Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange

A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (∼2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for ∼50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.     

Association of the ScaI atrial natriuretic peptide gene polymorphism with nonfatal myocardial infarction and extent of coronary artery disease

Background There is growing evidence from recent studies that atrial natriuretic peptide (ANP) plays an important part in coronary blood flow regulation and in atherosclerosis. Transition T2238→C in the atrial natriuretic peptide (ANP) precursor gene, which leads potentially to the translation of ANP with 2 additional arginines, has been suggested to be associated with salt-sensitive hypertension. According to our knowledge, this study is the first to look for the potential association of the ScaI ANP gene polymorphism with the history of nonfatal myocardial infarction and the extent of coronary artery disease (CAD).Methods The study was performed in 847 consecutive, white patients (719 men and 128 women) with significant coronary artery stenosis confirmed by means of elective coronary angiography (at least 1 coronary artery with ≥50% lumen narrowing). Screening for the T2238→C substitution was performed by means of polymerase chain reaction of genomic DNA, followed by ScaI digestion and agarose gel electrophoresis.Results We found a significant association of the A2A2 ScaI ANP genotype with a higher incidence of positive history of nonfatal myocardial infarction (odds ratio 1.85, 95% CI 1.33-2.58) and multiple-vessel CAD (odds ratio 1.45, 95% CI 1.02-2.06). The ScaI ANP genotype distribution did not differ with age, sex, body mass index, plasma lipids, hypertension, diabetes mellitus, and family history of CAD in studied groups.Conclusions Our results suggest that the ScaI ANP polymorphism may be associated with nonfatal myocardial infarction and the extent of CAD. However, the precise mechanism of this association remains to be determined. (Am Heart J 2003;145:125-31.)     

Treatment of heart failure: guidelines compared to clinical practice

Last decade brought great development in the treatment of patients with heart failure (HF). General use of angiotensin-converting-enzyme inhibitors (ACE-I) in patients with asymptomatic left ventricular dysfunction or with HF significantly reduced morbidity and mortality. The aim of this study was to assess how the specialists from Cardiology Department and Gastroenterology Department think that heart failure should be managed, how they implement their knowledge and if it is consistent with the recommendation of European Society of Cardiology (ESC) and whether differences exists in practice between specialists. In the first phase the specialists, cardiologists and diabetologists, answered the questions about the management of different stages of HF. In second phase we analysed medical documentation of 345 patients aged between 38 and 98 years, hospitalised in Cardiology and Gastroenterology Departments from October 2000 to February 2002 by reason of coronary artery disease, hypertension and dilated cardiomyopathy. In the third phase we compared the knowledge of heart failure management from questionnaire and its implementation, the compliance with ESC recommendation and finally whether differences in clinical practice exist between cardiologist and diabetologists. RESULTS: ACE-I were prescribed in all NYHA classes of HF. In over 50% patients in II NYHA class to 94% in IV NYHA class in Cardiology Department. Differences between the Departments in prescribing of ACE-I were observed. Beta-blockers (BB) were used with the same frequency in all NYHA classes, more often in Cardiology Department. Frequency of the administration of digoxin, diuretics, aldosterone receptor blocker was increasing starting with II NYHA class. The highest compliance between declarations from questionnaire and clinical practice concerned the use of BB and ACE-I combination.     

Analysis of efficacy and safety of multiple intravenous infusion of anti-tumor necrosis factor-alpha monoclonal antibody (Remicade) combined with methotrexate compared with sodium aurothiomalate and intramuscular depot methylprednisolone in rheumatoid arthritis

