RESUMO
The timing of DNA replication in mammals is crucial for minimizing errors and influenced by genome usage and chromatin states. Replication timing in the newly formed mammalian embryo remains poorly understood. Here, we have investigated replication timing in mouse zygotes and 2-cell embryos, revealing that zygotes lack a conventional replication timing program, which then emerges in 2-cell embryos. This program differs from embryonic stem cells and generally correlates with transcription and genome compartmentalization of both parental genomes. However, consistent and systematic differences existed between the replication timing of the two parental genomes, including considerably later replication of maternal pericentromeric regions compared to paternal counterparts. Moreover, maternal chromatin modified by Polycomb Repressive Complexes in the oocyte, undergoes early replication, despite belonging to the typically late-replicating B-compartment of the genome. This atypical and asynchronous replication of the two parental genomes may advance our understanding of replication stress in early human embryos and trigger strategies to reduce errors and aneuploidies.
Assuntos
Replicação do DNA , Embrião de Mamíferos , Zigoto , Animais , Feminino , Camundongos , Zigoto/metabolismo , Masculino , Embrião de Mamíferos/metabolismo , Cromatina/metabolismo , Cromatina/genética , Oócitos/metabolismo , Período de Replicação do DNA , Genoma , Desenvolvimento Embrionário/genética , Camundongos Endogâmicos C57BLRESUMO
Endomucin (EMCN) currently represents the only hematopoietic stem cell (HSC) marker expressed by both murine and human HSCs. Here, we report that EMCN+ long-term repopulating HSCs (LT-HSCs; CD150+CD48-LSK) have a higher long-term multi-lineage repopulating capacity compared to EMCN- LT-HSCs. Cell cycle analyses and transcriptional profiling demonstrated that EMCN+ LT-HSCs were more quiescent compared to EMCN- LT-HSCs. Emcn-/- and Emcn+/+ mice displayed comparable steady-state hematopoiesis, as well as frequencies, transcriptional programs, and long-term multi-lineage repopulating capacity of their LT-HSCs. Complementary functional analyses further revealed increased cell cycle entry upon treatment with 5-fluorouracil and reduced granulocyte colony-stimulating factor (GCSF) mobilization of Emcn-/- LT-HSCs, demonstrating that EMCN expression by LT-HSCs associates with quiescence in response to hematopoietic stress and is indispensable for effective LT-HSC mobilization. Transplantation of wild-type bone marrow cells into Emcn-/- or Emcn+/+ recipients demonstrated that EMCN is essential for endothelial cell-dependent maintenance/self-renewal of the LT-HSC pool and sustained blood cell production post-transplant.
Assuntos
Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Movimento Celular , Fluoruracila/farmacologia , Humanos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Ciclo Celular , Células Endoteliais/metabolismoRESUMO
Mass spectrometry (MS)-based proteomics workflows typically involve complex, multi-step processes, presenting challenges with sample losses, reproducibility, requiring substantial time and financial investments, and specialized skills. Here we introduce One-Tip, a proteomics methodology that seamlessly integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. One-Tip substantially simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells. The versatility of One-Tip is highlighted by nDIA identification of ~6000 proteins in single cells from early mouse embryos. Additionally, the study incorporates the Uno Single Cell Dispenser™, demonstrating the capability of One-Tip in single-cell proteomics with >3000 proteins identified per HeLa cell. We also extend One-Tip workflow to analysis of extracellular vesicles (EVs) extracted from blood plasma, demonstrating its high sensitivity by identifying >3000 proteins from 16 ng EV preparation. One-Tip expands capabilities of proteomics, offering greater depth and throughput across a range of sample types.
