RESUMO
The term 'insight' is generically defined in English language as the ability to perceive deeper truths about people and situations. In clinical practice, patient insight is known to have important implications in treatment compliance and clinical outcomes, and can be assessed clinically by looking for the presence of illness awareness, correct attribution of symptoms to underlying condition, and acceptance of treatment. In this article, we suggest that cultivating insight is actually a highly important, yet often overlooked, component of medical training, which may explain why some consistently learn well, communicate effectively, and quickly attain clinical competency, while others struggle throughout their clinical training and may even be difficult to remediate. We herein define 'insight' in the context of medical training as having an astute perception of personal cognitive processes, motivations, emotions, and ability (strengths, weaknesses, and limitations) that should drive self-improvement and effective behavioural regulation. We then describe the utility of cultivating 'insight' in medical training through three lenses of (i) promoting self-regulated, lifelong clinical learning, (ii) improving clinical competencies and person-centred care, and (iii) enhancing physician mental health and well-being. In addition, we review educational pedagogies that are helpful to create a medical eco-system that promotes the cultivation of insight among its trainees and practitioners. Finally, we highlight several tell-tale signs of poor insight and discuss psychological and non-psychological interventions that may help those severely lacking in insight to become more amenable to change and remediation.
Assuntos
Educação Médica , Aprendizagem , Saúde Mental , Humanos , Competência Clínica , Assistência Centrada no PacienteRESUMO
PURPOSE: Generative AI will become an integral part of education in future. The potential of this technology in different disciplines should be identified to promote effective adoption. This study evaluated the performance of ChatGPT in tutorial and case-based learning questions in physiology and biochemistry for medical undergraduates. Our study mainly focused on the performance of GPT-3.5 version while a subgroup was comparatively assessed on GPT-3.5 and GPT-4 performances. MATERIALS AND METHODS: Answers were generated in GPT-3.5 for 44 modified essay questions (MEQs) in physiology and 43 MEQs in biochemistry. Each answer was graded by two independent examiners. Subsequently, a subset of 15 questions from each subject were selected to represent different score categories of the GPT-3.5 answers; responses were generated in GPT-4, and graded. RESULTS: The mean score for physiology answers was 74.7 (SD 25.96). GPT-3.5 demonstrated a statistically significant (p = .009) superior performance in lower-order questions of Bloom's taxonomy in comparison to higher-order questions. Deficiencies in the application of physiological principles in clinical context were noted as a drawback. Scores in biochemistry were relatively lower with a mean score of 59.3 (SD 26.9) for GPT-3.5. There was no statistically significant difference in the scores for higher and lower-order questions of Bloom's taxonomy. The deficiencies highlighted were lack of in-depth explanations and precision. The subset of questions where the GPT-4 and GPT-3.5 were compared demonstrated a better overall performance in GPT-4 responses in both subjects. This difference between the GPT-3.5 and GPT-4 performance was statistically significant in biochemistry but not in physiology. CONCLUSIONS: The differences in performance across the two versions, GPT-3.5 and GPT-4 across the disciplines are noteworthy. Educators and students should understand the strengths and limitations of this technology in different fields to effectively integrate this technology into teaching and learning.
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The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.
Assuntos
Redes Reguladoras de Genes/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Fatores de Transcrição NFI/genética , Transdução de Sinais/genética , Animais , Apoptose/genética , Mapeamento Cromossômico , Biologia Computacional , Primers do DNA/genética , Elementos de DNA Transponíveis/genética , Proteínas Hedgehog/genética , Humanos , Camundongos , Camundongos Transgênicos , Mutagênese Insercional/métodos , Receptores Patched , Receptor Patched-1 , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/genética , Análise de Sequência de DNA , Transposases/genéticaRESUMO
Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. Here we describe a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction.
