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1.
Int J Mol Sci ; 25(19)2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-39408922

RESUMEN

Retrotransposons are mobile DNA elements that are more active with increasing age and exacerbate aging phenotypes in multiple species. We previously reported an unexpected extension of chronological lifespan in the yeast, Saccharomyces paradoxus, due to the presence of Ty1 retrotransposons when cells were aged under conditions of mild stress. In this study, we tested a subset of genes identified by RNA-seq to be differentially expressed in S. paradoxus strains with a high-copy number of Ty1 retrotransposons compared with a strain with no retrotransposons and additional candidate genes for their contribution to lifespan extension when cells were exposed to a moderate dose of hydroxyurea (HU). Deletion of ADE8, NCS2, or TRM9 prevented lifespan extension, while deletion of CDD1, HAC1, or IRE1 partially prevented lifespan extension. Genes overexpressed in high-copy Ty1 strains did not typically have Ty1 insertions in their promoter regions. We found that silencing genomic copies of Ty1 prevented lifespan extension, while expression of Ty1 from a high-copy plasmid extended lifespan in medium with HU or synthetic medium. These results indicate that cells adapt to expression of retrotransposons by changing gene expression in a manner that can better prepare them to remain healthy under mild stress.


Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN de Transferencia , Retroelementos , Estrés Fisiológico , Retroelementos/genética , Estrés Fisiológico/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Nucleótidos/genética , Nucleótidos/metabolismo , Hidroxiurea/farmacología , Saccharomyces/genética , Saccharomyces/metabolismo
2.
Cell Rep ; 43(7): 114471, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-38996069

RESUMEN

Low-oxygen conditions (hypoxia) have been associated primarily with cell-cycle arrest in dividing cells. Macrophages are typically quiescent in G0 but can proliferate in response to tissue signals. Here we show that hypoxia (1% oxygen tension) results in reversible entry into the cell cycle in macrophages. Cell cycle progression is largely limited to G0-G1/S phase transition with little progression to G2/M. This cell cycle transitioning is triggered by an HIF2α-directed transcriptional program. The response is accompanied by increased expression of cell-cycle-associated proteins, including CDK1, which is known to phosphorylate SAMHD1 at T592 and thereby regulate antiviral activity. Prolyl hydroxylase (PHD) inhibitors are able to recapitulate HIF2α-dependent cell cycle entry in macrophages. Finally, tumor-associated macrophages (TAMs) in lung cancers exhibit transcriptomic profiles representing responses to low oxygen and cell cycle progression at the single-cell level. These findings have implications for inflammation and tumor progression/metastasis where low-oxygen environments are common.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Ciclo Celular , Hipoxia de la Célula , Macrófagos , Macrófagos/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Macrófagos Asociados a Tumores/metabolismo
3.
Cell ; 186(23): 4996-5014.e24, 2023 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-37949056

RESUMEN

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.


Asunto(s)
Células Madre Embrionarias , Ingeniería Genética , Haplorrinos , Animales , Femenino , Embarazo , Haplorrinos/genética , Nacimiento Vivo , Mamíferos , Células Madre Pluripotentes , Primates , Ingeniería Genética/métodos
4.
Cell Stem Cell ; 30(9): 1235-1245.e6, 2023 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-37683604

RESUMEN

Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Porcinos , Animales , Mesonefro , Embrión de Mamíferos , Blastocisto , Mamíferos , Proteínas de Homeodominio
7.
Cell ; 185(10): 1777-1792.e21, 2022 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-35512705

RESUMEN

Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.


Asunto(s)
Organogénesis , Transcriptoma , Animales , ADN/genética , Embrión de Mamíferos , Femenino , Perfilación de la Expresión Génica/métodos , Mamíferos/genética , Ratones , Organogénesis/genética , Embarazo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/genética
8.
Nature ; 605(7909): 315-324, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35314832

RESUMEN

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.


