Differential Diffusional Properties in Loose and Tight Docking Prior to Membrane Fusion

Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps, including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study because of their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here, we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion.     

A Chimeric 18L1-45RG1 Virus-Like Particle Vaccine Cross-Protects against Oncogenic Alpha-7 Human Papillomavirus Types

Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV.     

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.     

Non-Invasive Genetic Mark-Recapture as a Means to Study Population Sizes and Marking Behaviour of the Elusive Eurasian Otter (Lutra lutra)

Quantifying population status is a key objective in many ecological studies, but is often difficult to achieve for cryptic or elusive species. Here, non-invasive genetic capture-mark-recapture (CMR) methods have become a very important tool to estimate population parameters, such as population size and sex ratio. The Eurasian otter (Lutra lutra) is such an elusive species of management concern and is increasingly studied using faecal-based genetic sampling. For unbiased sex ratios or population size estimates, the marking behaviour of otters has to be taken into account. Using 2132 otter faeces of a wild otter population in Upper Lusatia (Saxony, Germany) collected over six years (2006–2012), we studied the marking behaviour and applied closed population CMR models accounting for genetic misidentification to estimate population sizes and sex ratios. We detected a sex difference in the marking behaviour of otters with jelly samples being more often defecated by males and placed actively exposed on frequently used marking sites. Since jelly samples are of higher DNA quality, it is important to not only concentrate on this kind of samples or marking sites and to invest in sufficiently high numbers of repetitions of non-jelly samples to ensure an unbiased sex ratio. Furthermore, otters seemed to increase marking intensity due to the handling of their spraints, hence accounting for this behavioural response could be important. We provided the first precise population size estimate with confidence intervals for Upper Lusatia (for 2012: N^ = 20 ± 2.1, 95% CI = 16–25) and showed that spraint densities are not a reliable index for abundances. We further demonstrated that when minks live in sympatry with otters and have comparably high densities, a non-negligible number of supposed otter samples are actually of mink origin. This could severely bias results of otter monitoring if samples are not genetically identified.     

Sex- and Time-Dependent Patterns in Risk Factors of End-Stage Renal Disease: A Large Austrian Cohort with up to 20 Years of Follow-Up

Objective

We investigated the association between metabolic factors and End-Stage Renal Disease (ESRD) and quantified the magnitude of their influence dependent on sex and time of exposure up to 20 years.

Material and Methods

A prospective cohort study was conducted to determine risk factors for the development of ESRD. From 1988 to 2005 185,341 persons (53.9% women) participated in the “Vorarlberg Health Monitoring and Promotion Programme” (VHM&PP). Data on body mass index (BMI), fasting blood glucose (FBG), systolic (BPsys) and diastolic (BPdia) blood pressure, total cholesterol (TC), triglycerides (TG), gamma-glutamyltransferase (GGT) and smoking status were collected. Data of the population-based VHM&PP were merged with the Austrian Dialysis and Transplant Registry. Cox proportional hazards models were applied to calculate hazard ratios (HRs) for ESRD, stratified by sex and 5-year time intervals.

Results

During a mean follow-up of 17.5 years 403 patients (39.1% women) developed ESRD. Significant risk factors were: BMI (per 1 kg/m²) HR 1.04 (95% CI 1.01–1.06), FBG (per 1 mmol/L) HR 1.09 (1.05–1.12), BPsys (per 5 mmHg) HR 1.10 (1.07–1.14), BPdia (per 5 mmHg) HR 1.09 (1.03–1.15), TG (per 1 mmol/L) HR 1.07 (1.02–1.13), TC (per 1 mmol/L) HR 1.22 (1.13–1.32). We observed a sex-specific risk pattern with an increased ESRD risk for men for increasing TG and smoking, and for women for increasing BMI and GGT. In time interval analyses BPsys and TC were associated with early ESRD onset, whereas BMI, FBG, BPdia and GGT were associated with later onset.

Conclusions

Anthropometric and metabolic factors are differentially associated with the long-term risk for ESRD in a sex- and time-dependent manner. Consideration of these patterns in preventive and therapeutic strategies could have an impact on ESRD incidence.     

Diverse Genotypes of Yersinia pestis Caused Plague in Madagascar in 2007

Background Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging.Conclusions/SignificancePlague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks.     

Keratin Dynamics: Modeling the Interplay between Turnover and Transport

Keratin are among the most abundant proteins in epithelial cells. Functions of the keratin network in cells are shaped by their dynamical organization. Using a collection of experimentally-driven mathematical models, different hypotheses for the turnover and transport of the keratin material in epithelial cells are tested. The interplay between turnover and transport and their effects on the keratin organization in cells are hence investigated by combining mathematical modeling and experimental data. Amongst the collection of mathematical models considered, a best model strongly supported by experimental data is identified. Fundamental to this approach is the fact that optimal parameter values associated with the best fit for each model are established. The best candidate among the best fits is characterized by the disassembly of the assembled keratin material in the perinuclear region and an active transport of the assembled keratin. Our study shows that an active transport of the assembled keratin is required to explain the experimentally observed keratin organization.