Assuntos
Proteoma , Zigoto , Humanos , Animais , Camundongos , Proteoma/análise , Células HeLa , Zigoto/química , Reprodutibilidade dos Testes , Espectrometria de Massas/métodosRESUMO
INTRODUCTION AND OBJECTIVES: Remote monitoring (RM) of cardiac implantable electronic devices (CIEDs) is considered more reliable, efficient, and safer than conventional in-person follow-up. However, the implementation of RM is still suboptimal. This study aimed to analyze the impact of the COVID-19 pandemic on the rates of CIED implants and RM activations in Spain. METHODS: The COVID-19 RM Spain Registry was used to analyze the monthly number of all CIED implantations and RM activations from January 2018 to December 2021. A descriptive analysis was performed using aggregated data from the five major CIED manufacturers. RESULTS: A total of 205 345 CIEDs were recorded. The number of implants decreased sharply (48.2%) during the pandemic lockdown (March-June 2020) but gradually increased thereafter, compensating for the previous reduction. However, pacemakers and implantable cardiac defibrillators (ICD) showed an aggregate loss of 7% and 3%, respectively, from the annual average during 2020-2021. In contrast, cardiac resynchronization therapy defibrillators (CRT-D) increased by 17%, and pacemakers (CRT-P) by 4.5% over the 2-year period. The percentage of RM activations increased from 24.5% in 2018 to 49.0% in 2021, with a sharp increase during the lockdown. The RM activation rates consistently increased during the lockdown for all devices: pacemakers (14.4% vs 37.2%; P <.001); ICD (75.6% vs 94.2%; P <.001); CRT-D/CRT-P (68.6-44.2% vs 81.6-61%; P <.001), and implantable loop recorders (50.2% vs 68.7%; P <.001). CONCLUSIONS: The significant decline in implants during the lockdown gradually recovered, except for pacemakers and ICD. However, the COVID-19 pandemic boosted RM for all CIEDs in Spain.
Assuntos
COVID-19 , Terapia de Ressincronização Cardíaca , Desfibriladores Implantáveis , Marca-Passo Artificial , Humanos , Pandemias , COVID-19/epidemiologia , Controle de Doenças TransmissíveisRESUMO
In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear. Here, we address mitotic retention of the hemogenic factors GATA2, GFI1B and FOS during haematopoietic specification. We show that GATA2 remains bound to chromatin throughout mitosis, in contrast to GFI1B and FOS, via C-terminal zinc finger-mediated DNA binding. GATA2 bookmarks a subset of its interphase targets that are co-enriched for RUNX1 and other regulators of definitive haematopoiesis. Remarkably, homozygous mice harbouring the cyclin B1 mitosis degradation domain upstream Gata2 partially phenocopy knockout mice. Degradation of GATA2 at mitotic exit abolishes definitive haematopoiesis at aorta-gonad-mesonephros, placenta and foetal liver, but does not impair yolk sac haematopoiesis. Our findings implicate GATA2-mediated mitotic bookmarking as critical for definitive haematopoiesis and highlight a dependency on bookmarkers for lineage commitment.
Assuntos
Cromatina , Fator de Transcrição GATA2 , Mitose , Animais , Camundongos , Cromossomos/metabolismo , DNA , Hematopoese/genética , Fator de Transcrição GATA2/genéticaRESUMO
Atrial fibrillation (AF) is explained by anatomical and electrophysiological changes in the atria determined by high pressure, dilatation, infiltration and inflammation in the myocardium. There are some biomarkers implicated in these processes, namely, NT-proBNP, high sensitivity troponin (Hs-Tn), urate, galectin-3, ST2, C reactive protein and fibrinogen. The aim of this study was to assess differences in these biomarkers between patients with AF and healthy controls. We designed a cross-sectional study consecutively including all patients undergoing electrical cardioversion in our hospital for persistent AF and matched healthy controls. We included 115 patients with persistent non-valvular AF and 33 healthy subjects. The biomarkers NT-proBNP, ST2 and Hs-Tn T were significantly related to the presence of AF (1054 ± 833.30 vs. 58.31 ± 59.40, p < 0.001; 35.43 ± 15.89 vs. 27.43 ± 10.95, p < 0.001 and 10.25 ± 6.11 vs. 8.42 ± 6.85, p < 0.001, respectively). NT-proBNP was the best biomarker differentiating AF patients (area under the curve 0.995). The best NT-proBNP cut-off point to differentiate AF was 102 pg/mL; for Hs-Tn T it was 11.5 ng/L and for ST2 it was 37.7 ng/mL. It is possible that these biomarkers intervene at the onset of AF and have no role in AF maintenance.