Assuntos
Anáfase/fisiologia , Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/enzimologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Proteína Quinase CDC2/metabolismo , Cromossomos Humanos/metabolismo , Ciclina B/metabolismo , Complexo Dinactina , Dineínas/metabolismo , Retroalimentação Fisiológica/fisiologia , Células HCT116 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica/fisiologiaRESUMO
Population variation in disease and other phenotype are partly attributed to single nucleotide polymorphisms (SNPs) in the human genome. Due to selection pressure, two individuals from the same ancestral population have more genetic similarity compared to individuals from further geographic regions. Here, we elucidated the genomic population differentiation pattern, by interrogating >22,000,000 SNPs. Majority of population-differentiated (pd) SNPs (~95%), including the potentially functional (pf) (~84%) subset reside in non-genic regions, compared to the proportion of all SNPs (58%) found in non-genic regions. This suggests that differences between populations are more likely due to differences in gene regulation rather than protein function. Actin Cytoskeleton, Axonal Guidance and Protein Kinase A signaling pathways are enriched with genes carrying at least three pdSNPs (enriched pdGenes), while Antigen Presentation, Hepatic Fibrosis and Huntington Disease Signalling pathways are over-represented by enriched pf-pdGenes. An inverse correlation between chromosome size and the proportion of pd-/pf-pdSNPs was observed. Smaller chromosomes have relatively more of such SNPs including genes carrying these SNPs. Genes associated with common diseases and enriched with these pd-/pfpdSNPs are localized to 11 different chromosomes, with immune-related disease pd/pf-pdGenes mainly residing in chromosome 6 while neurological disease pd/pf-pdGenes residing in smaller chromosomes including chromosome 21/22. The associated diseases were reported to show population differences in incidence, severity and/or etiology. In summary, this study highlights the non-sporadic nature of population differentiation footprint in the human genome, which can potentially lead to the identification of genomic regions that play roles in the manifestation of phenotypic differences, including in disease predisposition and drug response.
Assuntos
Genoma Humano , Polimorfismo de Nucleotídeo Único , Citoesqueleto de Actina/genética , Regulação da Expressão Gênica/genética , Genética Populacional , Humanos , Transdução de Sinais/genéticaRESUMO
AIMS: Multiple myeloma (MM) is a heterogeneous disease characterised by genetically complex abnormalities. The classical mutational spectrum includes recurrent chromosomal aberrations and gene-level mutations. Recurrent translocations involving the IGH gene such as t(11;14), t(4;14) and t(14;16) are well known. However, the presence of complex genetic abnormalities raises the possibility that fusions other than the recurrent IGH translocations exist. We therefore employed a targeted RNA-sequencing panel to identify novel putative fusions in a local cohort of MM. METHODS: Targeted RNA-sequencing was performed on 21 patient samples using the Illumina TruSight RNA Pan-Cancer Panel (comprising 1385 genes). Fusion calls were generated from the Illumina RNA-Sequencing Alignment software (V.1.0.0). These samples had conventional cytogenetic and fluorescence in situ hybridisation data for the common recurrent chromosomal abnormalities (t(11;14), t(4;14), t(14;16) and 17p13 deletion). The MMRF CoMMpass dataset was analysed using the TopHat-fusion pipeline. RESULTS: A total of 10 novel fusions were identified by the TruSight RNA Pan-Cancer Panel. Two of these fusions, HGF/CACNA2D1 and SMC3/MXI1, were validated by reverse transcription PCR and Sanger sequencing as they involve genes that may have biological relevance in MM genesis. Four of these (MAP2K4/MAP2K4P1) are likely to be spurious secondary to misalignment of reads to a pseudogene. One record of the HGF/CACNA2D1 fusion was identified from the MMRF CoMMpass dataset. CONCLUSIONS: The identification of novel fusions offers insights into the biology of MM and might have clinical relevance. Further functional studies are required to determine the biological and clinical relevance of these novel fusions.
