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1. The Multifaceted Antibacterial Mechanisms of the Pioneering Peptide Antibiotics Tyrocidine and Gramicidin S

Author: Michaela Wenzel, Marina Rautenbach, J. Arnold Vosloo, Tjalling Siersma, Christopher H. M. Aisenbrey, Ekaterina Zaitseva, Wikus E. Laubscher, Wilma van Rensburg, Jan C. Behrends, Burkhard Bechinger, and Leendert W. Hamoen
Subjects: antibiotics, antimicrobial peptides, bacterial cell biology, bacterial cytological profiling, cell membranes, mode of action, Microbiology, QR1-502
Abstract: ABSTRACT Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent. IMPORTANCE Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of β-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them.
Published: 2018
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2. Races to Modernity: Metropolitan Aspirations in Eastern Europe, 1890–1940

Author: Jan C. Behrends, Martin Kohlrausch, Jan C. Behrends, Martin Kohlrausch
Published: 2014

3. The radiosensitizing effect of platinum nanoparticles in proton irradiations is not caused by an enhanced proton energy deposition at the macroscopic scale

Author: C Behrends, C M Bäcker, I Schilling, S Zwiehoff, J Weingarten, K Kröninger, C Rehbock, S Barcikowski, J Wulff, C Bäumer, and B Timmermann
Subjects: Radiation-Sensitizing Agents, Radiological and Ultrasound Technology, Medizin, Chemie, Metal Nanoparticles, Radiosensitizing effect, Proton therapy, Gelatin, Energy deposition, Radiology, Nuclear Medicine and imaging, Protons, Reactive Oxygen Species, Noble metal nanoparticles, Platinum
Abstract: Objective. Due to the radiosensitizing effect of biocompatible noble metal nanoparticles (NPs), their administration is considered to potentially increase tumor control in radiotherapy. The underlying physical, chemical and biological mechanisms of the NPs' radiosensitivity especially when interacting with proton radiation is not conclusive. In the following work, the energy deposition of protons in matter containing platinum nanoparticles (PtNPs) is experimentally investigated. Approach. Surfactant-free monomodal PtNPs with a mean diameter of (40 ± 10) nm and a concentration of 300 μg ml−1, demonstrably leading to a substantial production of reactive oxygen species (ROS), were homogeneously dispersed into cubic gelatin samples serving as tissue-like phantoms. Gelatin samples without PtNPs were used as control. The samples' dimensions and contrast of the PtNPs were verified in a clinical computed tomography scanner. Fields from a clinical proton machine were used for depth dose and stopping power measurements downstream of both samples types. These experiments were performed with a variety of detectors at a pencil beam scanning beam line as well as a passive beam line with proton energies from about 56–200 MeV. Main results. The samples' water equivalent ratios in terms of proton stopping as well as the mean proton energy deposition downstream of the samples with ROS-producing PtNPs compared to the samples without PtNPs showed no differences within the experimental uncertainties of about 2%. Significance. This study serves as experimental proof that the radiosensitizing effect of biocompatible PtNPs is not due to a macroscopically increased proton energy deposition, but is more likely caused by a catalytic effect of the PtNPs. Thus, these experiments provide a contribution to the highly discussed radiobiological question of the proton therapy efficiency with noble metal NPs and facilitate initial evidence that the dose calculation in treatment planning is straightforward and not affected by the presence of sensitizing PtNPs., Physics in medicine and biology;67(15)
Published: 2022
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4. Kinetic Analysis of Single Molecule Electrodiffusion in a Biological Nanopore with Two Binding Sites

Author: Murugappan Muthukumar, Norbert Ankri, Gerhard Baaken, Mordjane Boukhet, Jan C. Behrends, Debanne, Dominique, Unité de Neurobiologie des canaux Ioniques et de la Synapse (UNIS - Inserm U1072), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Saclay (COmUE), University of Massachusetts [Amherst] (UMass Amherst), University of Massachusetts System (UMASS), Institut für Silizium-Photovoltaik, and Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB)
Subjects: 0303 health sciences, Chemistry, [SDV]Life Sciences [q-bio], Kinetic analysis, Biophysics, [SDV] Life Sciences [q-bio], 03 medical and health sciences, Nanopore, 0302 clinical medicine, Molecule, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Binding site, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: International audience; When single short adenine oligonucleotides (d.p. 3-5) block the pore formed by the bacterial toxin aerolysin, the resultant resistive pulses are complex with two levels (short and deep vs. long and shallow) in a variety of configurations, the probability of which depends on voltage. We account for this by a four-state kinetic mechanism with two undistinguishable open states i,o and two blocked states m1 (short-deep) and m2 (long-shallow) linked as om1m2->i. M1 is directly accessible from o which designates the presence of the nucleotide at the pore0s cis-side mouth, while m2 is inaccessible from state i, as nucleotides are unable to enter from the trans-side irrespective of voltage. In this framework, several experimentally accessible statistical quantities such as the frequency or probability of returns to m1 from m2, the fraction of restive pulses ending in m1and the mean dwell times in m2 as well as the mean total duration of resistive pulses acquire mechanistic significance and allow direct kinetic predictions using a Q-matrix approach. For A3, the data are consistent with a charged particle moving through a one-dimensional energy landscape with two minimain an electrically biased random walk. For longer nucleotides (e.g. A5) the success rate for translocation is higher than predicted by the rate constants determined from the other observables. It appears likely that this is due to an ability of the longer oligomers to simultaneously link with the two binding sites, producing an excess of returns from m2 to m1, which, however, does not entail a propensity to result in translocation failures. Such double tethering of DNA might promote translocation of longer chains by producing an extended conformation and may also contribute to the strong rectification of transport observed for aerolysin.
Published: 2019

5. Transgenic short-QT syndrome 1 rabbits mimic the human disease phenotype with QT/action potential duration shortening in the atria and ventricles and increased ventricular tachycardia/ventricular fibrillation inducibility

Author: David Ziupa, Heiko Bugger, Anna Ryan de Medeiros, Marius Menza, Corinna N. Lang, Stefanie Perez-Feliz, Jan C. Behrends, Michael Brunner, Naga Deepa Pantulu, Lea Mettke, Konstantin Michaelides, Zoltán Doleschall, Raphaela Rieke, Rémi Peyronnet, Geneviève Jolivet, Christoph Bode, Axel zur Hausen, Hannah E Fürniss, Ilona Bodi, Manfred Zehender, Katja E. Odening, Johannes Steinfurt, Gerlind Franke, Department of Cardiology and Angiology I, University Heart Centre Freiburg - Bad Krozingen, University of Freiburg [Freiburg], Department of Pathology, University of Alabama at Birmingham [ Birmingham] (UAB), Department of Pathogenetics, National Institute of Oncology, Biologie du Développement et Reproduction (BDR), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), Université Paris Saclay (COmUE), St. Josefskrankenhaus, German Research Foundation DFG-BR2107/4-1, MUMC+: DA Klinische Pathologie (5), Pathologie, and RS: GROW - R2 - Basic and Translational Cancer Biology
Subjects: Male, Action Potentials, 030204 cardiovascular system & hematology, Ventricular tachycardia, Animals, Genetically Modified, Electrocardiography, 0302 clinical medicine, MUTATION, [SDV.BDD]Life Sciences [q-bio]/Development Biology, biology, Quinidine, animal models, Phenotype, Ion channels, Cardiology, Female, Rabbits, medicine.symptom, Cardiology and Cardiovascular Medicine, Anti-Arrhythmia Agents, medicine.drug, Heart Defects, Congenital, medicine.medical_specialty, Heart Ventricles, hERG, arrhythmia, QT interval, Sudden death, 03 medical and health sciences, SUDDEN-DEATH, Heart Conduction System, Internal medicine, medicine, Animals, Humans, Heart Atria, Fibrillation, business.industry, Arrhythmias, Cardiac, Short QT syndrome, 030229 sport sciences, medicine.disease, electrical remodelling, Disease Models, Animal, Ventricular fibrillation, biology.protein, cardiac repolarization, business, short-QT syndrome
Abstract: AIMS Short-QT syndrome 1 (SQT1) is an inherited channelopathy with accelerated repolarization due to gain-of-function in HERG/IKr. Patients develop atrial fibrillation, ventricular tachycardia (VT), and sudden cardiac death with pronounced inter-individual variability in phenotype. We generated and characterized transgenic SQT1 rabbits and investigated electrical remodelling. METHODS AND RESULTS Transgenic rabbits were generated by oocyte-microinjection of β-myosin-heavy-chain-promoter-KCNH2/HERG-N588K constructs. Short-QT syndrome 1 and wild type (WT) littermates were subjected to in vivo ECG, electrophysiological studies, magnetic resonance imaging, and ex vivo action potential (AP) measurements. Electrical remodelling was assessed using patch clamp, real-time PCR, and western blot. We generated three SQT1 founders. QT interval was shorter and QT/RR slope was shallower in SQT1 than in WT (QT, 147.8 ± 2 ms vs. 166.4 ± 3, P
Published: 2019
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6. Translocation of Sequence-Controlled Synthetic Polymers through Biological Nanopores

Author: Jean-François Lutz, Jan C. Behrends, Abdelaziz Al Ouahabi, Gerhard Baaken, Niklas Felix König, Mordjane Boukhet, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Saclay (COmUE), Institut Charles Sadron (ICS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut für Silizium-Photovoltaik, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Matériaux et nanosciences d'Alsace (FMNGE), and Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: chemistry.chemical_classification, 0303 health sciences, Chemistry, Biophysics, Chromosomal translocation, Polymer, 03 medical and health sciences, Nanopore, 0302 clinical medicine, [CHIM.POLY]Chemical Sciences/Polymers, [CHIM]Chemical Sciences, 030217 neurology & neurosurgery, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Sequence (medicine)
Abstract: International audience
Published: 2018
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7. Translocation of Precision Polymers through Biological Nanopores

Author: Abdelaziz Al Ouahabi, Mordjane Boukhet, Gerhard Baaken, Jan C. Behrends, Jean-François Lutz, Niklas Felix König, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Saclay (COmUE), Institut Charles Sadron (ICS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut für Silizium-Photovoltaik, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, and Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Pore Forming Cytotoxic Proteins, Staphylococcus aureus, Materials science, Polymers and Plastics, Polymers, Entropy, Bacterial Toxins, Aerolysin, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Hemolysin Proteins, Nanopores, Materials Chemistry, Molecule, [CHIM]Chemical Sciences, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, Organic Chemistry, Nucleic acid sequence, Polymer, 021001 nanoscience & nanotechnology, Organophosphates, Aeromonas hydrophila, 0104 chemical sciences, Nanopore, Crystallography, [CHIM.POLY]Chemical Sciences/Polymers, chemistry, Phosphodiester bond, Biophysics, Nanopore sequencing, Threading (protein sequence), 0210 nano-technology
Abstract: Nanopore analysis, which is, currently, chiefly used for DNA sequencing, is also an appealing technique for characterizing abiotic polymers. As a first step toward this goal, nanopore detection of non-natural monodispersed poly(phosphodiester)s as candidate backbone structures is reported herein. Two model homopolymers containing phosphopropyl repeat units (i.e., 56 or 104 r.u.) and a short thymidine nucleotide sequence are analyzed in the present work. They are tested in two different biological nanopores, α-hemolysin from Staphylococcus aureus, and aerolysin from Aeromonas hydrophila. These recordings are performed in aqueous medium at different KCl concentrations and various driving voltages. The data show a complex interaction with evidence for voltage dependence and threading, and underline the influence of the molecular structure and orientation of the precision poly(phosphodiester)s on the observed residual current signal as well as on the translocation dynamics. In particular, they suggest a dominant entropic contribution due to the high flexibility of the phosphodiester homopolymer.
Published: 2017
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8. Tuning Polymer-Protein Interaction with Salt

Author: Aleksandra Dylewska, Monasadat Talarimoghari, Jean-François Lutz, Dalila Chouikhi, Gerhard Baaken, Jan C. Behrends, Marcel Hoffmann, Institut Charles Sadron (ICS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut für Silizium-Photovoltaik, and Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB)
Subjects: chemistry.chemical_classification, 0303 health sciences, Chemistry, Biophysics, Analytical chemistry, Ionic bonding, Electrolyte, Polymer, 010402 general chemistry, Mass spectrometry, 01 natural sciences, 0104 chemical sciences, Ion, 03 medical and health sciences, Nanopore, [CHIM.POLY]Chemical Sciences/Polymers, PEG ratio, Molecule, [CHIM]Chemical Sciences, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract: Biological nanopores are known to interact with synthetic and biological polymers, enabling their use in label-free single-molecule analytical tasks such as sequencing and/or mass discrimination. The latter, called nanopore-based single molecule mass spectrometry (Np-SMMS) has, to date, only been shown for one synthetic polymer, poly(ethyleneglycol) (PEG). It is based on the fact that binding of PEG inside the pore gives rise to blocks of ionic current, with the degree of block being detectably different for PEG molecules differing in size by only one monomer1,2.In order to extend the range of application of Np-SMMS, and to obtain information on the mechanism underying mass sensitivity of polymer-induced pore block, we have begun to test the interaction of other neutral polymers with biological nanoores. Here, we report on poly(dimethylacrylamide) (PDMA), a water-soluble neutral polymer. Under conditions used for Np-SMMS of PEG with alpha-hemolysine (4M KCl, +40 mV), we obtained current blocks with polydisperse PDMA (Mn 1500 g/mol by MALDI) that were low in frequency (
Published: 2017
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9. High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore

Author: Juan Pelta, Abdelghani Oukhaled, Francoise Discala, Marie-France Breton, Jan C. Behrends, Hadjer Ouldali, Fabien Piguet, Laboratoire de Physique Théorique et Modélisation (LPTM - UMR 8089), Centre National de la Recherche Scientifique (CNRS)-CY Cergy Paris Université (CY), Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), University of Freiburg [Freiburg], and Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Materials science, [SDV]Life Sciences [q-bio], 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Article, chemistry.chemical_compound, Nanopores, Single-molecule biophysics, PEG ratio, Molecule, Solubility, chemistry.chemical_classification, Multidisciplinary, Molar mass, technology, industry, and agriculture, Polymer, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Nanopore, Monomer, chemistry, Chemical engineering, [PHYS.PHYS.PHYS-CHEM-PH]Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], 0210 nano-technology, Ethylene glycol
Abstract: We explore the effect of temperature on the interaction of polydisperse mixtures of nonionic poly(ethylene glycol) (PEG) polymers of different average molar masses with the biological nanopore α-hemolysin. In contrast with what has been previously observed with various nanopores and analytes, we find that, for PEGs larger than a threshold molar mass (2000 g/mol, PEG 2000), increasing temperature increases the duration of the PEG/nanopore interaction. In the case of PEG 3400 the duration increases by up to a factor of 100 when the temperature increases from 5 °C to 45 °C. Importantly, we find that increasing temperature extends the polymer size range of application of nanopore-based single-molecule mass spectrometry (Np-SMMS)-type size discrimination. Indeed, in the case of PEG 3400, discrimination of individual molecular species of different monomer number is impossible at room temperature but is achieved when the temperature is raised to 45 °C. We interpret our observations as the consequence of a decrease of PEG solubility and a collapse of PEG molecules with higher temperatures. In addition to expanding the range of application of Np-SMMS to larger nonionic polymers, our findings highlight the crucial role of the polymer solubility for the nanopore detection.
Published: 2016
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10. Electrophoresis and Electroosmosis in Aerolysin and Hemolysin Nanopores

