Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores.

Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

Inclination-dependent changes of the critical shoulder angle significantly influence superior glenohumeral joint stability.

BACKGROUND The critical shoulder angle combines the acromion index and glenoid inclination and has potential to discriminate between shoulders at risk for rotator cuff tear or osteoarthritis and those that are asymptomatic. However, its biomechanics, and particularly the role of the glenoid inclination, are not yet fully understood. METHODS A shoulder simulator was used to analyze the independent influence of glenoid inclination during abduction from 0 to 60°. Spindle motors transferred tension forces by a cable-pulley on human cadaveric humeri. A six-degree-of-freedom force transducer was mounted directly behind the polyethylene glenoid to measure shear and compressive joint reaction force and calculate the instability ratio (ratio of shear and compressive joint reaction force) with the different force ratios of the deltoid and supraspinatus muscles (2:1 and 1:1). A stepwise change in the inclination by 5° increments allowed simulation of a critical shoulder angle range of 20° to 45°. FINDINGS Tilting the glenoid to cranial (increasing the critical shoulder angle) increases the shear joint reaction force and therefore the instability ratio. A balanced force ratio (1:1) between the deltoid and the supraspinatus allowed larger critical shoulder angles before cranial subluxation occurred than did the deltoid-dominant ratio (2:1). INTERPRETATION Glenoid inclination-dependent changes of the critical shoulder angle have a significant impact on superior glenohumeral joint stability. The increased compensatory activity of the rotator cuff to keep the humeral head centered may lead to mechanical overload and could explain the clinically observed association between large angles and degenerative rotator cuff tears.

Management of a complication with a fractured zirconia implant abutment in the esthetic zone

Technical complications in implant prosthetic cases represent a major challenge in dentistry. This case report describes minimally invasive management to recover an implant with a fractured remnant of a zirconia abutment, including provisional rehabilitation during a sequential treatment protocol in the esthetic zone. A patient was treated with a screw-retained one-piece implant-supported reconstruction made of a customized zirconia abutment with direct ceramic veneering in the maxillary right central incisor position. During the prosthetic try-in, a fracture in the apical portion of the abutment was evident. The first rescue attempt led to fracture of the retrieval instrument. Immediately, an individualized wired construction was applied to bond the existing fractured reconstruction to the neighboring teeth to maintain the peri-implant mucosal architecture. Because the implant screw canal was blocked, a customized round bur had to be manufactured and was placed in the implant axis with a specific bracket tool from the service set to protect the interior implant threads. Then, the drills of the service set were guided by the newly created access to remove the fractured remnants. The implant screw was retapped and the area rinsed with chlorhexidine solution. All remnants were removed without the need for surgical intervention. Neither the implant connection nor the bone-to-implant interface was damaged. The stepwise treatment approach with the customized round bur combined with the system-specific drills of the service set saved the blocked implant so that the patient could be successfully rehabilitated with a new implant reconstruction.

Effect of Chronic Kidney Disease in Women Undergoing Percutaneous Coronary Intervention With Drug-Eluting Stents: A Patient-Level Pooled Analysis of Randomized Controlled Trials.

OBJECTIVES This study sought to evaluate: 1) the effect of impaired renal function on long-term clinical outcomes in women undergoing percutaneous coronary intervention (PCI) with drug-eluting stent (DES); and 2) the safety and efficacy of new-generation compared with early-generation DES in women with chronic kidney disease (CKD). BACKGROUND The prevalence and effect of CKD in women undergoing PCI with DES is unclear. METHODS We pooled patient-level data for women enrolled in 26 randomized trials. The study population was categorized by creatinine clearance (CrCl) <45 ml/min, 45 to 59 ml/min, and ≥60 ml/min. The primary endpoint was the 3-year rate of major adverse cardiovascular events (MACE). Participants for whom baseline creatinine was missing were excluded from the analysis. RESULTS Of 4,217 women included in the pooled cohort treated with DES and for whom serum creatinine was available, 603 (14%) had a CrCl <45 ml/min, 811 (19%) had a CrCl 45 to 59 ml/min, and 2,803 (66%) had a CrCl ≥60 ml/min. A significant stepwise gradient in risk for MACE was observed with worsening renal function (26.6% vs. 15.8% vs. 12.9%; p < 0.01). Following multivariable adjustment, CrCl <45 ml/min was independently associated with a higher risk of MACE (adjusted hazard ratio: 1.56; 95% confidence interval: 1.23 to 1.98) and all-cause mortality (adjusted hazard ratio: 2.67; 95% confidence interval: 1.85 to 3.85). Compared with older-generation DES, the use of newer-generation DES was associated with a reduction in the risk of cardiac death, myocardial infarction, or stent thrombosis in women with CKD. The effect of new-generation DES on outcomes was uniform, between women with or without CKD, without evidence of interaction. CONCLUSIONS Among women undergoing PCI with DES, CKD is a common comorbidity associated with a strong and independent risk for MACE that is durable over 3 years. The benefits of newer-generation DES are uniform in women with or without CKD.

