2,853 research outputs found
Assembly and Disassembly of DNA Polymerase Holoenzyme
The complex task of genomic replication requires a large collection of proteins properly assembled within the close confines of the replication fork. The mechanism and dynamics of holoenzyme assembly and disassembly have been investigated using steady state and pre-steady state methods as opposed to structural studies, primarily due to the intrinsic transient nature of these protein complexes during DNA replication. The key step in bacteriophage T4 holoenzyme assembly involves ATP hydrolysis, whereas disassembly is mediated by subunit dissociation of the clamp protein in an ATP-independent manner
Assembly and Disassembly of DNA Polymerase Holoenzyme
The complex task of genomic replication requires a large collection of proteins properly assembled within the close confines of the replication fork. The mechanism and dynamics of holoenzyme assembly and disassembly have been investigated using steady state and pre-steady state methods as opposed to structural studies, primarily due to the intrinsic transient nature of these protein complexes during DNA replication. The key step in bacteriophage T4 holoenzyme assembly involves ATP hydrolysis, whereas disassembly is mediated by subunit dissociation of the clamp protein in an ATP-independent manner
Protein-Protein and Protein-DNA Interactions at The Bacteriophage T4 DNA Replication Fork. Characterization of a Fluorescently Labeled DNA Polymerase Sliding Clamp
The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine 5′-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis. A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis. Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA
Incidence and outcome of Staphylococcus aureus bacteremia in hemodialysis patients
Incidence and outcome of Staphylococcus aureus bacteremia in hemodialysis patients.Background. Staphylococcus aureusbacteremia is frequently associated with metastatic complications and infective endocarditis (IE). The Duke criteria for the diagnosis of IE utilize echocardiographic techniques and are more sensitive than previous criteria. The documentation of IE in patients undergoing hemodialysis (HD) has become increasingly important in order to avoid the overuse of empiric vancomycin and the emergence of antibiotic resistance.MethodsPatients who developed S. aureus bacteremia while undergoing HD at a tertiary medical center or one of four affiliated outpatient HD units were identified. Clinical outcome (death, metastatic complications, IE, and microbiologic recurrence) was assessed during hospitalization and at three months after discharge. Transthoracic and transesophageal echocardiograms were performed and the Duke criteria were used to diagnose IE. Pulse field gel electrophoresis was performed to confirm genetic similarity of recurrent isolates.ResultsFour hundred and forty-five patients underwent hemodialysis for 5431.8 patient-months. Sixty-two developed 65 episodes of S. aureus bacteremia (1.2 episodes/100 patient-months). Complications occurred in 27 (44%) patients. Bacteremia recurred in patients who dialyzed through polytetrafluorethylene grafts (44.4% vs. 7.1%, P = 0.0.01), and there was a trend to increased recurrence in patients who received only vancomycin (19.5% vs. 7.1%, P = 0.4). IE was diagnosed in 8 patients (12%), six of whom had normal transthoracic echocardiograms.ConclusionsSensitive echocardiographic techniques and the Duke criteria for the diagnosis of IE should be used to determine the proper duration of antibiotic therapy in hemodialysis patients with S. aureus bacteremia. This diagnostic approach, coupled with early removal of hardware, may assist in improving outcomes
Clinical and Financial Outcomes Due to Methicillin Resistant Staphylococcus aureus Surgical Site Infection: A Multi-Center Matched Outcomes Study
The clinical and financial outcomes of SSIs directly attributable to MRSA and methicillin-resistance are largely uncharacterized. Previously published data have provided conflicting conclusions.We conducted a multi-center matched outcomes study of 659 surgical patients. Patients with SSI due to MRSA were compared with two groups: matched uninfected control patients and patients with SSI due to MSSA. Four outcomes were analyzed for the 90-day period following diagnosis of the SSI: mortality, readmission, duration of hospitalization, and hospital charges. Attributable outcomes were determined by logistic and linear regression.In total, 150 patients with SSI due to MRSA were compared to 231 uninfected controls and 128 patients with SSI due to MSSA. SSI due to MRSA was independently predictive of readmission within 90 days (OR = 35.0, 95% CI 17.3-70.7), death within 90 days (OR = 7.27, 95% CI 2.83-18.7), and led to 23 days (95% CI 19.7-26.3) of additional hospitalization and 24,113 (95% 4,521-43,704) of additional charges.The attributable impact of S. aureus and methicillin-resistance on outcomes of surgical patients is substantial. Preventing a single case of SSI due to MRSA can save hospitals as much as $60,000
The Correlation Between Neonatal Intensive Care Unit Safety Culture and Quality of Care
OBJECTIVES: Key validated clinical metrics are being used individually and in aggregate (Baby-MONITOR) to monitor the performance of neonatal intensive care units (NICUs). The degree to which perceptions of key components of safety culture, safety climate, and teamwork are related to aspects of NICU quality of care is poorly understood. The objective of this study was to test whether NICU performance on key clinical metrics correlates with caregiver perceptions of safety culture.
