gathering data from the EMPReSSslim pipeline (http://www.empress.har.mrc.ac.uk/) which is performed on inbred mouse strains and knock-out lines arising from the EUCOMM project. The EuroPhenome interface allows the user to access the data via the phenotype or genotype. It also allows the user to access the data in a variety of ways, including graphical display, statistical analysis and access to the raw data via web services. The raw phenotyping data captured in EuroPhenome is annotated by an annotation pipeline which automatically identifies statistically different mutants from the appropriate baseline and assigns ontology terms for that specific test. Mutant phenotypes can be quickly identified using two EuroPhenome tools: PhenoMap, a graphical representation of statistically relevant phenotypes, and mining for a mutant using ontology terms. To assist with data definition and cross-database comparisons, phenotype data is annotated using combinations of terms from biological ontologies.
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Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Proteínas , Animais , Biologia Computacional/tendências , Armazenamento e Recuperação da Informação/métodos , Internet , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Fenótipo , Linguagens de Programação , Estrutura Terciária de Proteína , SoftwareRESUMO
BACKGROUND/OBJECTIVE: The COVID-19 pandemic has brought the use of telehealth to the fore, as many people have been unable to interact directly with healthcare professionals (HCP). For community palliative care (CPC) services, this has meant a sudden change from a predominantly face-to-face model of care to one that incorporates telehealth. Understanding patient and HCP experiences with telehealth and how telehealth compares to 'usual' care will be crucial in planning future CPC services. METHODOLOGY: All patients of the Barwon Health CPC service between 1 April and 31 May 2020 were invited to complete a questionnaire evaluating their interactions with the palliative care service and specifically their involvement with telehealth consultations. Palliative care HCP who provided clinical services during the same time period were also surveyed. RESULTS/CONCLUSION: Seventy-four patients (response rate 36%) and 22 HCP returned surveys. Both groups felt comfortable using telehealth, however, also encountered a range of issues when undertaking telehealth consultations. Despite reporting issues, the preference of both groups was for a CPC service model, which combined face-to-face and telehealth consultations. This study is one of the first to directly ask this question and as such provides useful guidance for health services when planning future CPC service models.
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A systematic approach to nonhousehold TB contact identified a similar number of LTBI cases to household screening over the same time period https://bit.ly/2Tq96LN.
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Contact tracing is central to the public health response to COVID-19, but the approach taken has received criticism for failing to make enough of an impact on disease transmission. We discuss what can be learned from contact tracing in other infections, and how the natural history of COVID-19 should shape the strategies used.
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COVID-19 , Busca de Comunicante , Saúde Pública , Humanos , SARS-CoV-2RESUMO
OBJECTIVES: The increasing social needs of people with Tuberculosis (TB), and the poor adherence to anti-TB therapy (ATT) associated with homelessness, drug or alcohol abuse, and prison history, led us to introduce a social care team (SCT) to support patient engagement with care within this low TB incidence setting. METHODS: Using a risk assessment, patients with social risk factors (SRF) for non-adherence to ATT are identified and a referral made to the SCT, who then provide intensive casework support for areas including homelessness, housing, benefits, debt and immigration. Retrospective data analysis of the social care database from 2017 to 2019 was conducted. Patients who were (nâ¯=â¯170) and were not referred to the SCT (nâ¯=â¯734) were compared. RESULTS: Patients referred were significantly more likely to complete treatment for TB than those not (88.2% versus 77.7% respectively, pâ¯=â¯0.0025), irrespective of receipt of Directly/Video Observed Therapy and adjusting for confounders. CONCLUSIONS: This paper demonstrates important evidence for the positive impact of a dedicated SCT within a TB service, and these improved treatment outcomes provide a strong argument for development of similar SCTs within UK TB services and similar healthcare settings.