The objective of the paper was compare the effects and tolerability of combined therapy of multiple intravenous infusions of anti-tumour necrosis factor-alfa (TNF-alfa) monoclonal antibody (Remicade) with methotrexate versus treatment with sodium aurothiomalate and intramuscular depot methylprednisolone in rheumatoid arthritis (RA). We investigate also the interval necessary to obtain the improvement in both treatment groups. 36 patients commencing intramuscular sodium aurothiomalate therapy with intramuscular depot methylprednisolone acetate at weeks 0, 4, 8 and 12 in addition to chrysotherapy were compared in retrospective analysis with 32 patients starting with multiple intravenous infusions of infliximab, anti-TNF-alfa monoclonal antibody (Remicade) and methotrexate at a stable dose. Patients were assessed by composite clinical score (DAS 28) and C-reactive protein during 22 weeks of therapy. At week 2 and 6 a significantly greater percentage of infliximab-treated than gold-treated RA patients achieved improvement in each clinical measurement of disease activity. At 22 week of treatment moderate and good response according to EULAR criteria was achieved in 91% of infliximab-treated patients and 58% gold treated patients (p < 0.001). Adverse events were more frequently observed in infliximab-treated patients, but only gold-treated patients discontinued treatment because adverse events (2 patients due to proteinuria, 2 patients due to mucocutaneous changes and one patient due to leucopenia). The higher percentage of adverse events in infliximab-treated patients was caused mainly by the occurrence of infusion reactions (23 reactions out of 160 infusions); most of them were mild (somnolentia and headache) and transient. Viral infections (including herpes simplex and zoster) were more common in patients treated with infliximab and methotrexate. Combination therapy of infliximab and methotrexate is more effective in reducing clinical and biochemical disease activity than gold with methylprednisolone treatment in RA patients during 22 weeks of treatment, especially in the first 6 weeks.     

Usefulness of intravascular low-power laser illumination in preventing restenosis after percutaneous coronary intervention

Despite the several years of studies, no factor that could reduce the restenosis rate without significant limitations has been introduced. The aim of the present study was to evaluate the influence of low-power 808-nm laser illumination of coronary vessels after percutaneous angioplasty in preventing restenosis. The procedure of laser intravascular illumination was performed on 52 patients (laser group), and another 49 patients formed the control group. All patients were monitored for major adverse cardiac events (MACE) at the 6- and 12-month follow-up points. The MACE rate after 6 and 12 months was 7.7% in the laser group at both points. The MACE rate was 14.3% and 18.5% at 6 and 12 months of follow-up in the control group, respectively (p = NS). Follow-up coronary angiography was performed after 6 months. The difference in the restenosis rate was insignificant (15.0% vs 32.4%); however, significant differences were observed in the minimal lumen diameter (2.18 ± 0.70 vs 1.76 ± 0.74 mm; p < 0.05), late lumen loss (0.53 ± 0.68 vs 0.76 ± 0.76 mm; p < 0.01), and the late lumen loss index (0.28 ± 0.39 vs 0.46 ± 0.43; p < 0.005) in favor of the laser group. In conclusion, the new therapy seemed effective and safe. Marked differences between late loss, late loss index, and minimal lumen diameter were observed. The late lumen loss in the laser group was only slightly greater than that in studies of drug-eluting stents, and MACE rate remained within very comparable ranges. This suggests that intravascular laser illumination could bring advantages comparable to those of drug-eluting stents without the risk of late thrombosis.     

The Influence of Annealing on the Optical Properties and Microstructure Recrystallization of the TiO2 Layers Produced by Means of the E-BEAM Technique

Titanium dioxide films, about 200 nm in thickness, were deposited using the e-BEAM technique at room temperature and at 227 °C (500K) and then annealed in UHV conditions (as well as in the presence of oxygen (at 850 °C). The fabricated dielectric films were examined using X-ray powder diffraction, Raman spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy, scanning electron microscopy, transmission electron microscopy, and spectroscopic ellipsometry. The applied experimental techniques allowed us to characterize the phase composition and the phase transformation of the fabricated TiO₂ coatings. The films produced at room temperature are amorphous but after annealing consist of anatase crystallites. The layers fabricated at 227 °C contain both anatase and rutile phases. In this case the anatase crystallites are accumulated near the substrate interface whilst the rutile crystallites were formed closer to the surface of the TiO₂ film. It should be emphasized that these two phases of TiO₂ are distinctly separated from each other.     

