RESUMEN
OBJECTIVE: Assess the accuracy of 3 early warning scores for predicting severe adverse events in postoperative inpatients. SUMMARY OF BACKGROUND DATA: Postoperative clinical deterioration on inpatient hospital services is associated with increased morbidity, mortality, and cost. Early warning scores have been developed to detect inpatient clinical deterioration and trigger rapid response activation, but knowledge regarding the application of early warning scores to postoperative inpatients is limited. METHODS: This was a retrospective cohort study of adult patients hospitalized on the wards after surgical procedures at an urban academic medical center from November, 2008 to January, 2016. The accuracies of the Modified Early Warning Score (MEWS), National Early Warning Score (NEWS), and the electronic cardiac arrest risk triage (eCART) score were compared in predicting severe adverse events (ICU transfer, ward cardiac arrest, or ward death) in the postoperative period using the area under the receiver operating characteristic curve (AUC). RESULTS: Of the 32,537 patient admissions included in the study, 3.8% (n = 1243) experienced a severe adverse outcome after the procedure. The accuracy for predicting the composite outcome was highest for eCART [AUC 0.79 (95% CI: 0.78-0.81)], followed by NEWS [AUC 0.76 (95% CI: 0.75-0.78)], and MEWS [AUC 0.75 (95% CI: 0.73-0.76)]. Of the individual vital signs and labs, maximum respiratory rate was the most predictive (AUC 0.67) and maximum temperature was an inverse predictor (AUC 0.46). CONCLUSION: Early warning scores are predictive of severe adverse events in postoperative patients. eCART is significantly more accurate in this patient population than both NEWS and MEWS.
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Paro Cardíaco/diagnóstico , Paro Cardíaco/etiología , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/etiología , Triaje , Adulto , Anciano , Registros Electrónicos de Salud , Femenino , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Curva ROC , Estudios Retrospectivos , Medición de Riesgo , Signos VitalesRESUMEN
As the population of patients with cancer and survivors grows, physician knowledge of oncology clinical care and research is increasingly important. Despite this patient population growth, medical students and non-oncology physicians report insufficient oncologic and survivorship care training. First-year students at a single US medical school completing a summer research experience were invited to participate in integrated Scholars in Oncology-Associated Research (SOAR) program. SOAR seeks to broaden students' understanding of multidisciplinary and interprofessional oncology clinical care and research. SOAR consists of three components: structured didactics, multidisciplinary tumor board attendance, and interprofessional shadowing. A mixed-methods approach investigated whether student knowledge improved after SOAR. Thirty-three students enrolled in SOAR (20 in 2015, 13 in 2016) and completed pre-assessments. Twenty-five (75.8%) students completed SOAR and post-assessments. Self-reported understanding of clinical (2[2, 3] vs. 4[4], p < 0.01) and research oncology (2[2, 3] vs. 4[4], p < 0.01) improved after SOAR. Understanding of individual disciplines also significantly improved. When describing clinical oncology, responses written post-SOAR were more comprehensive, averaging 3.7 themes per response vs. 2.8 on pre-assessments (p = 0.03). There were more references to "survivorship" as a component of oncology on post-assessments (0[0.0%] vs. 7[28.0%], p < 0.01) and "screening/prevention" (2[6.1%] vs. 7[28.0%], p = 0.03). Additionally, students more often described cancer care as a continuum on post-assessments (4[12.1%] vs. 11[44.0%], p = 0.01). A structured didactic and experiential introduction to oncology, SOAR, was successfully piloted. SOAR improved participant understanding of oncology and its distinct clinical and research disciplines. Future work will focus on expanding SOAR into a longitudinal oncology curriculum.
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Curriculum/normas , Educación de Pregrado en Medicina/normas , Estudios Interdisciplinarios , Oncología Médica/educación , Atención Dirigida al Paciente/métodos , Facultades de Medicina/normas , Estudiantes de Medicina/estadística & datos numéricos , Adulto , Educación de Pregrado en Medicina/métodos , Evaluación Educacional/métodos , Femenino , Humanos , Relaciones Interprofesionales , Masculino , Proyectos Piloto , Adulto JovenRESUMEN
Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.
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Anilidas/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Cisteína/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Anilidas/síntesis química , Anilidas/química , Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/metabolismo , Muerte Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Cisteína/metabolismo , Daño del ADN , Humanos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/químicaRESUMEN
Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 3' end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 â CDK9 and Ctk1 â CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.