Author: Abdelghani Oukhaled, Mordjane Boukhet, Jan C. Behrends, Juan Pelta, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), University of Freiburg [Freiburg], and Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0303 health sciences, Chromatography, Chemistry, [SDV]Life Sciences [q-bio], Biophysics, Analytical chemistry, Aerolysin, Ionic bonding, Electrolyte, 03 medical and health sciences, Nanopore, Electrophoresis, Dwell time, 0302 clinical medicine, PEG ratio, [PHYS.PHYS.PHYS-CHEM-PH]Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], 030217 neurology & neurosurgery, 030304 developmental biology, Macromolecule
Abstract: Voltage-dependent interaction of macromolecules with biological nanopores can arise from electroosmotic (EOF) or electrophoretic (EPF) forces. Both poly(ethylene-glycol) (PEG), and cyclodextrins (β- and α-CD, respectively) block the ionic current through α-hemolysin (αHL) and Aerolysin (AeL) pore-forming proteins with well-documented voltage-dependence. Here, we attempt to relate differential effects of the electrolytes KCl and LiCl on the voltage-dependence of frequency and dwell-time of blocks to the relative contributions of EOF and EPF.For AeL, irrespective of electrolyte, blocks by α-CD only occurred from the pore's cis-side and with trans-positive voltages. Blocks in LiCl were much longer than those in KCl (11ms vs. 760μs at 90mV) but dwell times showed a positive, linear voltage dependence in both cases (LiCl:6fold/100mV; KCl:3fold/100mV). In KCl, cis-side PEG blocked AeL with trans-negative voltages only.1 Surprisingly, in LiCl, blocks occurred only at trans-positive voltages and at more than 1000 fold PEG-concentration but we still are in a very diluted regime. Blocks were shorter than in KCl by a factor of 5 (60 vs. 320μs at 100mV), but frequency (3fold/100mV) and dwell time (2fold/100mV) depended linearly on voltage, reminiscent of the behavior of α-CD in both electrolytes.Linear voltage dependence of block frequency might indicate dominance of EOF for pore entry of PEG in LiCl and for α-CD generally, while an exponential voltage-dependence would be expected for an electrophoretic force. Indeed, for PEG in KCl, which may be positively charged due to K+-chelation (see Ref.1 for discussion), we found an exponential increase in frequency for blocks of AeL and αHL with increasing trans-negative and trans-positive voltages, respectively. We cannot, however, exclude voltage and ion-dependent effects on the binding reaction, which, rather than partitioning, may be rate-limiting for the electrophysiologically detectable blockages.(1)Baaken, G.; et al. ACS Nano 2015, 6443-6449
Published: 2016
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11. Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

Author: Klionsky, D.J. Abdelmohsen, K. Abe, A. Abedin, M.J. Abeliovich, H. Arozena, A.A. Adachi, H. Adams, C.M. Adams, P.D. Adeli, K. Adhihetty, P.J. Adler, S.G. Agam, G. Agarwal, R. Aghi, M.K. Agnello, M. Agostinis, P. Aguilar, P.V. Aguirre-Ghiso, J. Airoldi, E.M. Ait-Si-Ali, S. Akematsu, T. Akporiaye, E.T. Al-Rubeai, M. Albaiceta, G.M. Albanese, C. Albani, D. Albert, M.L. Aldudo, J. Algül, H. Alirezaei, M. Alloza, I. Almasan, A. Almonte-Beceril, M. Alnemri, E.S. Alonso, C. Altan-Bonnet, N. Altieri, D.C. Alvarez, S. Alvarez-Erviti, L. Alves, S. Amadoro, G. Amano, A. Amantini, C. Ambrosio, S. Amelio, I. Amer, A.O. Amessou, M. Amon, A. An, Z. Anania, F.A. Andersen, S.U. Andley, U.P. Andreadi, C.K. Andrieu-Abadie, N. Anel, A. Ann, D.K. Anoopkumar-Dukie, S. Antonioli, M. Aoki, H. Apostolova, N. Aquila, S. Aquilano, K. Araki, K. Arama, E. Aranda, A. Araya, J. Arcaro, A. Arias, E. Arimoto, H. Ariosa, A.R. Armstrong, J.L. Arnould, T. Arsov, I. Asanuma, K. Askanas, V. Asselin, E. 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Xia, W. Xia, Y. Xiao, H. Xiao, J. Xiao, S. Xiao, W. Xie, C.-M. Xie, Z. Xie, Z. Xilouri, M. Xiong, Y. Xu, C. Xu, C. Xu, F. Xu, H. Xu, H. Xu, J. Xu, J. Xu, J. Xu, L. Xu, X. Xu, Y. Xu, Y. Xu, Z.-X. Xu, Z. Xue, Y. Yamada, T. Yamamoto, A. Yamanaka, K. Yamashina, S. Yamashiro, S. Yan, B. Yan, B. Yan, X. Yan, Z. Yanagi, Y. Yang, D.-S. Yang, J.-M. Yang, L. Yang, M. Yang, P.-M. Yang, P. Yang, Q. Yang, W. Yang, W.Y. Yang, X. Yang, Y. Yang, Y. Yang, Z. Yang, Z. Yao, M.-C. Yao, P.J. Yao, X. Yao, Z. Yao, Z. Yasui, L.S. Ye, M. Yedvobnick, B. Yeganeh, B. Yeh, E.S. Yeyati, P.L. Yi, F. Yi, L. Yin, X.-M. Yip, C.K. Yoo, Y.-M. Yoo, Y.H. Yoon, S.-Y. Yoshida, K.-I. Yoshimori, T. Young, K.H. Yu, H. Yu, J.J. Yu, J.-T. Yu, J. Yu, L. Yu, W.H. Yu, X.-F. Yu, Z. Yuan, J. Yuan, Z.-M. Yue, B.Y.J.T. Yue, J. Yue, Z. Zacks, D.N. Zacksenhaus, E. Zaffaroni, N. Zaglia, T. Zakeri, Z. Zecchini, V. Zeng, J. Zeng, M. Zeng, Q. Zervos, A.S. Zhang, D.D. Zhang, F. Zhang, G. Zhang, G.-C. Zhang, H. Zhang, H. Zhang, H. Zhang, J. Zhang, J. Zhang, J. Zhang, J.-P. Zhang, L. Zhang, L. Zhang, L. Zhang, M.-Y. Zhang, X. Zhang, X.D. Zhang, Y. Zhang, Y. Zhang, Y. Zhang, Y. Zhang, Y. Zhao, M. Zhao, W.-L. Zhao, X. Zhao, Y.G. Zhao, Y. Zhao, Y. Zhao, Y.-X. Zhao, Z. Zhao, Z.J. Zheng, D. Zheng, X.-L. Zheng, X. Zhivotovsky, B. Zhong, Q. Zhou, G.-Z. Zhou, G. Zhou, H. Zhou, S.-F. Zhou, X.-J. Zhu, H. Zhu, H. Zhu, W.-G. Zhu, W. Zhu, X.-F. Zhu, Y. Zhuang, S.-M. Zhuang, X. Ziparo, E. Zois, C.E. Zoladek, T. Zong, W.-X. Zorzano, A. Zughaier, S.M.
Published: 2016

12. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Author: Klionsky, D.J. Abdelmohsen, K. Abe, A. Abedin, M.J. Abeliovich, H. Arozena, A.A. Adachi, H. Adams, C.M. Adams, P.D. Adeli, K. Adhihetty, P.J. Adler, S.G. Agam, G. Agarwal, R. Aghi, M.K. Agnello, M. Agostinis, P. Aguilar, P.V. Aguirre-Ghiso, J. Airoldi, E.M. Ait-Si-Ali, S. Akematsu, T. Akporiaye, E.T. Al-Rubeai, M. Albaiceta, G.M. Albanese, C. Albani, D. Albert, M.L. Aldudo, J. Algül, H. Alirezaei, M. Alloza, I. Almasan, A. Almonte-Beceril, M. Alnemri, E.S. Alonso, C. Altan-Bonnet, N. Altieri, D.C. Alvarez, S. Alvarez-Erviti, L. Alves, S. Amadoro, G. Amano, A. Amantini, C. Ambrosio, S. Amelio, I. Amer, A.O. Amessou, M. Amon, A. An, Z. Anania, F.A. Andersen, S.U. Andley, U.P. Andreadi, C.K. Andrieu-Abadie, N. Anel, A. Ann, D.K. Anoopkumar-Dukie, S. Antonioli, M. Aoki, H. Apostolova, N. Aquila, S. Aquilano, K. Araki, K. Arama, E. Aranda, A. Araya, J. Arcaro, A. Arias, E. Arimoto, H. Ariosa, A.R. Armstrong, J.L. Arnould, T. Arsov, I. Asanuma, K. Askanas, V. Asselin, E. Atarashi, R. Atherton, S.S. Atkin, J.D. Attardi, L.D. Auberger, P. Auburger, G. Aurelian, L. Autelli, R. Avagliano, L. Avantaggiati, M.L. Avrahami, L. Azad, N. Awale, S. Bachetti, T. Backer, J.M. Bae, D.-H. Bae, J.-S. Bae, O.-N. Bae, S.H. Baehrecke, E.H. Baek, S.-H. Baghdiguian, S. Bagniewska-Zadworna, A. Bai, H. Bai, J. Bai, X.-Y. Bailly, Y. Balaji, K.N. Balduini, W. Ballabio, A. Balzan, R. Banerjee, R. Bánhegyi, G. Bao, H. Barbeau, B. Barrachina, M.D. Barreiro, E. Bartel, B. Bartolomé, A. Bassham, D.C. Bassi, M.T. Bast, R.C., Jr. Basu, A. Batista, M.T. Batoko, H. Battino, M. Bauckman, K. Baumgarner, B.L. Bayer, K.U. Beale, R. Beaulieu, J.-F. Beck, G.R., Jr. Becker, C. Beckham, J.D. Bédard, P.-A. Bednarski, P.J. Begley, T.J. Behl, C. Behrends, C. Behrens, G.M.N. Behrns, K.E. Bejarano, E. Belaid, A. Belleudi, F. Bénard, G. Berchem, G. Bergamaschi, D. Bergami, M. Berkhout, B. Berliocchi, L. Bernard, A. Bernard, M. Bernassola, F. Bertolotti, A. Bess, A.S. Besteiro, S. Bettuzzi, S. Bhalla, S. Bhattacharyya, S. Bhutia, S.K. Biagosch, C. Bianchi, M.W. Biard-Piechaczyk, M. Billes, V. Bincoletto, C. Bingol, B. Bird, S.W. Bitoun, M. Bjedov, I. Blackstone, C. Blanc, L. Blanco, G.A. Blomhoff, H.K. Boada-Romero, E. Böckler, S. Boes, M. Boesze-Battaglia, K. Boise, L.H. Bolino, A. Boman, A. Bonaldo, P. Bordi, M. Bosch, J. Botana, L.M. Botti, J. Bou, G. Bouché, M. Bouchecareilh, M. Boucher, M.-J. Boulton, M.E. Bouret, S.G. Boya, P. Boyer-Guittaut, M. Bozhkov, P.V. Brady, N. Braga, V.M.M. Brancolini, C. Braus, G.H. Bravo-San-Pedro, J.M. Brennan, L.A. Bresnick, E.H. Brest, P. Bridges, D. Bringer, M.-A. Brini, M. Brito, G.C. Brodin, B. Brookes, P.S. Brown, E.J. Brown, K. Broxmeyer, H.E. Bruhat, A. Brum, P.C. Brumell, J.H. Brunetti-Pierri, N. Bryson-Richardson, R.J. Buch, S. Buchan, A.M. Budak, H. Bulavin, D.V. Bultman, S.J. Bultynck, G. Bumbasirevic, V. Burelle, Y. Burke, R.E. Burmeister, M. Bütikofer, P. Caberlotto, L. Cadwell, K. Cahova, M. Cai, D. Cai, J. Cai, Q. Calatayud, S. Camougrand, N. Campanella, M. Campbell, G.R. Campbell, M. Campello, S. Candau, R. Caniggia, I. Cantoni, L. Cao, L. Caplan, A.B. Caraglia, M. Cardinali, C. Cardoso, S.M. Carew, J.S. Carleton, L.A. Carlin, C.R. Carloni, S. Carlsson, S.R. Carmona-Gutierrez, D. Carneiro, L.A.M. Carnevali, O. Carra, S. Carrier, A. Carroll, B. Casas, C. Casas, J. Cassinelli, G. Castets, P. Castro-Obregon, S. Cavallini, G. Ceccherini, I. Cecconi, F. Cederbaum, A.I. Ceña, V. Cenci, S. Cerella, C. Cervia, D. Cetrullo, S. Chaachouay, H. Chae, H.-J. Chagin, A.S. Chai, C.-Y. Chakrabarti, G. Chamilos, G. Chan, E.Y.W. Chan, M.T.V. Chandra, D. Chandra, P. Chang, C.-P. Chang, R.C.-C. Chang, T.Y. Chatham, J.C. Chatterjee, S. Chauhan, S. Che, Y. Cheetham, M.E. Cheluvappa, R. Chen, C.-J. Chen, G. Chen, G.-C. Chen, G. Chen, H. Chen, J.W. Chen, J.-K. Chen, M. Chen, M. Chen, P. Chen, Q. Chen, Q. Chen, S.-D. Chen, S. Chen, S.S.-L. Chen, W. Chen, W.-J. Chen, W.Q. Chen, W. Chen, X. Chen, Y.-H. Chen, Y.-G. Chen, Y. Chen, Y. Chen, Y. Chen, Y.-J. Chen, Y.-Q. Chen, Y. Chen, Z. Chen, Z. Cheng, A. Cheng, C.H.K. Cheng, H. Cheong, H. Cherry, S. Chesney, J. Cheung, C.H.A. Chevet, E. Chi, H.C. Chi, S.-G. Chiacchiera, F. Chiang, H.-L. Chiarelli, R. Chiariello, M. Chieppa, M. Chin, L.-S. Chiong, M. Chiu, G.N.C. Cho, D.-H. Cho, S.-G. Cho, W.C. Cho, Y.-Y. Cho, Y.-S. Choi, A.M.K. Choi, E.-J. Choi, E.-K. Choi, J. Choi, M.E. Choi, S.-I. Chou, T.-F. Chouaib, S. Choubey, D. Choubey, V. Chow, K.-C. Chowdhury, K. Chu, C.T. Chuang, T.-H. Chun, T. Chung, H. Chung, T. Chung, Y.-L. Chwae, Y.-J. Cianfanelli, V. Ciarcia, R. Ciechomska, I.A. Ciriolo, M.R. Cirone, M. Claerhout, S. Clague, M.J. Clària, J. Clarke, P.G.H. Clarke, R. Clementi, E. Cleyrat, C. Cnop, M. Coccia, E.M. Cocco, T. Codogno, P. Coers, J. Cohen, E.E.W. Colecchia, D. Coletto, L. Coll, N.S. Colucci-Guyon, E. Comincini, S. Condello, M. Cook, K.L. Coombs, G.H. Cooper, C.D. Cooper, J.M. Coppens, I. Corasaniti, M.T. Corazzari, M. Corbalan, R. Corcelle-Termeau, E. Cordero, M.D. Corral-Ramos, C. Corti, O. Cossarizza, A. Costelli, P. Costes, S. Cotman, S.L. Coto-Montes, A. Cottet, S. Couve, E. Covey, L.R. Cowart, L.A. Cox, J.S. Coxon, F.P. Coyne, C.B. Cragg, M.S. Craven, R.J. Crepaldi, T. Crespo, J.L. Criollo, A. Crippa, V. Cruz, M.T. Cuervo, A.M. Cuezva, J.M. Cui, T. Cutillas, P.R. Czaja, M.J. Czyzyk-Krzeska, M.F. Dagda, R.K. Dahmen, U. Dai, C. Dai, W. Dai, Y. Dalby, K.N. Valle, L.D. Dalmasso, G. D'amelio, M. Damme, M. Darfeuille-Michaud, A. Dargemont, C. Darley-Usmar, V.M. Dasarathy, S. Dasgupta, B. Dash, S. Dass, C.R. Davey, H.M. Davids, L.M. Dávila, D. Davis, R.J. Dawson, T.M. Dawson, V.L. Daza, P. de Belleroche, J. de Figueiredo, P. de Figueiredo, R.C.B.Q. de la Fuente, J. De Martino, L. De Matteis, A. De Meyer, G.R.Y. De Milito, A. De Santi, M. de Souza, W. De Tata, V. De Zio, D. Debnath, J. Dechant, R. Decuypere, J.-P. Deegan, S. Dehay, B. Del Bello, B. Del Re, D.P. Delage-Mourroux, R. Delbridge, L.M.D. Deldicque, L. Delorme-Axford, E. Deng, Y. Dengjel, J. Denizot, M. Dent, P. Der, C.J. Deretic, V. Derrien, B. Deutsch, E. Devarenne, T.P. Devenish, R.J. Di Bartolomeo, S. Di Daniele, N. Di Domenico, F. Di Nardo, A. Di Paola, S. Di Pietro, A. Di Renzo, L. Di Antonio, A. Díaz-Araya, G. Díaz-Laviada, I. Diaz-Meco, M.T. Diaz-Nido, J. Dickey, C.A. Dickson, R.C. Diederich, M. Digard, P. Dikic, I. Dinesh-Kumar, S.P. Ding, C. Ding, W.-X. Ding, Z. Dini, L. Distler, J.H.W. Diwan, A. Djavaheri-Mergny, M. Dmytruk, K. Dobson, R.C.J. Doetsch, V. Dokladny, K. Dokudovskaya, S. Donadelli, M. Dong, X.C. Dong, X. Dong, Z. Donohue, T.M., Jr. Donohue-Jr, T.M. Doran, K.S. D'orazi, G. Dorn, G.W., II Dosenko, V. Dridi, S. Drucker, L. Du, J. Du, L.-L. Du, L. du Toit, A. Dua, P. Duan, L. Duann, P. Dubey, V.K. Duchen, M.R. Duchosal, M.A. Duez, H. Dugail, I. Dumit, V.I. Duncan, M.C. Dunlop, E.A. Dunn, W.A., Jr. Dupont, N. Dupuis, L. Durán, R.V. Durcan, T.M. Duvezin-Caubet, S. Duvvuri, U. Eapen, V. Ebrahimi-Fakhari, D. Echard, A. Eckhart, L. Edelstein, C.L. Edinger, A.L. Eichinger, L. Eisenberg, T. Eisenberg-Lerner, A. Eissa, N.T. El-Deiry, W.S. El-Khoury, V. Elazar, Z. Eldar-Finkelman, H. Elliott, C.J.H. Emanuele, E. Emmenegger, U. Engedal, N. Engelbrecht, A.-M. Engelender, S. Enserink, J.M. Erdmann, R. Erenpreisa, J. Eri, R. Eriksen, J.L. Erman, A. Escalante, R. Eskelinen, E.-L. Espert, L. Esteban-Martínez, L. Evans, T.J. Fabri, M. Fabrias, G. Fabrizi, C. Facchiano, A. Færgeman, N.J. Faggioni, A. Fairlie, W.D. Fan, C. Fan, D. Fan, J. Fang, S. Fanto, M. Fanzani, A. Farkas, T. Faure, M. Favier, F.B. Fearnhead, H. Federici, M. Fei, E. Felizardo, T.C. Feng, H. Feng, Y. Feng, Y. Ferguson, T.A. Fernández, Á.F. Fernandez-Barrena, M.G. Fernandez-Checa, J.C. Fernández-López, A. Fernandez-Zapico, M.E. Feron, O. Ferraro, E. Ferreira-Halder, C.V. Fesus, L. Feuer, R. Fiesel, F.C. Filippi-Chiela, E.C. Filomeni, G. Fimia, G.M. Fingert, J.H. Finkbeiner, S. Finkel, T. Fiorito, F. Fisher, P.B. Flajolet, M. Flamigni, F. Florey, O. Florio, S. Floto, R.A. Folini, M. Follo, C. Fon, E.A. Fornai, F. Fortunato, F. Fraldi, A. Franco, R. Francois, A. François, A. Frankel, L.B. Fraser, I.D.C. Frey, N. Freyssenet, D.G. Frezza, C. Friedman, S.L. Frigo, D.E. Fu, D. Fuentes, J.M. Fueyo, J. Fujitani, Y. Fujiwara, Y. Fujiya, M. Fukuda, M. Fulda, S. Fusco, C. Gabryel, B. Gaestel, M. Gailly, P. Gajewska, M. Galadari, S. Galili, G. Galindo, I. Galindo, M.F. Galliciotti, G. Galluzzi, L. Galluzzi, L. Galy, V. Gammoh, N. Gandy, S. Ganesan, A.K. Ganesan, S. Ganley, I.G. Gannagé, M. Gao, F.-B. Gao, F. Gao, J.-X. Nannig, L.G. Véscovi, E.G. Garcia-Macía, M. Garcia-Ruiz, C. Garg, A.D. Garg, P.K. Gargini, R. Gassen, N.C. Gatica, D. Gatti, E. Gavard, J. Gavathiotis, E. Ge, L. Ge, P. Ge, S. Gean, P.-W. Gelmetti, V. Genazzani, A.A. Geng, J. Genschik, P. Gerner, L. Gestwicki, J.E. Gewirtz, D.A. Ghavami, S. Ghigo, E. Ghosh, D. Giammarioli, A.M. Giampieri, F. Giampietri, C. Giatromanolaki, A. Gibbings, D.J. Gibellini, L. Gibson, S.B. Ginet, V. Giordano, A. Giorgini, F. Giovannetti, E. Girardin, S.E. Gispert, S. Giuliano, S. Gladson, C.L. Glavic, A. Gleave, M. Godefroy, N. Gogal, R.M., Jr. Gokulan, K. Goldman, G.H. Goletti, D. Goligorsky, M.S. Gomes, A.V. Gomes, L.C. Gomez, H. Gomez-Manzano, C. Gómez-Sánchez, R. Gonçalves, D.A.P. Goncu, E. Gong, Q. Gongora, C. Gonzalez, C.B. Gonzalez-Alegre, P. Gonzalez-Cabo, P. González-Polo, R.A. Goping, I.S. Gorbea, C. Gorbunov, N.V. Goring, D.R. Gorman, A.M. Gorski, S.M. Goruppi, S. Goto-Yamada, S. Gotor, C. Gottlieb, R.A. Gozes, I. Gozuacik, D. Graba, Y. Graef, M. Granato, G.E. Grant, G.D. Grant, S. Gravina, G.L. Green, D.R. Greenhough, A. Greenwood, M.T. Grimaldi, B. Gros, F. Grose, C. Groulx, J.-F. Gruber, F. Grumati, P. Grune, T. Guan, J.-L. Guan, K.-L. Guerra, B. Guillen, C. Gulshan, K. Gunst, J. Guo, C. Guo, L. Guo, M. Guo, W. Guo, X.-G. Gust, A.A. Gustafsson, Å.B. Gutierrez, E. Gutierrez, M.G. Gwak, H.-S. Haas, A. Haber, J.E. Hadano, S. 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Tavernarakis, N. Taylor, A. Taylor, G.S. Taylor, G.A. Taylor, J.P. Taylor, M.J. Tchetina, E.V. Tee, A.R. Teixeira-Clerc, F. Telang, S. Tencomnao, T. Teng, B.-B. Teng, R.-J. Terro, F. Tettamanti, G. Theiss, A.L. Theron, A.E. Thomas, K.J. Thomé, M.P. Thomes, P.G. Thorburn, A. Thorner, J. Thum, T. Thumm, M. Thurston, T.L.M. Tian, L. Till, A. Ting, J.P.-Y. Ting, J.P.Y. Titorenko, V.I. Toker, L. Toldo, S. Tooze, S.A. Topisirovic, I. Torgersen, M.L. Torosantucci, L. Torriglia, A. Torrisi, M.R. Tournier, C. Towns, R. Trajkovic, V. Travassos, L.H. Triola, G. Tripathi, D.N. Trisciuoglio, D. Troncoso, R. Trougakos, I.P. Truttmann, A.C. Tsai, K.-J. Tschan, M.P. Tseng, Y.-H. Tsukuba, T. Tsung, A. Tsvetkov, A.S. Tu, S. Tuan, H.-Y. Tucci, M. Tumbarello, D.A. Turk, B. Turk, V. Turner, R.F.B. Tveita, A.A. Tyagi, S.C. Ubukata, M. Uchiyama, Y. Udelnow, A. Ueno, T. Umekawa, M. Umemiya-Shirafuji, R. Underwood, B.R. Ungermann, C. Ureshino, R.P. Ushioda, R. Uversky, V.N. Uzcátegui, N.L. Vaccari, T. Vaccaro, M.I. Váchová, L. Vakifahmetoglu-Norberg, H. Valdor, R. Valente, E.M. Vallette, F. Valverde, A.M. Van den Berghe, G. Van Den Bosch, L. van den Brink, G.R. van der Goot, F.G. van der Klei, I.J. van der Laan, L.J.W. van Doorn, W.G. van Egmond, M. van Golen, K.L. Van Kaer, L. Campagne, M.L. Vandenabeele, P. Vandenberghe, W. Vanhorebeek, I. Varela-Nieto, I. Vasconcelos, M.H. Vasko, R. Vavvas, D.G. Vega-Naredo, I. Velasco, G. Velentzas, A.D. Velentzas, P.D. Vellai, T. Vellenga, E. Vendelbo, M.H. Venkatachalam, K. Ventura, N. Ventura, S. Veras, P.S.T. Verdier, M. Vertessy, B.G. Viale, A. Vidal, M. Vieira, H.L.A. Vierstra, R.D. Vigneswaran, N. Vij, N. Vila, M. Villar, M. Villar, V.H. Villarroya, J. Vindis, C. Viola, G. Viscomi, M.T. Vitale, G. Vogl, D.T. Voitsekhovskaja, O.V. von Haefen, C. von Schwarzenberg, K. Voth, D.E. Vouret-Craviari, V. Vuori, K. Vyas, J.M. Waeber, C. Walker, C.L. Walker, M.J. Walter, J. Wan, L. Wan, X. Wang, B. Wang, C. Wang, C.-Y. Wang, C. Wang, C. Wang, C. Wang, D. Wang, F. Wang, F. Wang, G. Wang, H.-J. Wang, H. Wang, H.-G. Wang, H. Wang, H.-D. Wang, J. Wang, J. Wang, M. Wang, M.-Q. Wang, P.-Y. Wang, P. Wang, R.C. Wang, S. Wang, T.-F. Wang, X. Wang, X.-J. Wang, X.-W. Wang, X. Wang, X. Wang, Y. Wang, Y. Wang, Y. Wang, Y.-J. Wang, Y. Wang, Y. Wang, Y.T. Wang, Y. Wang, Z.-N. Wappner, P. Ward, C. Ward, D.M.V. Warnes, G. Watada, H. Watanabe, Y. Watase, K. Weaver, T.E. Weekes, C.D. Wei, J. Weide, T. Weihl, C.C. Weindl, G. Weis, S.N. Wen, L. Wen, X. Wen, Y. Westermann, B. Weyand, C.M. White, A.R. White, E. Whitton, J.L. Whitworth, A.J. Wiels, J. Wild, F. Wildenberg, M.E. Wileman, T. Wilkinson, D.S. Wilkinson, S. Willbold, D. Williams, C. Williams, K. Williamson, P.R. Winklhofer, K.F. Witkin, S.S. Wohlgemuth, S.E. Wollert, T. Wolvetang, E.J. Wong, E. Wong, G.W. Wong, R.W. Wong, V.K.W. Woodcock, E.A. Wright, K.L. Wu, C. Wu, D. Wu, G.S. Wu, J. Wu, J. Wu, M. Wu, M. Wu, S. Wu, W.K.K. Wu, Y. Wu, Z. Xavier, C.P.R. Xavier, R.J. Xia, G.-X. Xia, T. 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Zhang, J. Zhang, J. Zhang, J.-P. Zhang, L. Zhang, L. Zhang, L. Zhang, M.-Y. Zhang, X. Zhang, X.D. Zhang, Y. Zhang, Y. Zhang, Y. Zhang, Y. Zhang, Y. Zhao, M. Zhao, W.-L. Zhao, X. Zhao, Y.G. Zhao, Y. Zhao, Y. Zhao, Y.-X. Zhao, Z. Zhao, Z.J. Zheng, D. Zheng, X.-L. Zheng, X. Zhivotovsky, B. Zhong, Q. Zhou, G.-Z. Zhou, G. Zhou, H. Zhou, S.-F. Zhou, X.-J. Zhu, H. Zhu, H. Zhu, W.-G. Zhu, W. Zhu, X.-F. Zhu, Y. Zhuang, S.-M. Zhuang, X. Ziparo, E. Zois, C.E. Zoladek, T. Zong, W.-X. Zorzano, A. Zughaier, S.M.
Published: 2016

13. Guidelines for the use and interpretation of assays for monitoring autophagy

Author: Klionsky, D.J. Abdalla, F.C. Abeliovich, H. Abraham, R.T. Acevedo-Arozena, A. Adeli, K. Agholme, L. Agnello, M. Agostinis, P. Aguirre-Ghiso, J.A. Ahn, H.J. Ait-Mohamed, O. Ait-Si-Ali, S. Akematsu, T. Akira, S. Al-Younes, H.M. Al-Zeer, M.A. Albert, M.L. Albin, R.L. Alegre-Abarrategui, J. Aleo, M.F. Alirezaei, M. Almasan, A. Almonte-Becerril, M. Amano, A. Amaravadi, R. Amarnath, S. Amer, A.O. Andrieu-Abadie, N. Anantharam, V. Ann, D.K. Anoopkumar-Dukie, S. Aoki, H. Apostolova, N. Arancia, G. Aris, J.P. Asanuma, K. Asare, N.Y.O. Ashida, H. Askanas, V. Askew, D.S. Auberger, P. Baba, M. Backues, S.K. Baehrecke, E.H. Bahr, B.A. Bai, X.-Y. Bailly, Y. Baiocchi, R. Baldini, G. Balduini, W. Ballabio, A. Bamber, B.A. Bampton, E.T.W. Bánhegyi, G. Bartholomew, C.R. Bassham, D.C. Bast Jr., R.C. Batoko, H. Bay, B.-H. Beau, I. Béchet, D.M. Begley, T.J. Behl, C. Behrends, C. Bekri, S. Bellaire, B. Bendall, L.J. Benetti, L. Berliocchi, L. Bernardi, H. Bernassola, F. Besteiro, S. Bhatia-Kissova, I. Bi, X. Biard-Piechaczyk, M. Blum, J.S. Boise, L.H. Bonaldo, P. Boone, D.L. Bornhauser, B.C. Bortoluci, K.R. Bossis, I. Bost, F. Bourquin, J.-P. Boya, P. Boyer-Guittaut, M. Bozhkov, P.V. Brady, N.R. Brancolini, C. Brech, A. Brenman, J.E. Brennand, A. Bresnick, E.H. Brest, P. Bridges, D. Bristol, M.L. Brookes, P.S. Brown, E.J. Brumell, J.H. Brunetti-Pierri, N. Brunk, U.T. Bulman, D.E. Bultman, S.J. Bultynck, G. Burbulla, L.F. Bursch, W. Butchar, J.P. Buzgariu, W. Bydlowski, S.P. Cadwell, K. Cahová, M. Cai, D. Cai, J. Cai, Q. Calabretta, B. Calvo-Garrido, J. Camougrand, N. Campanella, M. Campos-Salinas, J. Candi, E. Cao, L. Caplan, A.B. Carding, S.R. Cardoso, S.M. Carew, J.S. Carlin, C.R. Carmignac, V. Carneiro, L.A.M. Carra, S. Caruso, R.A. Casari, G. Casas, C. Castino, R. Cebollero, E. Cecconi, F. Celli, J. Chaachouay, H. Chae, H.-J. Chai, C.-Y. Chan, D.C. Chan, E.Y. Chang, R.C.-C. Che, C.-M. Chen, C.-C. Chen, G.-C. Chen, G.-Q. Chen, M. Chen, Q. Chen, S.S.-L. Chen, W. Chen, X. Chen, X. Chen, X. Chen, Y.-G. Chen, Y. Chen, Y. Chen, Y.-J. Chen, Z. Cheng, A. Cheng, C.H.K. Cheng, Y. Cheong, H. Cheong, J.-H. Cherry, S. Chess-Williams, R. Cheung, Z.H. Chevet, E. Chiang, H.-L. Chiarelli, R. Chiba, T. Chin, L.-S. Chiou, S.-H. Chisari, F.V. Cho, C.H. Cho, D.-H. Choi, A.M.K. Choi, D. Choi, K.S. Choi, M.E. Chouaib, S. Choubey, D. Choubey, V. Chu, C.T. Chuang, T.-H. Chueh, S.-H. Chun, T. Chwae, Y.-J. Chye, M.-L. Ciarcia, R. Ciriolo, M.R. Clague, M.J. Clark, R.S.B. Clarke, P.G.H. Clarke, R. Codogno, P. Coller, H.A. Colombo, M.I. Comincini, S. Condello, M. Condorelli, F. Cookson, M.R. Coombs, G.H. Coppens, I. Corbalan, R. Cossart, P. Costelli, P. Costes, S. Coto-Montes, A. Couve, E. Coxon, F.P. Cregg, J.M. Crespo, J.L. Cronjé, M.J. Cuervo, A.M. Cullen, J.J. Czaja, M.J. D'Amelio, M. Darfeuille-Michaud, A. Davids, L.M. Davies, F.E. De Felici, M. De Groot, J.F. De Haan, C.A.M. De Martino, L. De Milito, A. De Tata, V. Debnath, J. Degterev, A. Dehay, B. Delbridge, L.M.D. Demarchi, F. Deng, Y.Z. Dengjel, J. Dent, P. Denton, D. Deretic, V. Desai, S.D. Devenish, R.J. Di Gioacchino, M. Di Paolo, G. Di Pietro, C. Díaz-Araya, G. Díaz-Laviada, I. Diaz-Meco, M.T. Diaz-Nido, J. Dikic, I. Dinesh-Kumar, S.P. Ding, W.-X. Distelhorst, C.W. Diwan, A. Djavaheri-Mergny, M. Dokudovskaya, S. Dong, Z. Dorsey, F.C. Dosenko, V. Dowling, J.J. Doxsey, S. Dreux, M. Drew, M.E. Duan, Q. Duchosal, M.A. Duff, K. Dugail, I. Durbeej, M. Duszenko, M. Edelstein, C.L. Edinger, A.L. Egea, G. Eichinger, L. Eissa, N.T. Ekmekcioglu, S. El-Deiry, W.S. Elazar, Z. Elgendy, M. Ellerby, L.M. Er Eng, K. Engelbrecht, A.-M. Engelender, S. Erenpreisa, J. Escalante, R. Esclatine, A. Eskelinen, E.-L. Espert, L. Espina, V. Fan, H. Fan, J. Fan, Q.-W. Fan, Z. Fang, S. Fang, Y. Fanto, M. Fanzani, A. Farkas, T. Farré, J.-C. Faure, M. Fechheimer, M. Feng, C.G. Feng, J. Feng, Q. Feng, Y. Fésüs, L. Feuer, R. Figueiredo-Pereira, M.E. Fimia, G.M. Fingar, D.C. Finkbeiner, S. Finkel, T. Finley, K.D. Fiorito, F. Fisher, E.A. Fisher, P.B. Flajolet, M. Florez-McClure, M.L. Florio, S. Fon, E.A. Fornai, F. Fortunato, F. Fotedar, R. Fowler, D.H. Fox, H.S. Franco, R. Frankel, L.B. Fransen, M. Fuentes, J.M. Fueyo, J. Fujii, J. Fujisaki, K. Fujita, E. Fukuda, M. Furukawa, R.H. Gaestel, M. Gailly, P. Gajewska, M. Galliot, B. Galy, V. Ganesh, S. Ganetzky, B. Ganley, I.G. Gao, F.-B. Gao, G.F. Gao, J. Garcia, L. Garcia-Manero, G. Garcia-Marcos, M. Garmyn, M. Gartel, A.L. Gatti, E. Gautel, M. Gawriluk, T.R. Gegg, M.E. Geng, J. Germain, M. Gestwicki, J.E. Gewirtz, D.A. Ghavami, S. Ghosh, P. Giammarioli, A.M. Giatromanolaki, A.N. Gibson, S.B. Gilkerson, R.W. Ginger, M.L. Ginsberg, H.N. Golab, J. Goligorsky, M.S. Golstein, P. Gomez-Manzano, C. Goncu, E. Gongora, C. Gonzalez, C.D. Gonzalez, R. González-Estévez, C. González-Polo, R.A. Gonzalez-Rey, E. Gorbunov, N.V. Gorski, S. Goruppi, S. Gottlieb, R.A. Gozuacik, D. Granato, G.E. Grant, G.D. Green, K.N. Gregorc, A. Gros, F. Grose, C. Grunt, T.W. Gual, P. Guan, J.-L. Guan, K.-L. Guichard, S.M. Gukovskaya, A.S. Gukovsky, I. Gunst, J. Gustafsson, A.B. Halayko, A.J. Hale, A.N. Halonen, S.K. Hamasaki, M. Han, F. Han, T. Hancock, M.K. Hansen, M. Harada, H. Harada, M. Hardt, S.E. Harper, J.W. Harris, A.L. Harris, J. Harris, S.D. Hashimoto, M. Haspel, J.A. Hayashi, S.-I. Hazelhurst, L.A. He, C. He, Y.-W. Hébert, M.-J. Heidenreich, K.A. Helfrich, M.H. Helgason, G.V. Henske, E.P. Herman, B. Herman, P.K. Hetz, C. Hilfiker, S. Hill, J.A. Hocking, L.J. Hofman, P. Hofmann, T.G. Höhfeld, J. Holyoake, T.L. Hong, M.-H. Hood, D.A. Hotamisligil, G.S. Houwerzijl, E.J. Høyer-Hansen, M. Hu, B. Hu, C.-A.A. Hu, H.-M. Hua, Y. Huang, C. Huang, J. Huang, S. Huang, W.-P. Huber, T.B. Huh, W.-K. Hung, T.-H. Hupp, T.R. Hur, G.M. Hurley, J.B. Hussain, S.N.A. Hussey, P.J. Hwang, J.J. Hwang, S. Ichihara, A. Ilkhanizadeh, S. Inoki, K. Into, T. Iovane, V. Iovanna, J.L. Ip, N.Y. Isaka, Y. Ishida, H. Isidoro, C. Isobe, K.-I. Iwasaki, A. Izquierdo, M. Izumi, Y. Jaakkola, P.M. Jäättelä, M. Jackson, G.R. Jackson, W.T. Janji, B. Jendrach, M. Jeon, J.-H. Jeung, E.-B. Jiang, H. Jiang, H. Jiang, J.X. Jiang, M. Jiang, Q. Jiang, X. Jiménez, A. Jin, M. Jin, S. Joe, C.O. Johansen, T. Johnson, D.E. Johnson, G.V.W. Jones, N.L. Joseph, B. Joseph, S.K. Joubert, A.M. Juhász, G. Juillerat-Jeanneret, L. Jung, C.H. Jung, Y.-K. Kaarniranta, K. Kaasik, A. Kabuta, T. Kadowaki, M. Kagedal, K. Kamada, Y. Kaminskyy, V.O. Kampinga, H.H. Kanamori, H. Kang, C. Kang, K.B. Il Kang, K. Kang, R. Kang, Y.-A. Kanki, T. Kanneganti, T.-D. Kanno, H. Kanthasamy, A.G. Kanthasamy, A. Karantza, V. Kaushal, G.P. Kaushik, S. Kawazoe, Y. Ke, P.-Y. Kehrl, J.H. Kelekar, A. Kerkhoff, C. Kessel, D.H. Khalil, H. Kiel, J.A.K.W. Kiger, A.A. Kihara, A. Kim, D.R. Kim, D.-H. Kim, D.-H. Kim, E.-K. Kim, H.-R. Kim, J.-S. Kim, J.H. Kim, J.C. Kim, J.K. Kim, P.K. Kim, S.W. Kim, Y.-S. Kim, Y. Kimchi, A. Kimmelman, A.C. King, J.S. Kinsella, T.J. Kirkin, V. Kirshenbaum, L.A. Kitamoto, K. Kitazato, K. Klein, L. Klimecki, W.T. Klucken, J. Knecht, E. Ko, B.C.B. Koch, J.C. Koga, H. Koh, J.-Y. Koh, Y.H. Koike, M. Komatsu, M. Kominami, E. Kong, H.J. Kong, W.-J. Korolchuk, V.I. Kotake, Y. Koukourakis, M.I. Kouri Flores, J.B. Kovács, A.L. Kraft, C. Krainc, D. Krämer, H. Kretz-Remy, C. Krichevsky, A.M. Kroemer, G. Krüger, R. Krut, O. Ktistakis, N.T. Kuan, C.-Y. Kucharczyk, R. Kumar, A. Kumar, R. Kumar, S. Kundu, M. Kung, H.-J. Kurz, T. Kwon, H.J. La Spada, A.R. Lafont, F. Lamark, T. Landry, J. Lane, J.D. Lapaquette, P. Laporte, J.F. László, L. Lavandero, S. Lavoie, J.N. Layfield, R. Lazo, P.A. Le, W. Le Cam, L. Ledbetter, D.J. Lee, A.J.X. Lee, B.-W. Lee, G.M. Lee, J. Lee, J.-H. Lee, M. Lee, M.-S. Lee, S.H. Leeuwenburgh, C. Legembre, P. Legouis, R. Lehmann, M. Lei, H.-Y. Lei, Q.-Y. Leib, D.A. Leiro, J. Lemasters, J.J. Lemoine, A. Lesniak, M.S. Lev, D. Levenson, V.V. Levine, B. Levy, E. Li, F. Li, J.-L. Li, L. Li, S. Li, W. Li, X.-J. Li, Y.-B. Li, Y.