RESUMO
Genotyping of ST2 and galectin-3 in atrial fibrillation (AF) is not well analyzed. The aim of our study was to analyze the possible relationship between levels of sST2 and galectin-3 and three polymorphisms in patients with AF. We included 125 patients with persistent AF undergoing electric cardioversion. We analyzed sST2 and galectin-3 levels and three polymorphisms in peripheral blood samples. Rs2274273 was significantly related with levels of galectin-3. Rs1558648 was associated with levels of sST2 but rs13019803 were not. None of the polymorphisms were connected to the variation of biomarkers levels during the follow up. We found a relationship between rs2274273 and galectin-3 levels and rs1558648 and sST2 levels in patients with AF.
Assuntos
Fibrilação Atrial/genética , Proteínas Sanguíneas/genética , Galectinas/genética , Predisposição Genética para Doença , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Idoso , Fibrilação Atrial/sangue , Fibrilação Atrial/patologia , Fibrilação Atrial/terapia , Biomarcadores/sangue , Cardioversão Elétrica/efeitos adversos , Feminino , Galectinas/sangue , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Dictyostelium discoideum is one of eight non-mammalian model organisms recognized by the National Institute of Health for the study of human pathology. The use of this slime mould is possible owing to similarities in cell structure, behaviour and intracellular signalling with mammalian cells. Its haploid set of chromosomes completely sequenced amenable to genetic manipulation, its unique and short life cycle with unicellular and multicellular stages, and phenotypic richness encoding many human orthologues, make Dictyostelium a representative and simple model organism to unveil cellular processes in human disease. Dictyostelium studies within the biomedical field have provided fundamental knowledge in the areas of bacterial infection, immune cell chemotaxis, autophagy/phagocytosis and mitochondrial and neurological disorders. Consequently, Dictyostelium has been used to the development of related pharmacological treatments. Herein, we review the utilization of Dictyostelium as a model organism in biomedicine.
Assuntos
Dictyostelium , Animais , Dictyostelium/genética , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Atrial fibrillation (AF) and heart failure (HF) with non-reduced left ventricle ejection fraction (LVEF) present a diagnostic overlap. In this paper, we analyze differences in biomarkers between patients with and without HF, in a cohort of patients presenting with symptomatic AF. Differences in biomarkers between patients with medium range ejection fraction HF (HFmrEF) and those with preserved ejection fraction HF (HFpEF) are also analyzed. METHODS: A total of 115 patients with symptomatic persistent AF were included. Seven biomarkers were measured: NT-proBNP, high sensitivity T troponin (hsTNT), galectin-3, ST2, fibrinogen, urate and C-reactive protein. RESULTS: Patients with non-reduced LVEF HF had significantly higher NT-proBNP levels than those without HF. This biomarker was the only variable independently related with the presence of non-reduced LVEF HF. Troponin was the only factor independently related with the presence of HFmrEF. CONCLUSIONS: NT-proBNP showed the best diagnostic accuracy for detecting the presence of non-reduced LVEF HF. We found higher diagnostic NT-proBNP cut-off values than those previously reported. Troponin was the most accurate biomarker differentiating HFmrEF from HFpEF.