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Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Western Blotting , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular , Mapeo Cromosómico , Ciclina T/genética , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Prueba de Complementación Genética , Células HeLa , Humanos , Microscopía Fluorescente , Mutación , Fosforilación , Proteínas Quinasas/genética , Interferencia de ARN , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3'-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a "2A" peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.
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Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Secuencia de Aminoácidos , Baculoviridae/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Descubrimiento de Drogas , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The RNA Polymerase II C-terminal domain (CTD) kinase CDK12 has been implicated as a tumor suppressor and regulator of DNA damage response genes. Although much has been learned about CDK12 and its activity, due to the lack of a specific inhibitor and the complications posed by long term RNAi depletion, much is still unknown about the particulars of CDK12 function. Therefore gaining a better understanding of CDK12's roles at the molecular level will be challenging without the development of additional tools. In order to address these issues we have used the CRISPR/Cas gene engineering system to create a mammalian cell line in which the only functional copy of CDK12 is selectively inhibitable by a cell-permeable adenine analog (analog-sensitive CDK12). Inhibition of CDK12 results in a perturbation of the phosphorylation patterns on the CTD and an arrest in cellular proliferation. This cell line should serve as a powerful tool for future studies.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Quinasas Ciclina-Dependientes/genética , Secuencia de Bases , ADN , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Focal and segmental glomerulosclerosis (FSGS) is a major cause of end-stage kidney disease. Recent advances in molecular genetics show that defects in the podocyte play a major role in its pathogenesis and mutations in inverted formin 2 (INF2) cause autosomal dominant FSGS. In order to delineate the role of INF2 mutations in familial and sporadic FSGS, we sought to identify variants in a large cohort of patients with FSGS. A secondary objective was to define an approach for genetic screening in families with autosomal dominant disease. A total of 248 individuals were identified with FSGS, of whom 31 had idiopathic disease. The remaining patients clustered into 64 families encompassing 15 from autosomal recessive and 49 from autosomal dominant kindreds. There were missense mutations in 8 of the 49 families with autosomal dominant disease. Three of the detected variants were novel and all mutations were confined to exon 4 of INF2, a regulatory region responsible for 90% of all changes reported in FSGS due to INF2 mutations. Thus, in our series, INF2 mutations were responsible for 16% of all cases of autosomal dominant FSGS, with these mutations clustered in exon 4. Hence, screening for these mutations may represent a rapid, non-invasive and cost-effective method for the diagnosis of autosomal dominant FSGS.
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Glomeruloesclerosis Focal y Segmentaria/genética , Proteínas de Microfilamentos/genética , Mutación , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Niño , Preescolar , Exones , Femenino , Forminas , Genes Dominantes , Genes Recesivos , Pruebas Genéticas , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Lactante , Masculino , Proteínas de Microfilamentos/química , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación Missense , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Adulto JovenRESUMEN
The cyclin-dependent kinase CDK12 has garnered interest as a cancer therapeutic target as DNA damage response genes are particularly suppressed by loss of CDK12 activity. In this study, we assessed the acute effects of CDK12 inhibition on transcription and RNA processing using nascent RNA Bru-seq and BruChase-seq. Acute transcriptional changes were overall small after CDK12 inhibition but over 600 genes showed intragenic premature termination, including DNA repair and cell cycle genes. Furthermore, many genes showed reduced transcriptional readthrough past the end of genes in the absence of CDK12 activity. RNA turnover was dramatically affected by CDK12 inhibition and importantly, caused increased degradation of many transcripts from DNA damage response genes. We also show that co-transcriptional splicing was suppressed by CDK12 inhibition. Taken together, these studies reveal the roles of CDK12 in regulating transcription elongation, transcription termination, co-transcriptional splicing, and RNA turnover.
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FSGS is a clinicopathologic entity characterized by nephrotic syndrome and progression to ESRD. Although the pathogenesis is unknown, the podocyte seems to play a central role in this disorder. Here, we present six kindreds with hereditary FSGS that did not associate with mutations in known causal genes, and we report a new locus for the disease on chromosome 2p15 in one kindred. We performed genome-wide linkage analysis and refined the linkage area with microsatellite markers and haplotype analysis to define the minimal candidate region. Genome-wide linkage analysis yielded a maximum two-point logarithm of odds (LOD) score of 3.6 for the six families on chromosome 2p. One family contributed the largest proportion of the additive score (LOD 2.02) at this locus. Multipoint parametric LOD score calculation in this family yielded a significant LOD score of 3.1 at markers D2S393 and D2S337, and fine mapping of this region with microsatellite markers defined a minimal candidate region of 0.9 Mb with observed recombinations at markers D2S2332 and RS1919481. We excluded the remaining five families from linkage to this region by haplotype analysis. These data support a new gene locus for familial FSGS on chromosome 2p15. Identification of the mutated gene at this locus may provide further insight into the disease mechanisms of FSGS.