Assuntos
Biomarcadores Tumorais/genética , Fusão Gênica , Mieloma Múltiplo/genética , RNA/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Canais de Cálcio/genética , Proteínas de Ciclo Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/genética , Perfilação da Expressão Gênica/métodos , Fator de Crescimento de Hepatócito/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Mieloma Múltiplo/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Transcriptoma , Translocação Genética , Proteínas Supressoras de Tumor/genéticaRESUMO
AIM: The presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption of next-generation sequencing (NGS) platforms, challenges with regard to amplification efficiency of CEBPA due to the high GC content may be encountered, potentially resulting in suboptimal coverage. Here, the performance of an amplicon-based NGS method using a laboratory-developed CEBPA-specific Nextera XT (CEBNX) was evaluated. METHODS: Mutational analyses of the CEBPA gene of 137 AML bone marrow or peripheral blood retrospective specimens were performed by the amplification of the CEBPA gene using the Expand Long Range dNTPack and the amplicons processed by CCS and NGS. CEBPA-specific libraries were then constructed using the Nextera XT V.2 kit. All FASTQ files were then processed with the MiSeq Reporter V.2.6.2.3 using the PCR Amplicon workflow via the customised CEBPA-specific manifest file. The variant calling format files were analysed using the Illumina Variant Studio V.2.2. RESULTS: A coverage per base of 3631X to 28184X was achieved. 22 samples (16.1%) were found to contain CEBPA mutations, with variant allele frequencies (VAF) ranging from 3.8% to 58.2%. Taking CCS as the 'gold standard', sensitivity and specificity of 97% and 97% was achieved. For the transactivation domain 2 polymorphism (c.584_589dupACCCGC/p.His195_Pro196dup), the CEBNX achieved 100% sensitivity and 100% specificity relative to CCS. CONCLUSIONS: Our laboratory-developed CEBNX workflow shows high coverage and thus overcomes the challenges associated with amplification efficiency and low coverage of CEBPA. Therefore, our assay is suitable for deployment in the clinical laboratory.
Assuntos
Biomarcadores Tumorais/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Mieloide Aguda/genética , Mutação , Linhagem Celular Tumoral , Frequência do Gene , Humanos , Leucemia Mieloide Aguda/diagnóstico , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fluxo de TrabalhoRESUMO
Progression through mitosis requires activation of cyclin B/Cdk1 and its downstream targets, including Polo-like kinase and the anaphase-promoting complex (APC), the ubiquitin ligase directing degradation of cyclins A and B. Recent evidence shows that APC activation requires destruction of the APC inhibitor Emi1. In prophase, phosphorylation of Emi1 generates a D-pS-G-X-X-pS degron to recruit the SCF(betaTrCP) ubiquitin ligase, causing Emi1 destruction and allowing progression beyond prometaphase, but the kinases directing this phosphorylation remain undefined. We show here that the polo-like kinase Plk1 is strictly required for Emi1 destruction and that overexpression of Plk1 is sufficient to trigger Emi1 destruction. Plk1 stimulates Emi1 phosphorylation, betaTrCP binding, and ubiquitination in vitro and cyclin B/Cdk1 enhances these effects. Plk1 binds to Emi1 in mitosis and the two proteins colocalize on the mitotic spindle poles, suggesting that Plk1 may spatially control Emi1 destruction. These data support the hypothesis that Plk1 activates the APC by directing the SCF-dependent destruction of Emi1 in prophase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/metabolismo , Anáfase , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Polaridade Celular , Ativação Enzimática , Proteínas F-Box , Humanos , Mitose , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/metabolismo , Fatores de Tempo , Ubiquitina/metabolismo , Proteínas de Xenopus , Xenopus laevis , Quinase 1 Polo-LikeRESUMO
Targeted next generation sequencing platforms have been increasingly utilised for identification of novel mutations in myeloid neoplasms, such as acute myeloid leukaemia (AML), and hold great promise for use in routine clinical diagnostics. In this study, we evaluated the utility of an open source variant caller in detecting large indels in a targeted sequencing of AML samples. While we found that this bioinformatics pipeline has the potential to accurately capture large indels (>20 bp) in patient samples, we highlighted the pitfall of a confounding ZRSR1 pseudogene that led to an erroneous ZRSR2 variant call. We further discuss possible clinical implications of the ZRSR1 pseudogene in myeloid neoplasms based on its molecular features. Knowledge of the confounding ZRSR1 pseudogene in ZRSR2 sequencing assays could be particularly important in AML diagnostics because the detection of ZRSR2 in AML patients is highly specific for an s-AML diagnosis.
Assuntos
Biomarcadores Tumorais/genética , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Mutação INDEL , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Proteínas Nucleares/genética , Pseudogenes , Ribonucleoproteínas/genética , Adulto , Idoso , Biologia Computacional , Bases de Dados Genéticas , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Fenótipo , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable.