Effect of Fine Size-Fractionated Sunflower Husk Biochar on Water Retention Properties of Arable Sandy Soil

Biochar application has been reported to improve the physical, chemical, and hydrological properties of soil. However, the information about the size fraction composition of the applied biochar as a factor that may have an impact on the properties of soil-biochar mixtures is often underappreciated. Our research shows how sunflower husk biochar (pyrolyzed at 650 °C) can modify the water retention characteristics of arable sandy soil depending on the biochar dose (up to 9.52 wt.%) and particle size (<50 µm, 50–100 µm, 100–250 µm). For comparison, we used soil samples mixed with biochar passed through 2 mm sieve and an unamended reference. The addition of sieved biochar to the soil caused a 30% increase in the available water content (AWC) in comparing to the soil without biochar. However, the most notable improvement (doubling the reference AWC value from 0.078 m³ m⁻³ to 0.157 m³ m⁻³) was observed at the lowest doses of biochar (0.95 and 2.24 wt.%) and for the finest size fractions (below 100 µm). The water retention effects on sandy soil are explained as the interplay between the dose, the size of biochar particles, and the porous properties of biochar fractions.     

Material Model Effect for Simulating a Single-Lap Joint with a Blind Rivet

This paper concerns the influence of the material modeling method on the results of strength analyses. The research object was a single lap joint with a blind rivet (ISO 12996). The results of numerical strength analysis for various configurations of material models with material and contact nonlinearity were compared not only with the experimental results of such a connection but also with the values estimated using classical analytical tools (pressure stress and Hertz stress). The research aimed to determine how the results of numerical analyses (FEMs) were influenced by the method of modeling the material model and how it relates to the experimental results. As part of the analyses, a discrete riveted model and material models were constructed. The analyses took into account various load cases (from 10 to 90% of the connection capacity) to better illustrate the relationship between the numerical and experimental results. As a result of the conducted analyses, it was determined that the linear-elastic model was an acceptable and suggested solution (with a load of up to 90% of the load capacity of the joint connection) for further tests. The work was summarized with general and specific conclusions relating to all cases of numerical modeling. In addition, the summary includes suggestions for future works.     

Theoretical and Experimental Identification of Frequency Characteristics and Control Signals of a Dynamic System in the Process of Turning

The article presents the results of the experimental validation of the developed static, time and frequency characteristics under interference and longitudinal feed control of a dynamic system in the process of turning axisymmetric parts. The experiments were conducted on a test bench, consisting of a 16B16P center lathe, a measuring system and a PC with a measurement card. The experiments were carried out to verify the assumptions of the baseline model of the turning process. As part of the study, we determined the static characteristics of the machining process, the time characteristics of the object under interference and under longitudinal feed rate control, and the frequency characteristics of the machine tool system under longitudinal feed rate control. During the experiments, we recorded the observed input and output signal curves and the observed characteristics of the interferences acting on the object, as well as the numerical values of the parameters of the equations describing the model, and in particular the gain of the elastic system, which is difficult to determine by analytical methods. The positive results of the experiments confirm the effectiveness of the proposed models and their usefulness for automation of machining processes.     

Does morphology of carotid plaque depend on patient's oxidative stress?

Objectives

This study explored the relationship between oxidative stress biomarkers and stability of carotid plaque. We decided to analyze the broad range of parameters describing oxidative stress in patients with carotid stenosis.

Design and methods

124 consecutive patients undergoing carotid endarterectomy were enrolled in the study group. The control group consisted of 49 patients without symptoms of atherosclerosis. The stability of carotid plaques was assessed using GSM (gray-scale median) scoring system and the study group was divided into three subgroups according to echogenicity of the plaque. The following parameters of oxidative stress/DNA damage were analyzed: i) urinary excretion of the products of oxidative DNA damage repair; ii) the background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine in leukocytes' DNA and in atherosclerotic plaques; and iii) the concentrations of antioxidant vitamins, uric acid and C-reactive protein in plasma.

Results

Oxidative stress (described by redox status) was higher in the patient group than in the control group. There is a correlation between oxidative stress of the patients and stability of the plaque, echolucent plaques (GSM < 25) being associated with the highest antioxidant level and lowest excretion of DNA repair markers.

Conclusions

The plaque formation/morphology may depend on local environment and is independent of oxidative stress/inflammation observed on the level of the whole body.