Assuntos
Pessoas Mal Alojadas , Tuberculose , Humanos , Incidência , Estudos Retrospectivos , Apoio Social , Tuberculose/tratamento farmacológico , Tuberculose/epidemiologiaRESUMO
Maintaining adherence to treatment for tuberculosis (TB) is essential if the disease is to be eliminated. As part of formative research to develop an intervention to improve adherence, we documented the lived experiences of adults receiving anti-TB treatment (ATT) in three UK cities and examined how personal, social, and structural circumstances interacted to impact on individuals' adherence to treatment. Using a topic guide that explored social circumstances and experiences of TB care, we conducted in-depth interviews with 18 adults (six women) who were being or had been treated for TB (patients) and four adults (all women) who were caring for a friend, relative, or partner being treated for TB (caregivers). We analysed transcripts using an adapted framework method that classified factors affecting adherence as personal, social, structural, health systems, or treatment-related. Eleven of 18 patients were born outside the UK (in South, Central, and East Asia, and Eastern and Southern Africa); among the seven who were UK-born, four were Black, Asian, or Minority Ethnic and three were White British. TB and its treatment were often disruptive: in addition to debilitating symptoms and side effects of ATT, participants faced job insecurity, unstable housing, stigma, social isolation, worsening mental health, and damaged relationships. Those who had a strong support network, stable employment, a routine that could easily be adapted, a trusting relationship with their TB team, and clear understanding of the need for treatment reported finding it easier to adhere to ATT. Changes in circumstances sometimes had dramatic effects on an individual's ability to take ATT; participants described how the impact of certain acute events (e.g., the onset of side effects or fatigue, episodes of stigmatisation, loss of income) were amplified by their timing or through their interaction with other elements of the individual's life. We suggest that the dynamic and fluctuating nature of these factors necessitates comprehensive and regular review of needs and potential problems, conducted before and during ATT; this, coupled with supportive measures that consider (and seek to mitigate) the influence of social and structural factors, may help improve adherence.
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A rising number of non-tuberculous mycobacterial (NTM) isolates are being identified in UK clinical practice. There are many uncertainties around the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD), including its epidemiology, diagnosis, treatment and prevention. Regional variations in how patients with NTM-PD are managed reflects the lack of standardised pathways in the UK. Service optimisation and multidisciplinary working can improve the quality of care for patients with NTM-PD, including (1) better identification of patients at risk of NTM-PD and modification of risk factors where applicable; (2) standardisation of reference laboratory testing to offer clinicians access to accurate and prompt information on NTM species and drug sensitivities; (3) development of recognised specialist NTM nursing care; (4) standardisation of NTM-PD imaging strategies for monitoring of treatment and disease progression; (5) establishment of a hub-and-spoke model of care, including clear referral and management pathways, dedicated NTM-PD multidisciplinary teams, and long-term patient follow-up; (6) formation of clinical networks to link experts who manage diseases associated with NTM; (7) enabling patients to access relevant support groups that can provide information and support for their condition; and (8) development of NTM research groups to allow patient participation in clinical trials and to facilitate professional education.
Assuntos
Infecções por Mycobacterium não Tuberculosas/epidemiologia , Infecções por Mycobacterium não Tuberculosas/terapia , Qualidade da Assistência à Saúde/organização & administração , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/terapia , Idoso , Pesquisa Biomédica/tendências , Progressão da Doença , Feminino , Humanos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/prevenção & controle , Micobactérias não Tuberculosas/isolamento & purificação , Qualidade da Assistência à Saúde/tendências , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/prevenção & controle , Reino UnidoRESUMO
Contact tracing is a key part of tuberculosis prevention and care, aiming to hasten diagnosis and prevent transmission. The proportion of case-contact pairs for which recent transmission occurred and the typical timespans between the index case and their contact accessing care are not known; we aimed to calculate these. We analysed individual-level TB contact tracing data, collected in London from 20/01/2011-31/12/2015, linked to tuberculosis surveillance and MIRU-VNTR 24-locus strain-typing information. Of pairs of index cases and contacts diagnosed with active tuberculosis, 85/314 (27%) had strain typing data available for both. Of these pairs, 79% (67/85) shared indistinguishable isolates, implying probable recent transmission. Of pairs in which both contact and the index case had a social risk factor, 11/11 (100%) shared indistinguishable isolates, compared to 55/75 (75%) of pairs in which neither had a social risk factor (P = 0.06). The median time interval between the index case and their contact accessing care was 42 days (IQR: 16, 96). As over 20% of pairs did probably not involve recent transmission between index case and contact, the effectiveness of contact tracing is not necessarily limited to those circumstances where the index case has transmitted disease to their close contacts.
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Busca de Comunicante , Transmissão de Doença Infecciosa , Tuberculose/epidemiologia , Tuberculose/transmissão , Londres/epidemiologia , Repetições Minissatélites , Epidemiologia Molecular , Tipagem Molecular , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.
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Perda Auditiva/genética , Mapas de Interação de Proteínas/genética , Animais , Conjuntos de Dados como Assunto , Testes Genéticos , Perda Auditiva/epidemiologia , Testes Auditivos , Camundongos , Camundongos Knockout , FenótipoRESUMO
Background: Identifying genes that are essential for mouse embryonic development and survival through term is a powerful and unbiased way to discover possible genetic determinants of human developmental disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Methods: Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant and 114 wild type embryos from 42 lethal and subviable lines, analysed at E14.5. Results: Virtually all mutant embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve.Conclusions: Overall, the most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates.
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The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.