-P. Liang, C. Liang, Q. Liao, Y.-F. Liberski, P.P. Lieberman, A. Lim, H.J. Lim, K.-L. Lim, K. Lin, C.-F. Lin, F.-C. Lin, J. Lin, J.D. Lin, K. Lin, W.-W. Lin, W.-C. Lin, Y.-L. Linden, R. Lingor, P. Lippincott-Schwartz, J. Lisanti, M.P. Liton, P.B. Liu, B. Liu, C.-F. Liu, K. Liu, L. Liu, Q.A. Liu, W. Liu, Y.-C. Liu, Y. Lockshin, R.A. Lok, C.-N. Lonial, S. Loos, B. Lopez-Berestein, G. López-Otín, C. Lossi, L. Lotze, M.T. Lõw, P. Lu, B. Lu, B. Lu, B. Lu, Z. Luciano, F. Lukacs, N.W. Lund, A.H. Lynch-Day, M.A. Ma, Y. Macian, F. MacKeigan, J.P. Macleod, K.F. Madeo, F. Maiuri, L. Maiuri, M.C. Malagoli, D. Malicdan, M.C.V. Malorni, W. Man, N. Mandelkow, E.-M. Manon, S. Manov, I. Mao, K. Mao, X. Mao, Z. Marambaud, P. Marazziti, D. Marcel, Y.L. Marchbank, K. Marchetti, P. Marciniak, S.J. Marcondes, M. Mardi, M. Marfe, G. Mariño, G. Markaki, M. Marten, M.R. Martin, S.J. Martinand-Mari, C. Martinet, W. Martinez-Vicente, M. Masini, M. Matarrese, P. Matsuo, S. Matteoni, R. Mayer, A. Mazure, N.M. McConkey, D.J. McConnell, M.J. McDermott, C. McDonald, C. McInerney, G.M. McKenna, S.L. McLaughlin, B. McLean, P.J. McMaster, C.R. McQuibban, G.A. Meijer, A.J. Meisler, M.H. Meléndez, A. Melia, T.J. Melino, G. Mena, M.A. Menendez, J.A. Menna-Barreto, R.F.S. Menon, M.B. Menzies, F.M. Mercer, C.A. Merighi, A. Merry, D.E. Meschini, S. Meyer, C.G. Meyer, T.F. Miao, C.-Y. Miao, J.-Y. Michels, P.A.M. Michiels, C. Mijaljica, D. Milojkovic, A. Minucci, S. Miracco, C. Miranti, C.K. Mitroulis, I. Miyazawa, K. Mizushima, N. Mograbi, B. Mohseni, S. Molero, X. Mollereau, B. Mollinedo, F. Momoi, T. Monastyrska, I. Monick, M.M. Monteiro, M.J. Moore, M.N. Mora, R. Moreau, K. Moreira, P.I. Moriyasu, Y. Moscat, J. Mostowy, S. Mottram, J.C. Motyl, T. Moussa, C.E.-H. Müller, S. Muller, S. Münger, K. Münz, C. Murphy, L.O. Murphy, M.E. Musarò, A. Mysorekar, I. Nagata, E. Nagata, K. Nahimana, A. Nair, U. Nakagawa, T. Nakahira, K. Nakano, H. Nakatogawa, H. Nanjundan, M. Naqvi, N.I. Narendra, D.P. Narita, M. Navarro, M. Nawrocki, S.T. Nazarko, T.Y. Nemchenko, A. Netea, M.G. Neufeld, T.P. Ney, P.A. Nezis, I.P. Nguyen, H.P. Nie, D. Nishino, I. Nislow, C. Nixon, R.A. Noda, T. Noegel, A.A. Nogalska, A. Noguchi, S. Notterpek, L. Novak, I. Nozaki, T. Nukina, N. Nürnberger, T. Nyfeler, B. Obara, K. Oberley, T.D. Oddo, S. Ogawa, M. Ohashi, T. Okamoto, K. Oleinick, N.L. Oliver, F.J. Olsen, L.J. Olsson, S. Opota, O. Osborne, T.F. Ostrander, G.K. Otsu, K. Ou, J.-H.J. Ouimet, M. Overholtzer, M. Ozpolat, B. Paganetti, P. Pagnini, U. Pallet, N. Palmer, G.E. Palumbo, C. Pan, T. Panaretakis, T. Pandey, U.B. Papackova, Z. Papassideri, I. Paris, I. Park, J. Park, O.K. Parys, J.B. Parzych, K.R. Patschan, S. Patterson, C. Pattingre, S. Pawelek, J.M. Peng, J. Perlmutter, D.H. Perrotta, I. Perry, G. Pervaiz, S. Peter, M. Peters, G.J. Petersen, M. Petrovski, G. Phang, J.M. Piacentini, M. Pierre, P. Pierrefite-Carle, V. Pierron, G. Pinkas-Kramarski, R. Piras, A. Piri, N. Platanias, L.C. Pöggeler, S. Poirot, M. Poletti, A. Poüs, C. Pozuelo-Rubio, M. Prætorius-Ibba, M. Prasad, A. Prescott, M. Priault, M. Produit-Zengaffinen, N. Progulske-Fox, A. Proikas-Cezanne, T. Przedborski, S. Przyklenk, K. Puertollano, R. Puyal, J. Qian, S.-B. Qin, L. Qin, Z.-H. Quaggin, S.E. Raben, N. Rabinowich, H. Rabkin, S.W. Rahman, I. Rami, A. Ramm, G. Randall, G. Randow, F. Rao, V.A. Rathmell, J.C. Ravikumar, B. Ray, S.K. Reed, B.H. Reed, J.C. Reggiori, F. Régnier-Vigouroux, A. Reichert, A.S. Reiners Jr., J.J. Reiter, R.J. Ren, J. Revuelta, J.L. Rhodes, C.J. Ritis, K. Rizzo, E. Robbins, J. Roberge, M. Roca, H. Roccheri, M.C. Rocchi, S. Rodemann, H.P. De Córdoba, S.R. Rohrer, B. Roninson, I.B. Rosen, K. Rost-Roszkowska, M.M. Rouis, M. Rouschop, K.M.A. Rovetta, F. Rubin, B.P. Rubinsztein, D.C. Ruckdeschel, K. Rucker III, E.B. Rudich, A. Rudolf, E. Ruiz-Opazo, N. Russo, R. Rusten, T.E. Ryan, K.M. Ryter, S.W. Sabatini, D.M. Sadoshima, J. Saha, T. Saitoh, T. Sakagami, H. Sakai, Y. Salekdeh, G.H. Salomoni, P. Salvaterra, P.M. Salvesen, G. Salvioli, R. Sanchez, A.M.J. Sánchez-Alcázar, J.A. Sánchez-Prieto, R. Sandri, M. Sankar, U. Sansanwal, P. Santambrogio, L. Saran, S. Sarkar, S. Sarwal, M. Sasakawa, C. Sasnauskiene, A. Sass, M. Sato, K. Sato, M. Schapira, A.H.V. Scharl, M. Schätzl, H.M. Scheper, W. Schiaffino, S. Schneider, C. Schneider, M.E. Schneider-Stock, R. Schoenlein, P.V. Schorderet, D.F. Schüller, C. Schwartz, G.K. Scorrano, L. Sealy, L. Seglen, P.O. Segura-Aguilar, J. Seiliez, I. Seleverstov, O. Sell, C. Seo, J.B. Separovic, D. Setaluri, V. Setoguchi, T. Settembre, C. Shacka, J.J. Shanmugam, M. Shapiro, I.M. Shaulian, E. Shaw, R.J. Shelhamer, J.H. Shen, H.-M. Shen, W.-C. Sheng, Z.-H. Shi, Y. Shibuya, K. Shidoji, Y. Shieh, J.-J. Shih, C.-M. Shimada, Y. Shimizu, S. Shintani, T. Shirihai, O.S. Shore, G.C. Sibirny, A.A. Sidhu, S.B. Sikorska, B. Silva-Zacarin, E.C.M. Simmons, A. Simon, A.K. Simon, H.-U. Simone, C. Simonsen, A. Sinclair, D.A. Singh, R. Sinha, D. Sinicrope, F.A. Sirko, A. Siu, P.M. Sivridis, E. Skop, V. Skulachev, V.P. Slack, R.S. Smaili, S.S. Smith, D.R. Soengas, M.S. Soldati, T. Song, X. Sood, A.K. Soong, T.W. Sotgia, F. Spector, S.A. Spies, C.D. Springer, W. Srinivasula, S.M. Stefanis, L. Steffan, J.S. Stendel, R. Stenmark, H. Stephanou, A. Stern, S.T. Sternberg, C. Stork, B. Strålfors, P. Subauste, C.S. Sui, X. Sulzer, D. Sun, J. Sun, S.-Y. Sun, Z.-J. Sung, J.J.Y. Suzuki, K. Suzuki, T. Swanson, M.S. Swanton, C. Sweeney, S.T. Sy, L.-K. Szabadkai, G. Tabas, I. Taegtmeyer, H. Tafani, M. Takács-Vellai, K. Takano, Y. Takegawa, K. Takemura, G. Takeshita, F. Talbot, N.J. Tan, K.S.W. Tanaka, K. Tanaka, K. Tang, D. Tang, D. Tanida, I. Tannous, B.A. Tavernarakis, N. Taylor, G.S. Taylor, G.A. Taylor, J.P. Terada, A.S. Terman, A. Tettamanti, G. Thevissen, K. Thompson, C.B. Thorburn, A. Thumm, M. Tian, F. Tian, Y. Tocchini-Valentini, G. Tolkovsky, A.M. Tomino, Y. Tönges, L. Tooze, S.A. Tournier, C. Tower, J. Towns, R. Trajkovic, V. Travassos, L.H. Tsai, T.-F. Tschan, M.P. Tsubata, T. Tsung, A. Turk, B. Turner, L.S. Tyagi, S.C. Uchiyama, Y. Ueno, T. Umekawa, M. Umemiya-Shirafuji, R. Unni, V.K. Vaccaro, M.I. Valente, E.M. Van Den Berghe, G. Van Der Klei, I.J. Van Doorn, W.G. Van Dyk, L.F. Van Egmond, M. Van Grunsven, L.A. Vandenabeele, P. Vandenberghe, W.P. Vanhorebeek, I. Vaquero, E.C. Velasco, G. Vellai, T. Vicencio, J.M. Vierstra, R.D. Vila, M. Vindis, C. Viola, G. Viscomi, M.T. Voitsekhovskaja, O.V. Von Haefen, C. Votruba, M. Wada, K. Wade-Martins, R. Walker, C.L. Walsh, C.M. Walter, J. Wan, X.-B. Wang, A. Wang, C. Wang, D. Wang, F. Wang, F. Wang, G. Wang, H. Wang, H.-G. Wang, H.-D. Wang, J. Wang, K. Wang, M. Wang, R.C. Wang, X. Wang, X. Wang, Y.-J. Wang, Y. Wang, Z. Wang, Z.C. Wang, Z. Wansink, D.G. Ward, D.M. Watada, H. Waters, S.L. Webster, P. Wei, L. Weihl, C.C. Weiss, W.A. Welford, S.M. Wen, L.-P. Whitehouse, C.A. Whitton, J.L. Whitworth, A.J. Wileman, T. Wiley, J.W. Wilkinson, S. Willbold, D. Williams, R.L. Williamson, P.R. Wouters, B.G. Wu, C. Wu, D.-C. Wu, W.K.K. Wyttenbach, A. Xavier, R.J. Xi, Z. Xia, P. Xiao, G. Xie, Z. Xie, Z. Xu, D.-Z. Xu, J. Xu, L. Xu, X. Yamamoto, A. Yamamoto, A. Yamashina, S. Yamashita, M. Yan, X. Yanagida, M. Yang, D.-S. Yang, E. Yang, J.-M. Yang, S.Y. Yang, W. Yang, W.Y. Yang, Z. Yao, M.-C. Yao, T.-P. Yeganeh, B. Yen, W.-L. Yin, J.-J. Yin, X.-M. Yoo, O.-J. Yoon, G. Yoon, S.-Y. Yorimitsu, T. Yoshikawa, Y. Yoshimori, T. Yoshimoto, K. You, H.J. Youle, R.J. Younes, A. Yu, L. Yu, L. Yu, S.-W. Yu, W.H. Yuan, Z.-M. Yue, Z. Yun, C.-H. Yuzaki, M. Zabirnyk, O. Silva-Zacarin, E. David Zacks, E. Zacksenhaus, L. Zaffaroni, N. Zakeri, Z. Zeh III, H.J. Zeitlin, S.O. Zhang, H. Zhang, H.-L. Zhang, J. Zhang, J.-P. Zhang, L. Zhang, L. Zhang, M.-Y. Zhang, X.D. Zhao, M. Zhao, Y.-F. Zhao, Y. Zhao, Z.J. Zheng, X. Zhivotovsky, B. Zhong, Q. Zhou, C.-Z. Zhu, C. Zhu, W.-G. Zhu, X.-F. Zhu, X. Zhu, Y. Zoladek, T. Zong, W.-X. Zorzano, A. Zschocke, J. Zuckerbraun, B.
Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field. © 2012 Landes Bioscience.
Published: 2012