RESUMO
Epigenetic changes, like DNA methylation, affect gene expression and in colorectal cancer (CRC), a distinct phenotype called the CpG island methylator phenotype ("CIMP") has significantly higher levels of DNA methylation at so-called "Type C loci" within the genome. We postulate that enhancer-gene pairs are coordinately controlled through DNA methylation in order to regulate the expression of key genes/biomarkers for a particular phenotype.Firstly, we found 24 experimentally-validated enhancers (VISTA enhancer browser) that contained statistically significant (FDR-adjusted q-value of <0.01) differentially methylated regions (DMRs) (1000bp) in a study of CIMP versus non-CIMP CRCs. Of these, the methylation of 2 enhancers, 1702 and 1944, were found to be very well correlated with the methylation of the genes Wnt3A and IGDCC3, respectively, in two separate and independent datasets.We show for the first time that there are indeed distinct and dynamic changes in the methylation pattern of specific enhancer-gene pairs in CRCs. Such a coordinated epigenetic event could be indicative of an interaction between (1) enhancer 1702 and Wnt3A and (2) enhancer 1944 and IGDCC3. Moreover, our study shows that the methylation patterns of these 2 enhancer-gene pairs can potentially be used as biomarkers to delineate CIMP from non-CIMP CRCs.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Moléculas de Adesão Celular Neuronais/genética , Ilhas de CpG/genética , Epigênese Genética , Humanos , Imunoglobulinas/genética , Transcriptoma , Proteína Wnt3A/genéticaRESUMO
AIMS: PCR amplicon-based next-generation sequencing (NGS) panels are increasingly used for clinical diagnostic assays. Amplification bias is a well-known limitation of PCR amplicon-based approaches. We sought to characterise lower-performance amplicons in an off-the-shelf NGS panel (TruSight Myeloid Sequencing Panel) for myeloid neoplasms and attempted to patch the low read depth for one of the affected genes, CEBPA. METHODS: We performed targeted NGS of 158 acute myeloid leukaemia samples and analysed the amplicon read depths across 568 amplicons to identify lower-performance amplicons. We also correlated the amplicon read depths with the template GC content. Finally, we attempted to patch the low read depth for CEBPA using a parallel library preparation (Nextera XT) workflow. RESULTS: We identified 16 lower-performance amplicons affecting nine genes, including CEBPA. There was a slight negative correlation between the amplicon read depths and template GC content. Addition of the separate CEBPA library generated a minimum read depth per base across the CEBPA gene ranging from 268x to 758x across eight samples. CONCLUSIONS: The identification of lower-performance amplicons will be informative to laboratories intending to use this panel. We have also demonstrated proof-of-concept that different libraries (TruSight Myeloid and Nextera XT) can be combined and sequenced on the same flow cell to generate additional reads for CEBPA.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide Aguda/genética , Reação em Cadeia da Polimerase/métodos , HumanosRESUMO
The ubiquitin-like modifier (UBL) family has recently generated much interest in the scientific community, as it is implicated to play important regulatory roles via novel protein-protein modification. FAT10 (diubiquitin) belongs to this family of proteins, comprising two ubiquitin-like moieties fused in tandem, and has been implicated to be involved in the maintenance of spindle integrity during mitosis. As FAT10 may play a role in the regulation of genomic stability, we examined if there is an association between FAT10 expression and hepatocellular carcinoma (HCC) or other cancers. Northern blot analyses revealed upregulation of FAT10 expression in the tumors of 90% of HCC patients. In situ hybridization as well as immunohistochemistry utilizing anti-FAT10 antibodies localized highest FAT10 expression in the nucleus of HCC hepatocytes rather than the surrounding immune and non-HCC cells. FAT10 expression was also found to be highly upregulated in other cancers of the gastrointestinal tract and female reproductive system. In conclusion, we demonstrated upregulation of FAT10 expression in various gastrointestinal and gynecological cancers. Its overexpression is unrelated to the general increase in protein synthesis or a general immune/inflammatory response to cancer. Rather, FAT10 may modulate tumorigenesis through its reported interaction with the MAD2 spindle-assembly checkpoint protein.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/metabolismo , Ubiquitinas , Núcleo Celular/metabolismo , Feminino , Neoplasias Gastrointestinais/metabolismo , Neoplasias dos Genitais Femininos/metabolismo , Humanos , Especificidade de Órgãos , Células Tumorais Cultivadas , Regulação para CimaRESUMO
In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the anaphase-promoting complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.
