19F-Festkörper-NMR-Untersuchungen zur Orientierung und Dynamik des antimikrobiellen Peptids PGLa in Lipidmembranen

Das antimikrobielle Peptid PGLa (21 Aminosäuren) aus der Haut des Krallenfroschs Xenopus laevis ist für die Permeabilisierung von Bakterienmembranen verantwortlich. Als Ansatz zur Aufklärung seines Wirkmechanismus wurde die Orientierung der [alpha]-helikalen Peptidstruktur relativ zur Membran und ihre Dynamik mit Hilfe der sensitiven 19F-Festkörper-NMR anhand von fünf verschiedenen 4-CF3-Phenylglycin-Markierungen in orientierten Proben untersucht. Nach der Peptid-Festphasensynthese, der Aufreinigung durch RP-HPLC ihrer Identifizierung durch ESI- bzw. MALDI-Massenspektrometrie bestätigten CD-Spektroskopie und antimikrobielle Wachstumsassays sowohl den geringen strukturellen Einfluss der CF3-Phenylglycin-Substitutionen als auch die vergleichbare biologische Wirksamkeit der Analoga zum Wildtyp-PGLa, mit Ausnahme der Substitution an Position Ala8. Eine X2-Analyse der gesammelten 19F-Dipolaufspaltungen ergab die Orientierung der idealisierten PGLa [alpha]-Helix in Bezug auf orientierte Lipidmembranen. Interessanterweise wurden zwei unterschiedliche Zustände je nach experimentellen Bedingungen (Konzentration und Temperatur) und Membranumgebung (DMPC, DLPC, DPPC, DOPC, DMPC:DPMG, DMPG:DMPE, E. coli-Lipidextrakte) gefunden: 1. im S-Zustand (S = surface) bei niedriger Peptidkonzentration legt sich die PGLa-Helix parallel auf die Membranoberfläche, und die Lysin-Seitenketten sind vom hydrophoben Membraninneren weg gerichtet; 2. im T-Zustand (T = tilt) bei hoher Peptidkonzentration (Lipid-Peptid-Verhältnisse von 50:1 bis 20:1) neigt sich die Helix um 30° gegen die Membranebene und dreht sich 25° um ihre Achse. Ein derartiger T-Zustand wird in der vorliegenden Arbeit erstmals beschrieben, und eine plausible Erklärung für die 30°-Orientierung liefert ein Dimerisierungsmodell zweier gegeneinander verkippter antiparalleler PGLa-Helices auf der Membranoberfläche.PGLa, a 21-residue member of the magainin family of antibiotic peptides isolated from frog skin of Xenopus laevis, is known to be responsible for permeabilizing bacterial membranes. In order to elucidate its mechanism of action, the orientation of the [alpha]-helical peptide structure with respect to the membrane and its dynamics were investigated by sensitive 19F-solid-state NMR measurements through five 4-CF3-phenylglycine labels in oriented samples. After solid-phase peptide synthesis, purification by RP-HPLC and their identification by ESI and MALDI mass spectrometry, CD spectroscopy confirmed the insignificant structural interference of the CF3-phenylglycine substitutions, and antimicrobial growth assays resulted in biological activity comparable to wild-type PGLa, with the exception of the substitution in position Ala8. A X2 analysis of 19F-dipolar couplings yielded the orientational state of the idealized PGLa helix with respect to the membrane surface of lipid membranes. Interestingly, this was found to differ depending on the experimental conditions (concentration and temperature) and membrane composition (DMPC, DLPC, DPPC, DOPC, DMPC:DPMG, DMPG:DMPE, E. coli-lipid extracts): 1. in the S-state (S = surface) at low peptide concentration the PGLa helix assumes a flat orientation on the membrane surface with the lysine side chains directed away from the hydrophobic membrane interior; 2. in the T-state (T = tilt) at high peptide concentration (lipid-to-peptide ratio from 50:1 to 20:1) the helix tilt changes by 30° relative to the membrane plane accompanied by a 25° rotation around its axis. The present thesis describes such a T-state for the first time, and a plausible explanation for the 30°-orientation may be provided by a dimeric model of two tilted PGLa helices aligned antiparallel and located on the membrane surface

Trafikmodeller til brug for analyse af transport i Øresundsregionen

I 2008 startede Projektet ”Infrastruktur- og Byudvikling Øresund” (IBU), som skal udrede spørgsmål vedrørende den fremtidige infrastruktur og transport i Øresundsregionen. Der findes i dag ingen tra- fikmodel, som alene kan håndtere analysebehovet. Nærværende artikel beskriver eksisterende danske, svenske og europæiske trafikmodeller, som vurderes relevante i forhold til brug ved analyser i IBU- projektet. På baggrund af en vurdering af fordele og ulemper i forhold til modellernes anvendelse i IBU-projektet anbefales det, at belyse den lokale og regionale persontrafik i Øresundsregionen med udgangspunkt i den svenske nationale trafikmodel (SAMPERS) og trafikmodellen for Hovedstads- området i Danmark (OTM). Artiklen beskriver, hvorledes de to modeller kan kombineres og udvides til at dække Region Skåne og Østdanmark. Den europæiske trafikmodel TRANS-TOOLS anbefales til beskrivelse af den internationale persontrafik, hvorimod godsmodellen for Øresundsregionen (GORM) forslås anvendt til analyse af godstransport

The Selective Autophagy Receptor p62 Forms a Flexible Filamentous Helical Scaffold

Published version also available at http://dx.doi.org/10.1016/j.celrep.2015.03.062The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo

Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1

Impact of radiotherapy and sequencing of systemic therapy on survival outcomes in melanoma patients with previously untreated brain metastasis: a multicenter DeCOG study on 450 patients from the prospective skin cancer registry ADOREG

BACKGROUND: Despite of various therapeutic strategies, treatment of patients with melanoma brain metastasis (MBM) still is a major challenge. This study aimed at investigating the impact of type and sequence of immune checkpoint blockade (ICB) and targeted therapy (TT), radiotherapy, and surgery on the survival outcome of patients with MBM. METHOD: We assessed data of 450 patients collected within the prospective multicenter real-world skin cancer registry ADOREG who were diagnosed with MBM before start of the first non-adjuvant systemic therapy. Study endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: Of 450 MBM patients, 175 (38.9%) received CTLA-4+PD-1 ICB, 161 (35.8%) PD-1 ICB, and 114 (25.3%) BRAF+MEK TT as first-line treatment. Additional to systemic therapy, 67.3% of the patients received radiotherapy (stereotactic radiosurgery (SRS); conventional radiotherapy (CRT)) and 24.4% had surgery of MBM. 199 patients (42.2%) received a second-line systemic therapy. Multivariate Cox regression analysis revealed the application of radiotherapy (HR for SRS: 0.213, 95% CI 0.094 to 0.485, p1 cm: 1.977, 95% CI 1.117 to 3.500, p=0.019), age (HR for age >65 years: 1.802, 95% CI 1.016 to 3.197, p=0.044), and ECOG performance status (HR for ECOG ≥2: HR: 2.615, 95% CI 1.024 to 6.676, p=0.044) as independent prognostic factors of OS on first-line therapy. The type of first-line therapy (ICB vs TT) was not independently prognostic. As second-line therapy BRAF+MEK showed the best survival outcome compared with ICB and other therapies (HR for CTLA-4+PD-1 compared with BRAF+MEK: 13.964, 95% CI 3.6 to 54.4, p<0.001; for PD-1 vs BRAF+MEK: 4.587 95% CI 1.3 to 16.8, p=0.022 for OS). Regarding therapy sequencing, patients treated with ICB as first-line therapy and BRAF+MEK as second-line therapy showed an improved OS (HR for CTLA-4+PD-1 followed by BRAF+MEK: 0.370, 95% CI 0.157 to 0.934, p=0.035; HR for PD-1 followed by BRAF+MEK: 0.290, 95% CI 0.092 to 0.918, p=0.035) compared with patients starting with BRAF+MEK in first-line therapy. There was no significant survival difference when comparing first-line therapy with CTLA-4+PD-1 ICB with PD-1 ICB. CONCLUSIONS: In patients with MBM, the addition of radiotherapy resulted in a favorable OS on systemic therapy. In BRAF-mutated MBM patients, ICB as first-line therapy and BRAF+MEK as second-line therapy were associated with a significantly prolonged OS