14. An encounter of the third kind without the usual suspects: coherent activation of asynchronous transmitter release independent of presynaptic excitation

Author: Jan C. Behrends
Subjects: Male, Patch-Clamp Techniques, Physiology, Postsynaptic Current, Presynaptic Terminals, Hypothalamus, Action Potentials, Tetrodotoxin, Neurotransmission, Inhibitory postsynaptic potential, Synaptic Transmission, Bursting, Postsynaptic potential, Premovement neuronal activity, Animals, Patch clamp, Rats, Wistar, gamma-Aminobutyric Acid, Neurons, Communication, Voltage-dependent calcium channel, business.industry, Chemistry, Miniature Postsynaptic Potentials, Excitatory Postsynaptic Potentials, Electrophysiological Phenomena, Rats, nervous system, Inhibitory Postsynaptic Potentials, Calcium, Calcium Channels, business, Neuroscience, Cadmium, Perspectives, Sodium Channel Blockers
Abstract: Synapses are rightly viewed as devices primarily specializing in rapid, synchronous exocytotic transmitter release closely linked to presynaptic electrical excitation by Ca2+ influx through selective voltage-dependent channels, and we are probably just beginning to understand the complex machinery which ensures that this link is rapid and temporally selective. Thus, following a presynaptic impulse, multiple vesicles at various presynaptic locations undergo exocytotic fusion so nearly simultaneously that the changes in postsynaptic membrane conductance mediated by the transmitter content of each vesicle (the so-called quantum) sum to produce a much larger, synchronous postsynaptic voltage or current response in which the quantal elements cannot be individuated. Responses to asynchronous release of individual quanta, on the other hand, were, of course, immediately observed and studied once synaptic potentials could be directly recorded (Fatt & Katz, 1952). Until now, two phenomenologically distinct types of asynchronous release have been distinguished. The first, spontaneous (or constitutive) transmitter release, persists in the absence of presynaptic excitation, i.e. when action potentials are blocked, and produces small and discrete postsynaptic responses in a strictly random time series, so-called miniatures or minis. In the classical view, their randomness in time reflects the probabilistic nature of quantal release, while a synchronous impulse-evoked postsynaptic response is simply due to a very short-lasting but vigorous increase in the probability of their occurrence triggered by voltage-dependent Ca2+ influx. (Katz, 1966). The idea that minis represent chance occurrences of the same quantal elements that sum to form the much larger, synchronous postsynaptic response following an action potential has, however, recently been challenged (reviewed in Sullivan, 2009). The second, called either ‘slow release’, ‘late release’ or ‘asynchronous delayed component of evoked release’ is a transient enhancement of the frequency of asynchronous quantal events following the fast, synchronous postsynaptic response to a presynaptic action potential and building up during trains of impulses. This kind of asynchronous release is, of course, abolished along with synchronous release when presynaptic excitation is made impossible. It is classically interpreted as a result of the buildup of residual Ca2+ in the terminal, but more recently, a second, high-affinity Ca2+ sensor has been implicated in this phenomenon, which may also be linked to Ca2+-dependent processes of short-term facilitation (Sakaba & Neher, 2008). With the debate on the mechanistic relationship of these two forms of asynchronous release with one another and with synchronous release ongoing, the report by Popescu et al. (2010) published in a recent issue of The Journal of Physiology introduces yet a third type of asynchronous release: this novel form is neither dependent on action potential firing or presynaptic voltage-dependent Ca2+ entry nor stochastic in the sense of carrying no temporal signature deviating from randomness, instead occurring in clusters or bursts. Recording in whole-cell patch clamp mode from magnocellular neurons in acute in vitro slice preparations of rat hypothalamus, in about half of their recordings the authors observed bursts of grouped GABAergic inhibitory postsynaptic currents (IPSCs) occurring on average once every 13 min (at 28°C) and lasting several seconds with a frequency of quantal-like IPSCs 10- to 100-fold over the stochastic background mini activity. Intriguingly, like the latter, the bursts were resistant to block of both voltage-dependent sodium and calcium channels by tetrodotoxin (TTX) and Cd2+, respectively. TTX did reduce their frequency by about 4-fold, halved their incidence, and abolished the occurrence of correlated bursts in simultaneously recorded neighbouring neurones, thus clearly showing some influence of neuronal activity. However, albeit at lower frequency, the IPSCs did continue to burst healthily in the absence of action potential firing and voltage-dependent Ca2+ entry. One notes with some relief, then, that there is one thing that does seem to affect them less subtly: Popescu et al. report that removal of extracellular Ca2+ abolished IPSC bursting altogether. Together with their finding that intracellular Ca2+ stores seem uninvolved, Ca2+ influx into the presynaptic GABAergic terminal through a pathway other than voltage-gated Ca2+ channels is apparently required to trigger bursts. Now the great question is: through which pathway does Ca2+ enter the terminal? The authors consider presynaptic Ca2+-permeable ligand-gated non-selective cation channels, of which a number have been described (Schicker et al. 2008). Of course, we know already that, wherever the ligand comes from, it cannot be from synaptic terminals operated in the conventional manner requiring both action potentials and voltage-dependent Ca2+ channels. A factor that is released by neurones or glial cells in a manner less strictly linked to neuronal activity would possibly be required to fit the bill. The second big question raised by this work concerns a possible role of the bursts in the control of the pulsatile output of oxytocin from the magnocellular axon terminals in the neurohypophysis during birth and lactation. More generally, their finding might be the first inkling of a novel mode of synaptic transmission independent of (or at least very loosely dependent on) neuronal excitability and possibly even of a specific functional mission of slow, asynchronous transmitter release in the operation of a synaptic circuit.
Published: 2010

15. Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents.

Author: Gerhard Baaken, Markus SondermannPresent address: Institute for Optics, Information and Photonics, University of Erlangen, Germany., Christian Schlemmer, Jürgen Rühe, and Jan C. Behrends
Subjects: MICROELECTRODES, FUNCTIONAL analysis, ETHYLENE glycol, FUNCTIONAL equations
Abstract: Increasing the throughput and resolution of electrical recording of currents through ion conducting channels and pores is an important technical challenge both for the functional analysis of ion channel proteins and for the application of nanoscale pores in single molecule analytical tasks. We present a novel design based on sub-picoliter-cavities arrayed in a polymer substrate and endowed with individual planar microelectrodes that allows low-noise and parallel electrical recording from ion channels and pores. Resolution of voltage-dependent current transitions of alamethicin channels as well as polyethylene-glycol-induced blocking events of alpha-hemolysin nanopores on the submillisecond time scale is demonstrated using this device. [ABSTRACT FROM AUTHOR]
Published: 2008
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16. Whole Cell Patch Clamp Recording Performed on a Planar Glass Chip

Author: Jan C. Behrends, Niels Fertig, and Robert H. Blick
Subjects: Microscope, Materials science, Voltage-gated ion channel, Pipette, Biophysics, Nanotechnology, Chip, law.invention, Planar, Automated patch clamp, law, Patch clamp, Ion channel, Biomedical engineering
Abstract: The state of the art technology for the study of ion channels is the patch clamp technique. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. The most popular (whole cell) variant of the technique detects the ensemble current over the entire cell membrane. Patch clamping is still a laborious process, requiring a skilled experimenter to micromanipulate a glass pipette under a microscope to record from one cell at a time. Here we report on a planar, microstructured quartz chip for whole cell patch clamp measurements without micromanipulation or visual control. A quartz substrate of 200μm thickness is perforated by wet etching techniques resulting in apertures with diameters of ∼1μm. The apertures replace the tip of glass pipettes commonly used for patch clamp recording. Cells are positioned onto the apertures from suspension by application of suction. Whole cell recordings from different cell types (CHO, N1E-115 neuroblastoma) are performed with microstructured chips studying K+ channels and voltage gated Ca2+ channels.
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17. Serotonin-Loading Increases Granule Size and Prolongs Fusion Pore Formation in Two Types of Rat Basophilic Leukaemia (RBL) Mast Cell Analogs

Author: Daniel Steinbrenner and Jan C. Behrends
Subjects: Cell type, Chromatography, Chemistry, 010401 analytical chemistry, Granule (cell biology), Kinetics, Biophysics, hemic and immune systems, 02 engineering and technology, 021001 nanoscience & nanotechnology, Mast cell, 01 natural sciences, Capacitance, 0104 chemical sciences, medicine.anatomical_structure, Membrane curvature, medicine, Serotonin, 0210 nano-technology, 5-HT receptor
Abstract: Whole-cell capacitance/admittance recordings resolving single granule fusion in RBL subclone −1 and −2H3 cells were analyzed to test for a relation between granule size and the kinetics of the exocytotic event. Compared to RBL-2H3 cells, capacitance steps in RBL-1 cells had larger median amplitude (68.78 vs. 9.46 fF) corresponding to larger median diameter (1.48 vs. 0.55 μm) and displayed a significantly higher percentage (35 vs. 1.6%) of capacitance fluctuations before full pore expansion (flicker). In both cell types, loading of granules with serotonin (5-HT) by overnight incubation (verified by amperometry) resulted in a significant increase in median granule size (RBL-1: 111.41 fF or 2.58 μm; RBL-2H3: 61.39 fF or 1.4 μm). Interestingly, in RBL-2H3 cells, the growth in granule diameter (to 250% of control) was accompanied by a strong increase in the prevalence of capacitance flicker (to 41.5%), while in RBL-1 cells with granule size of 170% of control, capacitance flicker occured in 41.5% of cases after 5HT loading. These results suggest that granule size may influence fusion pore kinetics e.g. as a result of membrane curvature favoring rapid pore expansion.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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18. Optical and Electrical Analysis of Suspended Lipid Bilayer Microarrays Formed by Mechanical Liquid Spreading on Horizontal Planar Microelectrode Cavity Arrays

Author: Juan Del Rio Martinez, Gerhard Baaken, and Jan C. Behrends
Subjects: Materials science, business.industry, Bilayer, Analytical chemistry, Biophysics, Nanopore, chemistry.chemical_compound, Microelectrode, Planar, chemistry, Gramicidin, Annulus (firestop), Optoelectronics, Wetting, business, Lipid bilayer
Abstract: Over the recent years, we have developed a chip-based platform containing a 4x4-array of Ag/AgCl-electrodes in microcavities (6-50μm diam., depth 8-20μm) structured in SU8 (Microelectrode-Cavity-Array, MECA) and demonstrated its application in parallel single molecule detection using biological nanopores in bilayers formed by manual painting (Muller-Rudin procedure). Exploring ways for automation, we found that by spreading lipid/solvent mixtures using a remotely actuated effector highly satisfactory results could be obtained. Here, we use electrical (voltage-clamp) and optical (fluorescence-microscopy) methods to study the formation and morphology of the resulting microbilayers. Despite the difference in geometry between standard “through-hole” apertures and “blind-hole” cavities, the resulting bilayers show classical morphology, i.e. including a lipid-solvent annulus that appears continuous with the lipid-solvent mixture wetting the SU8-surface (Fig.1). It is likely, that these microbilayers are also formed by the thinning process described for standard apertures. This thinning process is, however, greatly accelerated: following automated bilayer formation in the presence of the pore-forming peptide gramicidin, channel events, signaling a bimolecular state of the lipid membrane, could be observed as early as 1050ms after the initial resistance increase (Fig.1).View Large Image | View Hi-Res Image | Download PowerPoint Slide
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19. Mixed Company in a Protein Nanopore: Transient Double Occupancy Enables Direct Exchange of Pore Ligand in Nanopore-Based Single Molecule Sensing

Author: Jan C. Behrends, Gerhard Baaken, and Anne K. Schuler
Subjects: Ligand, Dispersity, Analytical chemistry, Biophysics, Ionic bonding, equipment and supplies, Nanopore, chemistry.chemical_compound, Monomer, chemistry, Chemical physics, PEG ratio, Mass spectrum, Molecule
Abstract: Partitioning of poly(ethylene-glycol) (PEG) into an alpha-hemolysin nanopore gives rise to long-lasting blockades (τ≈0.1-10 ms) of ionic current. The depth of blockade shows exquisite sensitivity for polymer length, yielding mass spectra with single monomer resolution in the range between MW≈700-2200 or 15-50 repeat units (r.u.). Unexpectedly, high-resolution recordings of single PEG blocking events using a mixture of two monodisperse species unequivocally identified direct transitions between levels corresponding to 28 and 32 r.u. without an intervening unblocked interval. Closer analysis revealed that these occur by the intermediary of shorter, deeper blocks. Based on statistics and current amplitude distributions, we are able to identify three such deeply blocked states, each corresponding to the simultaneous presence in the pore of two ligands: either 2xPEG-28, 2xPEG-32 or PEG-28+PEG-32 (mixed double occupancy). Direct PEG28->PEG32 transitions (or vice versa) are observed only with an intervening mixed occupancy block, which, however, can also result in return to the first blocked level. We conclude that the alpha-hemolysin pore is capable of accomodating two PEG oligomers, with ligand exchange occurring by displacement and translocation of the first blocker.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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20. Double Occupancy of a Protein Pore as an Intermediate State of Competition at the Single Molecule Level

Author: Gerhard Baaken, Marcel Hoffmann, Anne-Katrin Schuler, and Jan C. Behrends
Subjects: Crystallography, Nanopore, Superposition principle, Chemistry, PEG ratio, Biophysics, Intermediate state, Molecule, State (functional analysis), Binding site, Degree of polymerization
Abstract: Alpha-hemolysin nanopores are used to detect single oligomers of poly(ethyleneglycol) (PEG) which under high salt conditions reside in the pore for extended periods of time (up to several ms) suggesting binding to the pore's inner wall. We study the interaction of two species of PEG of degree of polymerization 28 and 32 which, following sequential entry, simultaneously reside in the pore. This doubly occupied (DO) state can result in direct replacement of the first occupant by the second. Analyzing the dwell times of the DO states, we ask whether binding as a first or second pore occupant is equivalent in terms of stability. We find that the DO state lifetimes are shorter than would be predicted by simple superposition (see Figure). We conclude that binding in the pore as the second occupant is highly unstable, suggesting that the more stable primary binding state cannot be attained by more than one PEG molecule. The doubly occupied state thus represents an intermediate state with possible general relevance for competitive interactions at binding sites to which access is gained through long channels, such as in some enzymes.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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21. Parallel Reconstitution of Bacterial Toxins, Porins and Ion Channels into Suspended Lipid Membrane Microarrays for High-Throughput Electrophysiology

Author: Bert van den Berg, Frank Bernhard, Ekaterina Zaitseva, Christopher Hein, Gerhard Baaken, Jan C. Behrends, and Liviu Movileanu
Subjects: Nanopore, Vesicle fusion, Chromatography, Chemistry, Bilayer, Protein reconstitution, KcsA potassium channel, Biophysics, Model lipid bilayer, Lipid bilayer, Ion channel
Abstract: High-throughput electrophysiological recordings in artificial membranes have a great potential for pharmaceutical screening and nanopore analytics. In addition to parallelization and automation of lipid bilayers formation, different protein reconstitution protocols are required depending on the concrete application.We report on functional reconstitutions of three classes of pore-forming proteins into suspended lipid bilayers on Micro Electrode Cavity Array (MECA). Three reconstitution strategies have been employed and optimized depending on the protein structure: self-insertion for pore-forming toxins, transfer from detergent micelles for porins and vesicle fusion for ion channels.By optimizing protein concentration and facilitating spontaneous insertion with short voltage pulses just below the electroporation threshold, a controlled insertion of alpha-hemolysin was achieved. Exactly one pore per bilayer was inserted in over 50% of bilayers in the array, which is substantially higher than the 37% predicted by the Poisson distribution.We investigated and optimized insertion of outer membrane porins OmpF, MspA and OccK1 into bilayer arrays via detergent dilution. Best strategy was to use mild nonionic detergents and sequentially dilute protein stock solution to minimize influence of the detergent on the membrane. Protein injection in the immediate vicinity of a bilayer array and thorough mixing of the solution were decisive factors for homogeneous distribution of functional porins.Reconstitution via detergent micelles was found to be problematic for ion channels, but proteovesicle fusion was successful. The tetrameric potassium channel KcsA was expressed in vitro with cotranslational insertion into lipid vesicles or nanodiscs. Transfer from nanodiscs into suspended bilayers was shown to be inefficient. Additionally, the presence of nanodiscs substantially destabilized the membranes. For proteovesicles best fusion rates were achieved upon addition of negatively charged phospholipids followed by extrusion of the recovered proteovesicles through a polycarbonate filter.
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22. Incontinent Pool of Primed Vesicles in Synaptotagmin-1-Deficient GABAergic Synapses

