DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain.

The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB-DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB-DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I-binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46 DnaB Leu160 is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I-bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA-DnaB-DnaC complex, which could be relevant to the process of DnaB loading onto oriC.

Accuracy and Trending Ability of Cardiac Index Measured by the CNAP System in Patients Undergoing Abdominal Aortic Aneurysm Surgery.

OBJECTIVES: The CNAP system is a noninvasive monitor that provides a continuous arterial pressure waveform using an inflatable finger cuff. The authors hypothesized that dramatic changes in systemic vascular resistance index during abdominal aortic aneurysm (AAA) surgery might affect the accuracy of noninvasive pulse contour monitors. The aim of this study was to evaluate the accuracy and trending ability of cardiac index derived by the CNAP system (CICN) in patients undergoing AAA surgery. DESIGN: Prospective clinical study. SETTING: Cardiac surgery operating room in a single cardiovascular center. PARTICIPANTS: Twenty patients who underwent elective AAA surgery. INTERVENTIONS: CICN and cardiac index measured using 3-dimensional images (CI3D) were determined simultaneously at 8 points during the surgery. At aortic clamping and unclamping, the authors tested the trending ability of CICN using 4-quadrant plot analysis and polar plot analysis. MEASUREMENTS AND MAIN RESULTS: The authors found a wide limit of agreement between CICN and CI3D (percentage error: 85.0%). The cubic splines, which show the relationship between systemic vascular resistance index and percentage CI discrepancy [(CICN-CI3D)/CI3D], were sloped positively. Four-quadrant plot analysis showed poor trending ability for CICN at both aortic clamping and unclamping (concordance rate: 29.4% and 57.9%, respectively). In the polar plot analysis, the concordance rates at aortic clamping and unclamping were 15.0% and 35.0%, respectively. CONCLUSIONS: CICN is not interchangeable with CI3D in patients undergoing AAA surgery. The trending ability for CICN at aortic clamping and unclamping was below the acceptable limit. These inaccuracies might be secondary to the high systemic vascular resistance index during AAA surgery.

Accuracy and Trending Ability of Blood Pressure and Cardiac Index Measured by ClearSight System in Patients With Reduced Ejection Fraction.

OBJECTIVES: To investigate the accuracy and trending ability of ClearSight (Edwards Lifesciences, Irvine, CA) in patients with reduced ejection fraction (<55%) undergoing off-pump coronary artery bypass graft (CABG) surgery by comparing the ClearSight-derived cardiac index (CICS) with the cardiac index measured with thermodilution using a pulmonary artery catheter. In addition, the accuracy and trending ability of ClearSight for blood pressure measurement was investigated by comparing the mean arterial pressure (MAP) derived by ClearSight (MAPcs) with invasive intra-arterial pressure. DESIGN: Prospective clinical study. DESIGN: Cardiac surgery operating room in a single cardiovascular center. PARTICIPANTS: The study comprised 20 patients who underwent elective CABG surgery. INTERVENTIONS: MAP and cardiac index were measured simultaneously at 6 time points intraoperatively. Trending ability was investigated at the following 2 points: (1) before and after placing the patient in the Trendelenburg position and (2) before and after atrial pacing with a targeted heart rate increase of 20%. MEASUREMENTS AND MAIN RESULTS: Bland-Altman analysis showed that the percentage error between CICS and the cardiac index measured with thermodilution was 40.2% and the percentage error between MAPcs and MAP was 24.6%. Four-quadrant plot analysis showed that the tracking ability of CICS with the Trendelenburg position and atrial pacing was below the good trending ability cutoff (92%). However, the concordance rate of the 4-quadrant plot analysis showed a good trending ability for MAPcs. The polar plot analysis showed the same trend. CONCLUSIONS: CICS was not sufficiently accurate in patients with reduced ejection fraction undergoing off-pump CABG surgery. However, ClearSight was clinically acceptable for MAP regarding its accuracy and trending ability in patients with reduced ejection fraction.

Accuracy of the ClearSight™ system in patients undergoing abdominal aortic aneurysm surgery.

PURPOSE: The ClearSight™ device monitors continuous pressure and cardiac output via pulse contour analysis. ClearSight™, however, may not be reliable in patients with reduced peripheral perfusion caused by high peripheral resistance. This study aimed to elucidate the accuracy and trending ability of ClearSight™ in patients undergoing abdominal aortic aneurysm (AAA) surgery by comparing the ClearSightTM-derived cardiac index (CICS) with that measured using three-dimensional echocardiography (CI3D). METHODS: The study included 20 patients who underwent elective AAA surgery. CICS and CI3D were measured simultaneously at eight time points during the surgery. Trending ability was investigated after aortic clamping and unclamping. We used CI3D as the reference method. RESULTS: Bland-Altman analysis showed a wide limit of agreement between CICS and CI3D (percentage error 41.3%). Subgroup analysis showed a lower percentage error (33.2%) in patients with CI ≥ 2.5 L/min/m2. The cubic splines related to the CI3D and CI discrepancy were negatively sloped, indicating that CI3D had significant influence on the CI discrepancy (p < 0.001). Four-quadrant plot analysis showed that the tracking ability of ClearSight™ after aortic clamping and declamping were clinically unacceptable (81.3% and 78.6%, respectively). Also, the polar plot analysis showed that the concordance rate of ClearSight™ after aortic clamping and declamping were clinically unacceptable (58.3% and 66.7%, respectively). CONCLUSIONS: ClearSight™ was not sufficiently accurate in patients undergoing AAA surgery. The tracking ability of ClearSight™ after aortic clamping was below the acceptable limit.

Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex.

In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.

Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding.

Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB-DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB-DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

Profiling nematode communities in unmanaged flowerbed and agricultural field soils in Japan by DNA barcode sequencing.

Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis.

Gestäo de Processos no Laboratório Central do Hospital das Clínicas da Faculdade de Medicina USP vista sob a ótica dos critérios de excelência do Prêmio Nacional da Qualidade (PNQ) / Process Management in Central Laboratory of Hospital das Clínicas da Faculdade de Medicina USP analyzed according to National Award Foundation criteria for performance excellence

O trabalho mostra a aplicaçäo do modelo de gestäo baseada no Prêmio Nacional da Qualidade na Divisäo de Laboratório Central HC-FMUSP, enfocando o critério de Gestäo de Processos. Esta instituiçäo possui a certificaçäo ISO 9002 desde 1997 e sua política de qualidade dos serviços, assim como interagir de modo salutar com o meio ambiente, garantindo o bem-estar da comunidade. Usando os critérios de excelência do Prêmio Nacional da Qualidade, uma instituiçäo pode modelar seu sistema de gestäo, realizar uma auto-avaliaçäo ou se candidatar ao Prêmio Nacional da Qualidade