Assuntos
Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/metabolismo , Fuso Acromático/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Ciclina B/metabolismo , Complexo Dinactina , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Proteína Fosfatase 2/genética , Subunidades Proteicas/genéticaRESUMO
Embryonic stem (ES) cells were first derived from inner cell mass (ICM) explants of preimplantation stage mouse blastocysts some 30 years ago. ES cells are of primary interest as they are used to genetically modify the genome of mice via gene targeting. Although many founder ES lines have been established, there is still a need to obtain new ES lines or their derivatives, often from new mutant mouse lines, to study the function of a mutated gene in different cell types. Existing methods for isolating ES cell lines are inefficient. Here, we describe a reproducible, efficient, and economical method to derive ES cells from different mouse strains using a defined serum-free, serum replacement (KO-SR) media, with 50-85% efficiency. We have derived over 100 ES lines, which when karyotyped>70% were euploid. Two of these lines, when tested, produced germ-line chimeras. We also present procedures for the routine maintenance and karyotyping of the ES cells.
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Técnicas de Cultura de Células/métodos , Diferenciação Celular , Separação Celular/métodos , Células-Tronco Embrionárias , Animais , Blastocisto/citologia , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Criopreservação/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Células Germinativas/citologia , Cariotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
Expression of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 is required for the accumulation of APC/C substrates crucial for DNA synthesis and mitotic entry. We show that in vivo Emi1 expression correlates with the proliferative status of the cellular compartment and that cells lacking Emi1 undergo cellular senescence. Emi1 depletion leads to strong decreases in E2F target mRNA and APC/C substrate protein abundances. However, cyclin E mRNA and cyclin E protein levels and associated kinase activities are increased. Cells lacking Emi1 undergo DNA damage, likely explained by replication stress upon deregulated cyclin E- and A-associated kinase activities. Inhibition of ATM kinase prevents induction of senescence, implying that senescence is a consequence of DNA damage. Surprisingly, no senescence or no extensive amount of senescence is evident upon depletion of the Emi1-stabilizing factor Evi5 or Pin1, respectively. Our data suggest that maintenance of a protein stabilization/mRNA expression positive-feedback circuit fueled by Emi1 is required for accurate cell cycle progression, maintenance of DNA integrity, and prevention of cellular senescence.
Assuntos
Senescência Celular , Dano ao DNA , Fatores de Transcrição E2F/metabolismo , Regulação da Expressão Gênica , Proteínas/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Linhagem Celular , Ciclina E/genética , Ciclina E/metabolismo , Fatores de Transcrição E2F/genética , Proteínas Ativadoras de GTPase , Humanos , Camundongos , Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Distribuição Tecidual , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Various reports have implicated the virally encoded HBx protein as a cofactor in hepatocarcinogenesis. However, direct evidence of the role of HBx as a promoter of oncogenesis in response to an initiating factor such as DNA damage remains inadequate. Here, we report the effects of HBx in HepG2 cells exposed to UV light-induced DNA damage. HBx expression was found not to affect the morphology, viability, and cell cycle/apoptotic profiles or DNA repair machinery of untreated cells. Nonetheless, upon UV treatment, HBx protein levels increased concomitantly with p53 levels. Both HBx and p53 proteins were found to interact and colocalize primarily in the nucleus. The binding of HBx to p53 modulated (but did not inhibit) the transcriptional activation function of p53. Notably, HBx-expressing cells exhibited increased sensitivity to UV damage, resulting in greater G2/M arrest and apoptosis of these cells. Additionally, these cells displayed a reduced DNA repair capacity in response to UV damage. In conclusion, this work suggests that DNA damage may be an initiating factor in hepatocarcinogenesis and that HBx may act as the promoting factor by inhibiting DNA repair. In hepatitis B virus-infected hepatocytes, a chronic infection may present the opportunity for such a DNA-damaging event to occur, and accumulated errors caused by the inhibition of DNA repair by HBx may result in oncogenesis.
Assuntos
Carcinoma Hepatocelular/etiologia , Dano ao DNA/efeitos da radiação , Neoplasias Hepáticas/etiologia , Transativadores/fisiologia , Raios Ultravioleta , Apoptose , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Fase G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Modelos Biológicos , Tolerância a Radiação , Transativadores/genética , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/biossíntese , Proteínas Virais Reguladoras e AcessóriasRESUMO
Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCF(betaTrCP) ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.