Effect of Multiple Freezing of Stallion Semen on Sperm Quality and Fertility

To increase the efficiency of equine semen, it could be useful to split the artificial insemination dose and refreeze the redundant spermatozoa. In experiment I, semen of 10 sires of the Hanoverian breed, with poor and good semen freezability, was collected by artificial vagina, centrifuged, extended in INRA82 at 400 × 106 sperm/mL, and automatically frozen. After this first routinely applied freezing program, semen from each stallion was thawed, resuspended in INRA82 at 40 × 106 sperm/mL, filled in 0.5-mL straws, and refrozen. These steps were repeated, and sperm concentration was adjusted to 20 × 106 sperm/mL after a third freezing cycle. Regardless of stallion freezability group, sperm motility and sperm membrane integrity (FITC/PNA-Syto-PI-stain) decreased stepwise after first, second, and third freezing (62.3% ± 9.35, 24.0% ± 15.4, 3.3% ± 4.34, P ≤ .05; 29.6% ± 8.64, 14.9% ± 6.38, 8.3% ± 3.24, P ≤ .05), whereas the percentage of acrosome-reacted cells increased (19.5% ± 7.59, 23.9% ± 8.51, 29.2% ± 6.58, P ≤ .05). Sperm chromatin integrity was unaffected after multiple freeze/thaw cycles (DFI value: 18.6% ± 6.6, 17.2% ± 6.84, 17.1% ± 7.21, P > .05). In experiment II estrous, Hanoverian warmblood mares were inseminated with a total of 200 × 106 spermatozoa of two stallions with either good or poor semen freezability originating from the first, second, and third freeze/thaw cycle. First-cycle pregnancy rates were 4/10, 40%; 1/10, 10%; and 0/10, 0%. In conclusion, as expected, sperm viability of stallion spermatozoa significantly decreases as a consequence of multiple freezing. However, sperm chromatin integrity was not affected. Pregnancy rates after insemination of mares with refrozen semen are reduced.

Clinical effectiveness of cognitive behavioral therapy for depression in routine care: A propensity score based comparison between randomized controlled trials and clinical practice

Background: The efficacy of cognitive behavioral therapy (CBT) for the treatment of depressive disorders has been demonstrated in many randomized controlled trials (RCTs). This study investigated whether for CBT similar effects can be expected under routine care conditions when the patients are comparable to those examined in RCTs. Method: N=574 CBT patients from an outpatient clinic were stepwise matched to the patients undergoing CBT in the National Institute of Mental Health Treatment of Depression Collaborative Research Program (TDCRP). First, the exclusion criteria of the RCT were applied to the naturalistic sample of the outpatient clinic. Second, propensity score matching (PSM) was used to adjust the remaining naturalistic sample on the basis of baseline covariate distributions. Matched samples were then compared regarding treatment effects using effect sizes, average treatment effect on the treated (ATT) and recovery rates. Results: CBT in the adjusted naturalistic subsample was as effective as in the RCT. However, treatments lasted significantly longer under routine care conditions. Limitations: The samples included only a limited amount of common predictor variables and stemmed from different countries. There might be additional covariates, which could potentially further improve the matching between the samples. Conclusions: CBT for depression in clinical practice might be equally effective as manual-based treatments in RCTs when they are applied to comparable patients. The fact that similar effects under routine conditions were reached with more sessions, however, points to the potential to optimize treatments in clinical practice with respect to their efficiency.

Oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine that potentially interacts with parasite ferritin and cystatin.

This study investigated the effects of oral treatments of Echinococcus multilocularis-infected mice with the antimalarial drug mefloquine (MEF) and identified proteins that bind to MEF in parasite extracts and human cells by affinity chromatography. In a pilot experiment, MEF treatment was applied 5 days per week and was intensified by increasing the dosage stepwise from 12.5 mg/kg to 200 mg/kg during 4 weeks followed by treatments of 100 mg/kg during the last 7 weeks. This resulted in a highly significant reduction of parasite weight in MEF-treated mice compared with mock-treated mice, but the reduction was significantly less efficacious compared with the standard treatment regimen of albendazole (ABZ). In a second experiment, MEF was applied orally in three different treatment groups at dosages of 25, 50 or 100 mg/kg, but only twice a week, for a period of 12 weeks. Treatment at 100 mg/kg had a profound impact on the parasite, similar to ABZ treatment at 200 mg/kg/day (5 days/week for 12 weeks). No adverse side effects were noted. To identify proteins in E. multilocularis metacestodes that physically interact with MEF, affinity chromatography of metacestode extracts was performed on MEF coupled to epoxy-activated Sepharose(®), followed by SDS-PAGE and in-gel digestion LC-MS/MS. This resulted in the identification of E. multilocularis ferritin and cystatin as MEF-binding proteins. In contrast, when human cells were exposed to MEF affinity chromatography, nicotinamide phosphoribosyltransferase was identified as a MEF-binding protein. This indicates that MEF could potentially interact with different proteins in parasites and human cells.

In vitro effects of new artemisinin derivatives in Neospora caninum-infected human fibroblasts.

From a panel of 34 artemisinin derivatives tested in vitro, artemisone, GC007 and GC012 were most efficacious at inhibiting Neospora caninum replication (IC50 values of 3-54nM), did not notably impair the invasiveness of tachyzoites and were non-toxic for human foreskin fibroblasts (HFFs). Transmission electron microscopy of drug-treated N. caninum-infected HFFs demonstrated severe alterations in the parasite cytoplasm, changes in the composition of the matrix of the parasitophorous vacuole (PV) and diminished integrity of the PV membrane. To exert parasiticidal activity, parasites had to be cultured continuously in the presence of 5μM artemisone or GC007 for 3 weeks. N. caninum tachyzoites readily adapted to a stepwise increase in concentrations (0.5-10μM) of GC012, but not to artemisone or GC007. Drugs induced the expression of elevated levels of NcBAG1 and NcSAG4 mRNA, but only NcBAG1 could be detected by immunofluorescence. Thus, artemisinin derivatives represent interesting leads that should be investigated further.

Radiomic Texture Analysis Mapping Predicts Areas of True Functional MRI Activity.

Individual analysis of functional Magnetic Resonance Imaging (fMRI) scans requires user-adjustment of the statistical threshold in order to maximize true functional activity and eliminate false positives. In this study, we propose a novel technique that uses radiomic texture analysis (TA) features associated with heterogeneity to predict areas of true functional activity. Scans of 15 right-handed healthy volunteers were analyzed using SPM8. The resulting functional maps were thresholded to optimize visualization of language areas, resulting in 116 regions of interests (ROIs). A board-certified neuroradiologist classified different ROIs into Expected (E) and Non-Expected (NE) based on their anatomical locations. TA was performed using the mean Echo-Planner Imaging (EPI) volume, and 20 rotation-invariant texture features were obtained for each ROI. Using forward stepwise logistic regression, we built a predictive model that discriminated between E and NE areas of functional activity, with a cross-validation AUC and success rate of 79.84% and 80.19% respectively (specificity/sensitivity of 78.34%/82.61%). This study found that radiomic TA of fMRI scans may allow for determination of areas of true functional activity, and thus eliminate clinician bias.