Author: Silvia Willadt and Jan C. Behrends
Subjects: Vesicle fusion, Postsynaptic potential, Vesicle, Biophysics, Long-term potentiation, Patch clamp, Anatomy, Biology, Inhibitory postsynaptic potential, Exocytosis, Synaptotagmin 1, Cell biology
Abstract: The coupling of neuronal excitation to release of chemical transmitter at synaptic endings is mediated by an electrically gated entry of Ca2+-ions that within a fraction of a millisecond leads to exocytotic fusion of vesicles with the presynaptic membrane. In this rapid process, members of the synaptotagmin (Syt) family of vesicular membrane proteins are likely to function as the Ca2+-sensing molecular trigger. The trigger mechanism employed, however, is currently under debate. Here, using high resolution dual simultaneous pre- and postsynaptic whole cell patch clamp recordings from Syt1-deficient GABAergic neurons, we report a strong potentiation of asynchronous release in the absence of synchronous release. In addition, we show that Syt1-deficient vesicles are hypersensitive to Ca2+-independent osmotic stimulation of exocytosis and that, unlike in controls, chemical priming into a highly fusion competent state directly results in their spontaneous exocytosis. Therefore, the primed vesicle pool is incontinent in these synapses. These findings argue strongly that an inhibitory, latch-like action by Ca2+-free Syt1 on SNARE-function is part of its physiological role in the mammalian nervous system to ensure transmitter release closely timed to the presynaptic action potential.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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23. Parallel Acquisition of High Resolution Polymer Mass-Spectra on a Nanopore Microbilayer Array

Author: Jan C. Behrends, Jürgen Rühe, Norbert Ankri, Gerhard Baaken, and Anne-Katrin Schuler
Subjects: chemistry.chemical_classification, Materials science, Resolution (mass spectrometry), 010405 organic chemistry, Bilayer, Biophysics, Nanotechnology, Polymer, 010402 general chemistry, Chip, 01 natural sciences, 0104 chemical sciences, Microelectrode, Nanopore, chemistry, Mass spectrum, Lipid bilayer
Abstract: A more widespread use of protein nanopores with their many attractive applications in chemical and biological analytics will depend on the availability of bilayer recording methods that enable rapid collection of large amounts of data and can be easily automated. Nanopore-based single-molecule analysis typically require require low noise and high frequency bandwidth recordings so that low-capacitance microbilayers on smaller-than-standard (e.g. < 100 μm) bilayers are required.We have formed lipid bilayers of
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24. Parallel and Automated Formation of Lipid Bilayers on Microstructured Chips for Ion Channel and Nanopore Recordings

Author: Matthias Beckler, Gerhard Baaken, Jan C. Behrends, Soenke Petersen, Astrid Seifert, Mohamed Kreir, Niels Fertig, Marcel Hoffmann, and Juan M. del Rio-Martinez
Subjects: chemistry.chemical_classification, Nanopore, Membrane, Membrane protein, Chemistry, Bilayer, Vesicle, Biophysics, Nanotechnology, Polymer, Lipid bilayer, Ion channel
Abstract: Bilayer recording is a well-established technique for in-depth studies of biophysical properties of ion channels and is particularly suited for functional studies on proteins residing in intracellular membranes. Moreover, this technique supports a host of powerful emerging analytical techniques using biological nanopores as molecular sensors. Despite its proven value, bilayer recording can be frustrating due to the capricious nature of lipid bilayers, which have to be formed manually one by one and which often lack stability. We here show a new approach and device, which speeds up the entire process by the rapid and simultaneous formation of 16, highly stable micrometer-sized bilayers using Micro-Electrode-Cavity-Array (MECA)-Chips. A study will be presented showing that the MECA supports high-resolution polymer sizing with a single biological nanopore in a parallel format (Fig.1). Additionally, data on a variety of channel proteins recorded from proteoliposomes will be shown. using a surface containing micron-sized apertures in glass substrates, the fusion of vesicles on the surface becomes an attractive method for electrophysiology and then to reconstitute membrane proteins into the lipid bilayer.Fig. 1MonoPEG-28-mediated blockages of HL-nanopore(s) on a MECA chip.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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25. Surface-Charge Influences Voltage-Dependent Pore Formation in Lipid Membranes by Quantum Dots

Author: Jan C. Behrends, Ali Zulqurnain, Wolfgang J. Parak, Srujan Kumar Dondapati, Gerhard Baaken, and Feng Zhang
Subjects: chemistry.chemical_compound, chemistry, Cadmium selenide, Chemical physics, Quantum dot, Bilayer, Biophysics, Nanoparticle, Nanotechnology, Surface charge, Lipid bilayer, Electrostatics, Zinc sulfide
Abstract: Semiconductor nanocrystals, such as quantum dots (QDs) have many biomedical applications. It is of great interest to understand the mechanism by which these nanoparticles cross the cell membrane. Here, QDs, such as cadmium selenide / zinc sulfide core shell (CdSe/ZnS) nanoparticles are shown to interact with lipid bilayers painted on picoliter microelectrode cavities and to produce rapid current bursts with half widths in the range of tens of microseconds. These bursts are voltage dependent and are observed in both polarities. The voltage-dependence of the burst frequency is strongly influenced by external solution parameters like pH and salt valency, charge of the lipids, bilayer size and also nanoparticle size and charge. Correlating these findings with results of dynamic light scattering (zetasizer), we present evidence that electrostatic interactions play a pivotal role in generating the current bursts. Future experiments will focus on optical characterization in order to obtain more insight into the mechanism of interaction.Figure 1(A) Scheme of the measurement set up. (B) Current bursts measured across the bilayer formed over 10 um electrode in the presence of QDs at - 200 mV.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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26. High Resolution Single Molecule Analysis using Nanopore Recording on Microelectrode Cavity Arrays

Author: Jan C. Behrends, Jürgen Rühe, Norbert Ankri, Gerhard Baaken, and Srujan Kumar Dondapati
Subjects: chemistry.chemical_classification, business.industry, Chemistry, Amplifier, Bandwidth (signal processing), Biophysics, Analytical chemistry, Polymer, Dielectric, Capacitance, Microelectrode, Nanopore, Amplitude, Optoelectronics, business
Abstract: Single molecule detection using biological nanopores in lipid bilayers is crucially limited by noise and bandwidth of the recording. We have tested a newly developed 16-channel microelectrode cavity array (MECA, Ref. 1) for single molecule detection using alpha-Hemolysin (alphaHL) nanopores. The device is based on subpicoliter cavities in a high-quality dielectric polymer adding less than 0.5 pF to the input capacitance of the amplifier (Axopach200B), thereby optimizing noise and bandwidth.View Large Image | View Hi-Res Image | Download PowerPoint SlideAn example trace is shown in Fig.1A with an open state rms noise of 0.65pA at 0-5 kHz. Note that for the blocked state, there is no significant difference between an all points histogram of a 5 kHz filtered trace (black) and that of a event amplitudes defined by averaging (grey). Fig.1B shows 35 single PEG induced blockages superimposed and aligned in time. They are detected as square pulses down to durations
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27. Adaptive Platform for Highly Parallel Low-Noise Recordings of Single Membrane Proteins

Author: Jan C. Behrends, Gerhard Baaken, Srujan Kumar Dondapati, and Juergen Ruehe
Subjects: Alamethicin, Chemistry, business.industry, Biophysics, Nanotechnology, Chip, Noise (electronics), Microelectrode, Electrophysiology, chemistry.chemical_compound, Resist, Microsystem, Optoelectronics, Lipid bilayer, business
Abstract: Highly parallel, low noise electrophysiological recordings of single ion channels are of interest both for basic research and drug development. Here, a microsystems approach is presented (see Fig.1A) which greatly simplifies the recording configuration and optimizes the electrical parameters governing noise. The lipid bilayer is formed on a picoliter cavity generated within a photochemical resist acting as a dielectric (see Fig. 1B). On the bottom of each cavity a microelectrode is placed.Fig. 1Patch16Chip (B)Connecting lines and cavities over the circular shaped electrodes. (C)Current traces of single alamethicin-channels, detected in parallel at 9 different cavities.View Large Image | View Hi-Res Image | Download PowerPoint SlideUsing standard photolithographical techniques this design allows for many such setups on one chip, and is therefore in principle well suited for highly parallel single channel recordings. Parallel recordings (16-electrode multiarray) of currents mediated by alamethicin are shown (see Fig.1C), illustrating the potential of this novel approach towards high-throughput measurements of single membrane proteins. Furthermore, the flexibility of this approach is outlined by the reconstitution of α-hemolysin in the lipid bilayer. The capability for molecular analysis is demonstrated by the detection of oligomers diffusing through the bacterial pores.
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28. Linear ubiquitination at damaged lysosomes induces local NFKB activation and controls cell survival.

Author: Zein L, Dietrich M, Balta D, Bader V, Scheuer C, Zellner S, Weinelt N, Vandrey J, Mari MC, Behrends C, Zunke F, Winklhofer KF, and Van Wijk SJL
Abstract: Lysosomes are the major cellular organelles responsible for nutrient recycling and degradation of cellular material. Maintenance of lysosomal integrity is essential for cellular homeostasis and lysosomal membrane permeabilization (LMP) sensitizes toward cell death. Damaged lysosomes are repaired or degraded via lysophagy, during which glycans, exposed on ruptured lysosomal membranes, are recognized by galectins leading to K48- and K63-linked poly-ubiquitination (poly-Ub) of lysosomal proteins followed by recruitment of the macroautophagic/autophagic machinery and degradation. Linear (M1) poly-Ub, catalyzed by the linear ubiquitin chain assembly complex (LUBAC) E3 ligase and removed by OTULIN (OTU deubiquitinase with linear linkage specificity) exerts important functions in immune signaling and cell survival, but the role of M1 poly-Ub in lysosomal homeostasis remains unexplored. Here, we demonstrate that L-leucyl-leucine methyl ester (LLOMe)-damaged lysosomes accumulate M1 poly-Ub in an OTULIN- and K63 Ub-dependent manner. LMP-induced M1 poly-Ub at damaged lysosomes contributes to lysosome degradation, recruits the NFKB (nuclear factor kappa B) modulator IKBKG/NEMO and locally activates the inhibitor of NFKB kinase (IKK) complex to trigger NFKB activation. Inhibition of lysosomal degradation enhances LMP- and OTULIN-regulated cell death, indicating pro-survival functions of M1 poly-Ub during LMP and potentially lysophagy. Finally, we demonstrate that M1 poly-Ub also occurs at damaged lysosomes in primary mouse neurons and induced pluripotent stem cell-derived primary human dopaminergic neurons. Our results reveal novel functions of M1 poly-Ub during lysosomal homeostasis, LMP and degradation of damaged lysosomes, with important implications for NFKB signaling, inflammation and cell death. Abbreviation : ATG: autophagy related; BafA1: bafilomycin A 1 ; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CRISPR: clustered regularly interspaced short palindromic repeats; CHUK/IKKA: component of inhibitor of nuclear factor kappa B kinase complex; CUL4A-DDB1-WDFY1: cullin 4A-damage specific DNA binding protein 1-WD repeat and FYVE domain containing 1; DGCs: degradative compartments; DIV: days in vitro ; DUB: deubiquitinase/deubiquitinating enzyme; ELDR: endo-lysosomal damage response; ESCRT: endosomal sorting complex required for transport; FBXO27: F-box protein 27; GBM: glioblastoma multiforme; IKBKB/IKKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; IKK: inhibitor of NFKB kinase; iPSC: induced pluripotent stem cell; KBTBD7: kelch repeat and BTB domain containing 7; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LCD: lysosomal cell death; LGALS: galectin; LMP: lysosomal membrane permeabilization; LLOMe: L-leucyl-leucine methyl ester; LOP: loperamide; LUBAC: linear ubiquitin chain assembly complex; LRSAM1: leucine rich repeat and sterile alpha motif containing 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; NFKBIA/IĸBα: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; OPTN: optineurin; ORAS: OTULIN-related autoinflammatory syndrome; OTULIN: OTU deubiquitinase with linear linkage specificity; RING: really interesting new gene; RBR: RING-in-between-RING; PLAA: phospholipase A2 activating protein; RBCK1/HOIL-1: RANBP2-type and C3HC4-type zinc finger containing 1; RNF31/HOIP: ring finger protein 31; SHARPIN: SHANK associated RH domain interactor; SQSTM1/p62: sequestosome 1; SR-SIM: super-resolution-structured illumination microscopy; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TH: tyrosine hydroxylase; TNF/TNFα: tumor necrosis factor; TNFRSF1A/TNFR1-SC: TNF receptor superfamily member 1A signaling complex; TRIM16: tripartite motif containing 16; Ub: ubiquitin; UBE2QL1: ubiquitin conjugating enzyme E2 QL1; UBXN6/UBXD1: UBX domain protein 6; VCP/p97: valosin containing protein; WIPI2: WD repeat domain, phosphoinositide interacting 2; YOD1: YOD1 deubiquitinase.
Published: 2025
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29. Nonvesicular lipid transfer drives myelin growth in the central nervous system.

Author: Wu J, Kislinger G, Duschek J, Durmaz AD, Wefers B, Feng R, Nalbach K, Wurst W, Behrends C, Schifferer M, and Simons M
Subjects: Animals, Mice, Carrier Proteins metabolism, Carrier Proteins genetics, Lipid Metabolism, Glycolipids metabolism, Mice, Inbred C57BL, Biological Transport, Myelin Sheath metabolism, Oligodendroglia metabolism, Oligodendroglia cytology, Central Nervous System metabolism, Central Nervous System growth & development, Mice, Knockout, Endoplasmic Reticulum metabolism
Abstract: Oligodendrocytes extend numerous cellular processes that wrap multiple times around axons to generate lipid-rich myelin sheaths. Myelin biogenesis requires an enormously productive biosynthetic machinery for generating and delivering these large amounts of newly synthesized lipids. Yet, a complete understanding of this process remains elusive. Utilizing volume electron microscopy, we demonstrate that the oligodendroglial endoplasmic reticulum (ER) is enriched in developing myelin, extending into and making contact with the innermost myelin layer where growth occurs. We explore the possibility of transfer of lipids from the ER to myelin, and find that the glycolipid transfer protein (GLTP), implicated in nonvesicular lipid transport, is highly enriched in the growing myelin sheath. Mice with a specific knockout of Gltp in oligodendrocytes exhibit ER pathology, hypomyelination and a decrease in myelin glycolipid content. In summary, our results demonstrate a role for nonvesicular lipid transport in CNS myelin growth, revealing a cellular pathway in developmental myelination., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)
Published: 2024
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30. A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.

