Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli

Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolutio

Feasibility study of computed tomography colonography using limited bowel preparation at normal and low-dose levels study

The purpose was to evaluate low-dose CT colonography without cathartic cleansing in terms of image quality, polyp visualization and patient acceptance. Sixty-one patients scheduled for colonoscopy started a low-fiber diet, lactulose and amidotrizoic-acid for fecal tagging 2 days prior to the CT scan (standard dose, 5.8–8.2 mSv). The original raw data of 51 patients were modified and reconstructed at simulated 2.3 and 0.7 mSv levels. Two observers evaluated the standard dose scan regarding image quality and polyps. A third evaluated the presence of polyps at all three mSv levels in a blinded prospective way. All observers were blinded to the reference standard: colonoscopy. At three times patients were given questionnaires relating to their experiences and preference. Image quality was sufficient in all patients, but significantly lower in the cecum, sigmoid and rectum. The two observers correctly identified respectively 10/15 (67%) and 9/15 (60%) polyps ≥10 mm, with 5 and 8 false-positive lesions (standard dose scan). Dose reduction down to 0.7 mSv was not associated with significant changes in diagnostic value (polyps ≥10 mm). Eighty percent of patients preferred CT colonography and 13% preferred colonoscopy (P<0.001). CT colonography without cleansing is preferred to colonoscopy and shows sufficient image quality and moderate sensitivity, without impaired diagnostic value at dose-levels as low as 0.7 mSv

Assembly Factor Omp85 Recognizes Its Outer Membrane Protein Substrates by a Species-Specific C-Terminal Motif

Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence

Rate control drugs differ in the prevention of progression of atrial fibrillation

AIMS: We hypothesize that in patients with paroxysmal atrial fibrillation (AF), verapamil is associated with lower AF progression compared to beta blockers or no rate control. METHODS AND RESULTS: In this pre-specified post hoc analysis of the RACE 4 randomized trial, the effect of rate control medication on AF progression in paroxysmal AF was analysed. Patients using Vaughan-Williams Class I or III antiarrhythmic drugs were excluded. The primary outcome was a composite of first electrical cardioversion (ECV), chemical cardioversion (CCV), or atrial ablation. Event rates are displayed using Kaplan–Meier curves and multivariable Cox regression analyses are used to adjust for baseline differences. Out of 666 patients with paroxysmal AF, 47 used verapamil, 383 used beta blockers, and 236 did not use rate control drugs. The verapamil group was significantly younger than the beta blocker group and contained more men than the no rate control group. Over a mean follow-up of 37 months, the primary outcome occurred in 17% in the verapamil group, 33% in the beta blocker group, and 33% in the no rate control group (P = 0.038). After adjusting for baseline characteristics, patients using verapamil have a significantly lower chance of receiving ECV, CCV, or atrial ablation compared to patients using beta blockers [hazard ratio (HR) 0.40, 95% confidence interval (CI) 0.19–0.83] and no rate control (HR 0.64, 95% CI 0.44–0.93). CONCLUSION: In patients with newly diagnosed paroxysmal AF, verapamil was associated with less AF progression, as compared to beta blockers and no rate control

Outflows from the youngest stars are mostly molecular

The formation of stars and planets is accompanied not only by the build-up of matter, namely accretion, but also by its expulsion in the form of highly supersonic jets that can stretch for several parsecs 1,2. As accretion and jet activity are correlated and because young stars acquire most of their mass rapidly early on, the most powerful jets are associated with the youngest protostars 3. This period, however, coincides with the time when the protostar and its surroundings are hidden behind many magnitudes of visual extinction. Millimetre interferometers can probe this stage but only for the coolest components 3. No information is provided on the hottest (greater than 1,000 K) constituents of the jet, that is, the atomic, ionized and high-temperature molecular gases that are thought to make up the jet’s backbone. Detecting such a spine relies on observing in the infrared that can penetrate through the shroud of dust. Here we report near-infrared observations of Herbig-Haro 211 from the James Webb Space Telescope, an outflow from an analogue of our Sun when it was, at most, a few times 104 years old. These observations\ua0reveal copious emission from hot molecules, explaining the origin of the ‘green fuzzies’ 4–7 discovered nearly two decades ago by the Spitzer Space Telescope 8. This outflow is found to be propagating slowly in comparison to its more evolved counterparts and, surprisingly, almost no trace of atomic or ionized emission is seen, suggesting its spine is almost purely molecular

Atrioventricular and interventricular delay optimization in cardiac resynchronization therapy: physiological principles and overview of available methods

In this review, the physiological rationale for atrioventricular and interventricular delay optimization of cardiac resynchronization therapy is discussed including the influence of exercise and long-term cardiac resynchronization therapy. The broad spectrum of both invasive and non-invasive optimization methods is reviewed with critical appraisal of the literature. Although the spectrum of both invasive and non-invasive optimization methods is broad, no single method can be recommend for standard practice as large-scale studies using hard endpoints are lacking. Current efforts mainly investigate optimization during resting conditions; however, there is a need to develop automated algorithms to implement dynamic optimization in order to adapt to physiological alterations during exercise and after anatomical remodeling

Study protocol: population screening for colorectal cancer by colonoscopy or CT colonography: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Colorectal cancer (CRC) is the second most prevalent type of cancer in Europe. Early detection and removal of CRC or its precursor lesions by population screening can reduce mortality. Colonoscopy and computed tomography colonography (CT colonography) are highly accurate exams and screening options that examine the entire colon. The success of screening depends on the participation rate. We designed a randomized trial to compare the uptake, yield and costs of direct colonoscopy population screening, using either a telephone consultation or a consultation at the outpatient clinic, versus CT colonography first, with colonoscopy in CT colonography positives.</p> <p>Methods and design</p> <p>7,500 persons between 50 and 75 years will be randomly selected from the electronic database of the municipal administration registration and will receive an invitation to participate in either CT colonography (2,500 persons) or colonoscopy (5,000 persons) screening. Those invited for colonoscopy screening will be randomized to a prior consultation either by telephone or a visit at the outpatient clinic. All CT colonography invitees will have a prior consultation by telephone. Invitees are instructed to consult their general practitioner and not to participate in screening if they have symptoms suggestive for CRC. After providing informed consent, participants will be scheduled for the screening procedure. The primary outcome measure of this study is the participation rate. Secondary outcomes are the diagnostic yield, the expected and perceived burden of the screening test, level of informed choice and cost-effectiveness of both screening methods.</p> <p>Discussion</p> <p>This study will provide further evidence to enable decision making in population screening for colorectal cancer.</p> <p>Trial registration</p> <p>Dutch trial register: NTR1829</p

Guideline for collection, analysis and presentation of safety data in clinical trials of vaccines in pregnant women.

Vaccination during pregnancy is increasingly being used as an effective approach for protecting both young infants and their mothers from serious infections. Drawing conclusions from published studies in this area can be difficult because of the inability to compare vaccine trial results across different studies and settings due to the heterogeneity in the definitions of terms used to assess the safety of vaccines in pregnancy and the data collected in such studies. The guidelines proposed in this document have been developed to harmonize safety data collection in all phases of clinical trials of vaccines in pregnant women and apply to data from the mother, fetus and infant. Guidelines on the prioritization of the data to be collected is also provided to allow applicability in various geographic, cultural and resource settings, including high, middle and low-income countries