Asunto(s)
Embrión de Mamíferos , Desarrollo Embrionario , Células Madre Pluripotentes , Cigoto , Humanos , Proteínas Cromosómicas no Histona/genética , Embrión de Mamíferos/citología , Proteínas de Homeodominio/genética , Células Madre Pluripotentes/citología , Factores de Transcripción/genética , Cigoto/citología
9.
iScience ; 25(3): 103971, 2022 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-35224470

RESUMEN

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

10.
Sci Rep ; 12(1): 3114, 2022 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-35210470

RESUMEN

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.


Asunto(s)
SARS-CoV-2
11.
EBioMedicine ; 77: 103878, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-35189575

RESUMEN

BACKGROUND: Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This presentation resembles the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. METHODS: Detailed B cell phenotyping was undertaken by flow-cytometry on longitudinal samples from patients with COVID-19 across a range of severities (NIHR Cambridge BioResource). The impact of hypoxia on the transcriptome was assessed by single-cell and whole blood RNA sequencing analysis. The direct effect of hypoxia on B cells was determined through immunisation studies in genetically modified and hypoxia-exposed mice. FINDINGS: We demonstrate the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes also observed in B cell VHL-deficient mice. These findings were associated with hypoxia-related transcriptional changes in COVID-19 patient B cells, and similar B cell abnormalities were seen in mice kept in hypoxic conditions. INTERPRETATION: Hypoxia may contribute to the pronounced and persistent B cell pathology observed in acute COVID-19 pneumonia. Assessment of the impact of early oxygen therapy on these immune defects should be considered, as their correction could contribute to improved outcomes. FUNDING: Evelyn Trust, Addenbrooke's Charitable Trust, UKRI/NIHR, Wellcome Trust.


Asunto(s)
COVID-19 , Neumonía , Animales , Humanos , Hipoxia , Ratones , Oxígeno , SARS-CoV-2
12.
Nat Commun ; 13(1): 751, 2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-35136068

RESUMEN

Understanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.


Asunto(s)
COVID-19/epidemiología , COVID-19/transmisión , SARS-CoV-2/genética , Universidades , COVID-19/prevención & control , COVID-19/virología , Trazado de Contacto , Genoma Viral/genética , Genómica , Humanos , Filogenia , ARN Viral/genética , Factores de Riesgo , SARS-CoV-2/clasificación , SARS-CoV-2/aislamiento & purificación , Estudiantes , Reino Unido/epidemiología , Universidades/estadística & datos numéricos
16.
Nat Genet ; 53(7): 1022-1035, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34155378

RESUMEN

Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Hipoxia/genética , Acetilación , Animales , Humanos , Hipoxia/metabolismo , Metilación , Ratones , Ratones Noqueados , Modelos Animales , Regiones Promotoras Genéticas , Unión Proteica
19.
Nat Immunol ; 21(11): 1408-1420, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32868930

RESUMEN

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.


Asunto(s)
Linfocitos B/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Linfopoyesis/genética , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Biomarcadores , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Inmunofenotipificación , Ratones , Ratones Noqueados , Edición de ARN , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Activación Transcripcional
20.
Mob DNA ; 11: 16, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32336995

RESUMEN

Genomics and other large-scale analyses have drawn increasing attention to the potential impacts of transposable elements (TEs) on their host genomes. However, it remains challenging to transition from identifying potential roles to clearly demonstrating the level of impact TEs have on genome evolution and possible functions that they contribute to their host organisms. I summarize TE content and distribution in four well-characterized yeast model systems in this review: the pathogens Candida albicans and Cryptococcus neoformans, and the nonpathogenic species Saccharomyces cerevisiae and Schizosaccharomyces pombe. I compare and contrast their TE landscapes to their lifecycles, genomic features, as well as the presence and nature of RNA interference pathways in each species to highlight the valuable diversity represented by these models for functional studies of TEs. I then review the regulation and impacts of the Ty1 and Ty3 retrotransposons from Saccharomyces cerevisiae and Tf1 and Tf2 retrotransposons from Schizosaccharomyces pombe to emphasize parallels and distinctions between these well-studied elements. I propose that further characterization of TEs in the pathogenic yeasts would enable this set of four yeast species to become an excellent set of models for comparative functional studies to address outstanding questions about TE-host relationships.

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