Assuntos
Fibrilação Atrial , Insuficiência Cardíaca , Fibrilação Atrial/diagnóstico , Biomarcadores , Insuficiência Cardíaca/diagnóstico , Humanos , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Prognóstico , Volume SistólicoRESUMO
PICH is a DNA translocase necessary for the resolution of ultrafine anaphase DNA bridges and to ensure the fidelity of chromosomal segregation. Here, we report the generation of an animal model deficient for PICH that allowed us to investigate its physiological relevance. Pich KO mice lose viability during embryonic development due to a global accumulation of DNA damage. However, despite the presence of chromosomal instability, extensive p53 activation, and increased apoptosis throughout the embryo, Pich KO embryos survive until day 12.5 of embryonic development. The absence of p53 failed to improve the viability of the Pich KO embryos, suggesting that the observed developmental defects are not solely due to p53-induced apoptosis. Moreover, Pich-deficient mouse embryonic fibroblasts exhibit chromosomal instability and are resistant to RASV12/E1A-induced transformation. Overall, our data indicate that PICH is essential to preserve chromosomal integrity in rapidly proliferating cells and is therefore critical during embryonic development and tumorigenesis.
Assuntos
Instabilidade Cromossômica , Desenvolvimento Embrionário/genética , Animais , Apoptose , Células Cultivadas , Dano ao DNA , DNA Helicases/metabolismo , Camundongos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non-obese diabetic)-derived mouse strains, such as NSG (NOD-Scid-il2Rg) or NRG (NOD-Rag1-il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell-derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre-implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata-2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%-100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD-based mouse models with the aim to improve the existing xenograft models.
Assuntos
Sistemas CRISPR-Cas , Linhagem Celular , Células-Tronco Embrionárias , Camundongos Endogâmicos NOD/genética , Animais , Fertilização in vitro , Fator de Transcrição GATA2/genética , Marcação de Genes , Hospedeiro Imunocomprometido/genética , Camundongos , Camundongos Endogâmicos NOD/imunologia , Modelos BiológicosRESUMO
Ageing associates with significant alterations in somatic/adult stem cells and therapies to counteract these might have profound benefits for health. In the blood, haematopoietic stem cell (HSC) ageing is linked to several functional shortcomings. However, besides the recent realization that individual HSCs might be preset differentially already from young age, HSCs might also age asynchronously. Evaluating the prospects for HSC rejuvenation therefore ultimately requires approaching those HSCs that are functionally affected by age. Here we combine genetic barcoding of aged murine HSCs with the generation of induced pluripotent stem (iPS) cells. This allows us to specifically focus on aged HSCs presenting with a pronounced lineage skewing, a hallmark of HSC ageing. Functional and molecular evaluations reveal haematopoiesis from these iPS clones to be indistinguishable from that associating with young mice. Our data thereby provide direct support to the notion that several key functional attributes of HSC ageing can be reversed.
Assuntos
Envelhecimento/fisiologia , Linhagem da Célula , Senescência Celular , Células Clonais/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Linhagem da Célula/genética , Reprogramação Celular/genética , Senescência Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos C57BL , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.
Assuntos
Células-Tronco Embrionárias/fisiologia , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Southern Blotting , Genes BRCA1 , Camundongos TransgênicosRESUMO
We present the case of a 66-year-old female with early onset deafness and seizures, who was diagnosed with epilepsy at the age of 2 years. She received antiepileptic drugs and was free of syncope episodes for 32 years. After a syncope at the age of 34, the ECG was characteristic of long-QT syndrome and was treated with antiarrhythmic drugs. Sequencing of the KCNQ1 gene identified two novel KCNQ1 variants interpreted to be pathogenic, and the patient was finally diagnosed with Jervell and Lange-Nielsen syndrome. © 2016 Wiley Periodicals, Inc.
Assuntos
Heterozigoto , Síndrome de Jervell-Lange Nielsen/diagnóstico , Síndrome de Jervell-Lange Nielsen/genética , Canal de Potássio KCNQ1/genética , Mutação , Idoso , Análise Mutacional de DNA , Diagnóstico Tardio , Eletrocardiografia , Éxons , Feminino , Estudos de Associação Genética , Humanos , Fenótipo , Análise de Sequência de DNARESUMO
Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i) or with knockout serum replacement (KOSR), but not serum, can generate high-level chimeras regardless of how these cells were derived. ESCs cultured in these conditions showed a transcriptional correlation with early pre-implantation embryos (E1.5-E3.5) and contributed to development from the 2-cell stage. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition.