Bartlomiej Bartkowiak

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 ↔ CDK9 and Ctk1 ↔ 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.

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.

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.