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Fallo Renal Crónico/etiología , Fallo Renal Crónico/genética , Síndrome Nefrótico/complicaciones , Síndrome Nefrótico/genética , Adolescente , Adulto , Niño , Preescolar , Cromosomas Humanos/genética , Progresión de la Enfermedad , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Escala de Lod , Masculino , Linaje , Adulto JovenRESUMEN
We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition.
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Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Células HeLa , Humanos , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
RNA polymerase II translocates across much of the genome and since it can be blocked by many kinds of DNA lesions, detects DNA damage proficiently; it thereby contributes to DNA repair and to normal levels of DNA damage resistance. However, the components and mechanisms that respond to polymerase blockage are largely unknown, except in the case of UV-induced damage that is corrected by nucleotide excision repair. Because elongating RNAPII carries with it numerous proteins that bind to its hyperphosphorylated CTD, we tested for effects of interfering with this binding. We find that expressing a decoy CTD-carrying protein in the nucleus, but not in the cytoplasm, leads to reduced DNA damage resistance. Likewise, inducing aberrant phosphorylation of the CTD, by deleting CTK1, reduces damage resistance and also alters rates of homologous recombination-mediated repair. In line with these results, extant data sets reveal a remarkable, highly significant overlap between phosphoCTD-associating protein genes and DNA damage-resistance genes. For one well-known phosphoCTD-associating protein, the histone methyltransferase Set2, we demonstrate a role in DNA damage resistance, and we show that this role requires the phosphoCTD binding ability of Set2; surprisingly, Set2's role in damage resistance does not depend on its catalytic activity. To explain all of these observations, we posit the existence of a CTD-Associated DNA damage Response (CAR) system, organized around the phosphoCTD of elongating RNAPII and comprising a subset of phosphoCTD-associating proteins.
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ARN Polimerasa II/metabolismo , Daño del ADN/genética , Mitosis/genética , Mitosis/fisiología , FosforilaciónRESUMEN
The C-terminal domain of RNA polymerase II undergoes a cycle of phosphorylation which allows it to temporally couple transcription with transcription-associated processes. The characterization of hitherto unrecognized metazoan elongation phase CTD kinase activities expands our understanding of this coupling. We discuss the circumstances that delayed the recognition of these kinase activities.
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Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA but also coordinates transcription-related processes via its unique C-terminal repeat domain (CTD). The CTD is an RNAPII-specific protein segment consisting of repeating heptads with the consensus sequence Y(1)S(2)P(3)T(4)S(5)P(6)S(7) that has been shown to be extensively post-transcriptionally modified in a coordinated, but complicated, manner. Recent discoveries of new modifications, kinases, and binding proteins have challenged previously established paradigms. In this paper, we examine results and implications of recent studies related to modifications of the CTD and the respective enzymes; we also survey characterizations of new CTD-binding proteins and their associated processes and new information regarding known CTD-binding proteins. Finally, we bring into focus new results that identify two additional CTD-associated processes: nucleocytoplasmic transport of mRNA and DNA damage and repair.
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Focal and segmental glomerulosclerosis (FSGS) is the most common glomerular cause of end-stage kidney disease (ESKD). Although the etiology of FSGS has not been fully elucidated, recent results from the positional cloning of genes mutated in nephrotic syndromes are now beginning to provide insight into the pathogenesis of these diseases. Mutations in PLCE1/NPHS3 have recently been reported as a cause of nephrotic syndrome characterized by diffuse mesangial sclerosis (DMS) histology. One single family with a missense mutation had late onset of the disease that was characterized by FSGS. To further define the role of PLCE1 mutations in the etiology of FSGS, we performed mutational analysis in 69 families with FSGS. A total of 69 families with 231 affected individuals were examined. The median age of disease onset was 26 years (range 1-66 years). Onset of ESKD was at a median age of 35.5 years. Seven variants leading to non-synonymous changes were found, of which only two are new variants (exon 4 c.1682 G>A R561Q, exon 31 c.6518A>G K2173R). No known disease-causing mutations were identified in the families screened. PLCE1/NPHS3 mutations are not a cause of FSGS in this cohort. The absence of mutations in PLCE1/NPHS3 in this study indicates that there are additional genetic causes of FSGS and that hereditary FSGS is a heterogeneous disease. Kindreds appropriate for genome-wide screening are currently being subjected to analysis with the aim of identifying other genetic causes of FSGS.