Exploring, exploiting and evolving diversity of aquatic ecosystem models: A community perspective

Here, we present a community perspective on how to explore, exploit and evolve the diversity in aquatic ecosystem models. These models play an important role in understanding the functioning of aquatic ecosystems, filling in observation gaps and developing effective strategies for water quality management. In this spirit, numerous models have been developed since the 1970s. We set off to explore model diversity by making an inventory among 42 aquatic ecosystem modellers, by categorizing the resulting set of models and by analysing them for diversity. We then focus on how to exploit model diversity by comparing and combining different aspects of existing models. Finally, we discuss how model diversity came about in the past and could evolve in the future. Throughout our study, we use analogies from biodiversity research to analyse and interpret model diversity. We recommend to make models publicly available through open-source policies, to standardize documentation and technical implementation of models, and to compare models through ensemble modelling and interdisciplinary approaches. We end with our perspective on how the field of aquatic ecosystem modelling might develop in the next 5–10 years. To strive for clarity and to improve readability for non-modellers, we include a glossary

Brain metastasis and survival outcomes after first-line therapy in metastatic melanoma: a multicenter DeCOG study on 1704 patients from the prospective skin cancer registry ADOREG

Background Despite the availability of effective systemic therapies, a significant number of advanced melanoma patients develops brain metastases. This study investigated differences in incidence and time to diagnosis of brain metastasis and survival outcomes dependent on the type of first-line therapy.Methods Patients with metastatic, non-resectable melanoma (AJCCv8 stage IIIC–V) without brain metastasis at start of first-line therapy (1L-therapy) were identified from the prospective multicenter real-world skin cancer registry ADOREG. Study endpoints were incidence of brain metastasis, brain metastasis-free survival (BMFS), progression-free survival (PFS), and overall survival (OS).Results Of 1704 patients, 916 were BRAF wild-type (BRAFwt) and 788 were BRAF V600 mutant (BRAFmut). Median follow-up time after start of 1L-therapy was 40.4 months. BRAFwt patients received 1L-therapy with immune checkpoint inhibitors (ICI) against CTLA-4+PD-1 (n=281) or PD-1 (n=544). In BRAFmut patients, 1L-therapy was ICI in 415 patients (CTLA-4+PD-1, n=108; PD-1, n=264), and BRAF+MEK targeted therapy (TT) in 373 patients. After 24 months, 1L-therapy with BRAF+MEK resulted in a higher incidence of brain metastasis compared with PD-1±CTLA-4 (BRAF+MEK, 30.3%; CTLA-4+PD-1, 22.2%; PD-1, 14.0%). In multivariate analysis, BRAFmut patients developed brain metastases earlier on 1L-therapy with BRAF+MEK than with PD-1±CTLA-4 (CTLA-4+PD-1: HR 0.560, 95% CI 0.332 to 0.945, p=0.030; PD-1: HR 0.575, 95% CI 0.372 to 0.888, p=0.013). Type of 1L-therapy, tumor stage, and age were independent prognostic factors for BMFS in BRAFmut patients. In BRAFwt patients, tumor stage was independently associated with longer BMFS; ECOG Performance status (ECOG-PS), lactate dehydrogenase (LDH), and tumor stage with OS. CTLA-4+PD-1 did not result in better BMFS, PFS, or OS than PD-1 in BRAFwt patients. For BRAFmut patients, multivariate Cox regression revealed ECOG-PS, type of 1L-therapy, tumor stage, and LDH as independent prognostic factors for PFS and OS. 1L-therapy with CTLA-4+PD-1 led to longer OS than PD-1 (HR 1.97, 95% CI 1.122 to 3.455, p=0.018) or BRAF+MEK (HR 2.41, 95% CI 1.432 to 4.054, p=0.001), without PD-1 being superior to BRAF+MEK.Conclusions In BRAFmut patients 1L-therapy with PD-1±CTLA-4 ICI resulted in a delayed and less frequent development of brain metastasis compared with BRAF+MEK TT. 1L-therapy with CTLA-4+PD-1 showed superior OS compared with PD-1 and BRAF+MEK. In BRAFwt patients, no differences in brain metastasis and survival outcomes were detected for CTLA-4+PD-1 compared with PD-1