STUDY DESIGN: Cross-sectional study of 6253 very low-birth-weight infants in 44 NICUs. We measured clinical quality via the Baby-MONITOR and its nine risk-adjusted and standardized subcomponents (antenatal corticosteroids, hypothermia, pneumothorax, healthcare-associated infection, chronic lung disease, retinopathy screen, discharge on any human milk, growth velocity, and mortality). A voluntary sample of 2073 of 3294 eligible professional caregivers provided ratings of safety and teamwork climate using the Safety Attitudes Questionnaire. We examined NICU-level variation across clinical and safety culture ratings and conducted correlation analysis of these dimensions.
RESULTS: We found significant variation in clinical and safety culture metrics across NICUs. Neonatal intensive care unit teamwork and safety climate ratings were correlated with absence of healthcare-associated infection (r = 0.39 [P = 0.01] and r = 0.29 [P = 0.05], respectively). None of the other clinical metrics, individual or composite, were significantly correlated with teamwork or safety climate.
CONCLUSIONS: Neonatal intensive care unit teamwork and safety climate were correlated with healthcare-associated infections but not with other quality metrics. Linkages to clinical measures of quality require additional research
Safety climate, safety climate strength, and length of stay in the NICU
BACKGROUND: Safety climate is an important marker of patient safety attitudes within health care units, but the significance of intra-unit variation of safety climate perceptions (safety climate strength) is poorly understood. This study sought to examine the standard safety climate measure (percent positive response (PPR)) and safety climate strength in relation to length of stay (LOS) of very low birth weight (VLBW) infants within California neonatal intensive care units (NICUs).
METHODS: Observational study of safety climate from 2073 health care providers in 44 NICUs. Consistent perceptions among a NICU\u27s respondents, i.e., safety climate strength, was determined via intra-unit standard deviation of safety climate scores. The relation between safety climate PPR, safety climate strength, and LOS among VLBW (\u3c 1500 g) infants was evaluated using log-linear regression. Secondary outcomes were infections, chronic lung disease, and mortality.
RESULTS: NICUs had safety climate PPRs of 66 ± 12%, intra-unit standard deviations 11 (strongest) to 23 (weakest), and median LOS 60 days. NICUs with stronger climates had LOS 4 days shorter than those with weaker climates. In interaction modeling, NICUs with weak climates and low PPR had the longest LOS, NICUs with strong climates and low PPR had the shortest LOS, and NICUs with high PPR (both strong and weak) had intermediate LOS. Stronger climates were associated with lower odds of infections, but not with other secondary outcomes.
CONCLUSIONS: Safety climate strength is independently associated with LOS and moderates the association between PPR and LOS among VLBW infants. Strength and PPR together provided better prediction than PPR alone, capturing variance in outcomes missed by PPR. Evaluations of NICU safety climate consider both positivity (PPR) and consistency of responses (strength) across individuals
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