Author: Mukhopadhyay U, Levantovsky S, Carusone TM, Gharbi S, Stein F, Behrends C, and Bhogaraju S
Subjects: Humans, Substrate Specificity, DNA-Binding Proteins metabolism, DNA-Binding Proteins chemistry, Proliferating Cell Nuclear Antigen metabolism, Proliferating Cell Nuclear Antigen chemistry, Biotinylation, Staining and Labeling methods, Protein Binding, Ubiquitin-Protein Ligases metabolism, Ubiquitin-Protein Ligases chemistry, Ubiquitin metabolism, Ubiquitination
Abstract: Over 600 E3 ligases in humans execute ubiquitination of specific target proteins in a spatiotemporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. We applied our method, which we named Ub-POD, to the really interesting new gene (RING) E3 ligase RAD18 and identified proliferating cell nuclear antigen and several other critical players in the DNA damage repair pathway. Furthermore, we successfully applied Ub-POD to the RING ubiquitin ligase tumor necrosis factor receptor-associated factor 6 and a U-box-type E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein. We anticipate that our method could be widely adapted to all classes of ubiquitin ligases to identify substrates.
Published: 2024
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31. A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice.

Author: Brenner D, Sieverding K, Srinidhi J, Zellner S, Secker C, Yilmaz R, Dyckow J, Amr S, Ponomarenko A, Tunaboylu E, Douahem Y, Schlag JS, Rodríguez Martínez L, Kislinger G, Niemann C, Nalbach K, Ruf WP, Uhl J, Hollenbeck J, Schirmer L, Catanese A, Lobsiger CS, Danzer KM, Yilmazer-Hanke D, Münch C, Koch P, Freischmidt A, Fetting M, Behrends C, Parlato R, and Weishaupt JH
Subjects: Animals, Humans, Mice, Motor Neurons pathology, Mutation, Neuroinflammatory Diseases, Phosphorylation, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Amyotrophic Lateral Sclerosis pathology, Frontotemporal Dementia genetics, Frontotemporal Dementia metabolism, Frontotemporal Dementia pathology
Abstract: Heterozygous mutations in the TBK1 gene can cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The majority of TBK1-ALS/FTD patients carry deleterious loss-of-expression mutations, and it is still unclear which TBK1 function leads to neurodegeneration. We investigated the impact of the pathogenic TBK1 missense variant p.E696K, which does not abolish protein expression, but leads to a selective loss of TBK1 binding to the autophagy adaptor protein and TBK1 substrate optineurin. Using organelle-specific proteomics, we found that in a knock-in mouse model and human iPSC-derived motor neurons, the p.E696K mutation causes presymptomatic onset of autophagolysosomal dysfunction in neurons precipitating the accumulation of damaged lysosomes. This is followed by a progressive, age-dependent motor neuron disease. Contrary to the phenotype of mice with full Tbk1 knock-out, RIPK/TNF-α-dependent hepatic, neuronal necroptosis, and overt autoinflammation were not detected. Our in vivo results indicate autophagolysosomal dysfunction as a trigger for neurodegeneration and a promising therapeutic target in TBK1-ALS/FTD., (© 2024 Brenner et al.)
Published: 2024
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32. Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.

Author: Gahlot P, Kravic B, Rota G, van den Boom J, Levantovsky S, Schulze N, Maspero E, Polo S, Behrends C, and Meyer H
Subjects: Humans, Autophagy physiology, Intracellular Membranes metabolism, Lipids, Ubiquitin genetics, Ubiquitin metabolism, Lysosomes metabolism, Macroautophagy
Abstract: Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Published: 2024
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33. NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62.

Author: Furthmann N, Bader V, Angersbach L, Blusch A, Goel S, Sánchez-Vicente A, Krause LJ, Chaban SA, Grover P, Trinkaus VA, van Well EM, Jaugstetter M, Tschulik K, Damgaard RB, Saft C, Ellrichmann G, Gold R, Koch A, Englert B, Westenberger A, Klein C, Jungbluth L, Sachse C, Behrends C, Glatzel M, Hartl FU, Nakamura K, Christine CW, Huang EJ, Tatzelt J, and Winklhofer KF
Subjects: Humans, alpha-Synuclein genetics, Ubiquitin metabolism, Autophagy genetics, NF-kappa B metabolism, I-kappa B Kinase genetics, I-kappa B Kinase metabolism
Abstract: NEMO is a ubiquitin-binding protein which regulates canonical NF-κB pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identify an NF-κB-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO-encoding IKBKG gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including α-synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at α-synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62., (© 2023. The Author(s).)
Published: 2023
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34. Author Correction: Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2.

Author: Nalbach K, Schifferer M, Bhattacharya D, Ho-Xuan H, Tseng WC, Williams LA, Stolz A, Lichtenthaler SF, Elazar Z, and Behrends C
Published: 2023
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35. Validating a double Gaussian source model for small proton fields in a commercial Monte-Carlo dose calculation engine.

Author: Kugel F, Wulff J, Bäumer C, Janson M, Kretschmer J, Brodbek L, Behrends C, Verbeek N, Looe HK, Poppe B, and Timmermann B
Subjects: Radiotherapy Dosage, Radiotherapy Planning, Computer-Assisted methods, Algorithms, Monte Carlo Method, Protons, Proton Therapy methods
Abstract: Purpose: The primary fluence of a proton pencil beam exiting the accelerator is enveloped by a region of secondaries, commonly called "spray". Although small in magnitude, this spray may affect dose distributions in pencil beam scanning mode e.g., in the calculation of the small field output, if not modelled properly in a treatment planning system (TPS). The purpose of this study was to dosimetrically benchmark the Monte Carlo (MC) dose engine of the RayStation TPS (v.10A) in small proton fields and systematically compare single Gaussian (SG) and double Gaussian (DG) modeling of initial proton fluence providing a more accurate representation of the nozzle spray., Methods: The initial proton fluence distribution for SG/DG beam modeling was deduced from two-dimensional measurements in air with a scintillation screen with electronic readout. The DG model was either based on direct fits of the two Gaussians to the measured profiles, or by an iterative optimization procedure, which uses the measured profiles to mimic in-air scan-field factor (SF) measurements. To validate the DG beam models SFs, i.e. relative doses to a 10 × 10 cm 2 field, were measured in water for three different initial proton energies (100MeV, 160MeV, 226.7MeV) and square field sizes from 1×1cm 2 to 10×10cm 2 using a small field ionization chamber (IBA CC01) and an IBA ProteusPlus system (universal nozzle). Furthermore, the dose to the center of spherical target volumes (diameters: 1cm to 10cm) was determined using the same small volume ionization chamber (IC). A comprehensive uncertainty analysis was performed, including estimates of influence factors typical for small field dosimetry deduced from a simple two-dimensional analytical model of the relative fluence distribution. Measurements were compared to the predictions of the RayStation TPS., Results: SFs deviated by more than 2% from TPS predictions in all fields <4×4cm 2 with a maximum deviation of 5.8% for SG modeling. In contrast, deviations were smaller than 2% for all field-sizes and proton energies when using the directly fitted DG model. The optimized DG model performed similarly except for slightly larger deviations in the 1×1cm 2 scan-fields. The uncertainty estimates showed a significant impact of pencil beam size variations (±5%) resulting in up to 5.0% standard uncertainty. The point doses within spherical irradiation volumes deviated from calculations by up to 3.3% for the SG model and 2.0% for the DG model., Conclusion: Properly representing nozzle spray in RayStation's MC-based dose engine using a DG beam model was found to reduce the deviation to measurements in small spherical targets to below 2%. A thorough uncertainty analysis shows a similar magnitude for the combined standard uncertainty of such measurements., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Martin Janson is employed by RaySearch Laboratories AB. The remaining authors have no conflict of interest to disclose., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)
Published: 2023
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36. Surface Chemistry and Specific Surface Area Rule the Efficiency of Gold Nanoparticle Sensitizers in Proton Therapy.

Author: Johny J, van Halteren CER, Cakir FC, Zwiehoff S, Behrends C, Bäumer C, Timmermann B, Rauschenbach L, Tippelt S, Scheffler B, Schramm A, Rehbock C, and Barcikowski S
Subjects: Humans, Gold chemistry, Reactive Oxygen Species, Proton Therapy, Metal Nanoparticles chemistry, Radiation-Sensitizing Agents
Abstract: Gold nanoparticles (AuNPs) are currently the most studied radiosensitizers in proton therapy (PT) applicable for the treatment of solid tumors, where they amplify production of reactive oxygen species (ROS). However, it is underexplored how this amplification is correlated with the AuNPs' surface chemistry. To clarify this issue, we fabricated ligand-free AuNPs of different mean diameters by laser ablation in liquids (LAL) and laser fragmentation in liquids (LFL) and irradiated them with clinically relevant proton fields by using water phantoms. ROS generation was monitored by the fluorescent dye 7-OH-coumarin. Our findings reveal an enhancement of ROS production driven by I) increased total particle surface area, II) utilization of ligand-free AuNPs avoiding sodium citrate as a radical quencher ligands, and III) a higher density of structural defects generated by LFL synthesis, indicated by surface charge density. Based on these findings it may be concluded that the surface chemistry is a major and underexplored contributor to ROS generation and sensitizing effects of AuNPs in PT. We further highlight the applicability of AuNPs in vitro in human medulloblastoma cells., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)
Published: 2023
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37. The COP9 signalosome reduces neuroinflammation and attenuates ischemic neuronal stress in organotypic brain slice culture model.

Author: Tian Y, Milic J, Monasor LS, Chakraborty R, Wang S, Yuan Y, Asare Y, Behrends C, Tahirovic S, and Bernhagen J
Subjects: Humans, Animals, Mice, COP9 Signalosome Complex, Cullin Proteins, Endothelial Cells, Brain, Inflammation drug therapy, Cytokines, Neuroinflammatory Diseases, NF-kappa B
Abstract: The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics., (© 2023. The Author(s).)
Published: 2023
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38. C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.

Author: Jülg J, Edbauer D, and Behrends C
Subjects: Humans, C9orf72 Protein genetics, C9orf72 Protein metabolism, Ubiquitin metabolism, Carrier Proteins metabolism, Proteins genetics, Proteins metabolism, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Molecular Chaperones metabolism, Frontotemporal Dementia genetics, Frontotemporal Dementia metabolism, Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis metabolism
Abstract: Hexanucleotide repeat expansions within C9orf72 are a frequent cause of amyotrophic lateral sclerosis and frontotemporal dementia. Haploinsufficiency leading to reduced C9orf72 protein contributes to disease pathogenesis. C9orf72 binds SMCR8 to form a robust complex that regulates small GTPases, lysosomal integrity, and autophagy. In contrast to this functional understanding, we know far less about the assembly and turnover of the C9orf72-SMCR8 complex. Loss of either subunit causes the concurrent ablation of the respective partner. However, the molecular mechanism underlying this interdependence remains elusive. Here, we identify C9orf72 as a substrate of branched ubiquitin chain-dependent protein quality control. We find that SMCR8 prevents C9orf72 from rapid degradation by the proteasome. Mass spectrometry and biochemical analyses reveal the E3 ligase UBR5 and the BAG6 chaperone complex as C9orf72-interacting proteins, which are components of the machinery that modifies proteins with K11/K48-linked heterotypic ubiquitin chains. Depletion of UBR5 results in reduced K11/K48 ubiquitination and increased C9orf72 when SMCR8 is absent. Our data provide novel insights into C9orf72 regulation with potential implication for strategies to antagonize C9orf72 loss during disease progression., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)
Published: 2023
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39. Similar additive effects of doxorubicin in combination with photon or proton irradiation in soft tissue sarcoma models.

Author: Bernardo T, Behrends C, Klein D, Kuntze A, Timmermann B, and von Neubeck C
Abstract: High-precision radiotherapy with proton beams is frequently used in the management of aggressive soft tissue sarcoma (STS) and is often combined with doxorubicin (Dox), the first-line chemotherapy for STS. However, current treatment approaches continue to result in high local recurrence rates often occurring within the treatment field. This strongly indicates the need of optimized treatment protocols taking the vast heterogeneity of STS into account, thereby fostering personalized treatment approaches. Here, we used preclinical STS models to investigate the radiation response following photon (X) or proton (H) irradiation alone and in combination with different treatment schedules of Dox. As preclinical models, fibrosarcoma (HT-1080), undifferentiated pleiomorphic sarcoma (GCT), and embryonal rhabdomyosarcoma (RD) cell lines were used; the latter two are mutated for TP53. The cellular response regarding clonogenic survival, apoptosis, cell-cycle distribution, proliferation, viability, morphology, and motility was investigated. The different STS cell types revealed a dose-dependent radiation response with reduced survival, proliferation, viability, and motility whereas G2/M phase arrest as well as apoptosis were induced. RD cells showed the most radiosensitive phenotype; the linear quadratic model fit could not be applied. In combined treatment schedules, Dox showed the highest efficiency when applied after or before and after radiation; Dox treatment only before radiation was less efficient. GCT cells were the most chemoresistant cell line in this study most probably due to their TP53 mutation status. Interestingly, similar additive effects could be observed for X or H irradiation in combination with Dox treatment. However, the additive effects were determined more frequently for X than for H irradiation. Thus, further investigations are needed to specify alternative drug therapies that display superior efficacy when combined with H therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bernardo, Behrends, Klein, Kuntze, Timmermann and von Neubeck.)
Published: 2023
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40. FACS-mediated isolation of native autophagic vesicles.

Author: Schmitt D, Bozkurt S, Henning-Domres P, Huesmann H, Eimer S, Bindila L, Behrends C, Boyle E, Wilfling F, Tascher G, Münch C, Behl C, and Kern A
Subjects: Proteomics, Autophagosomes metabolism, Lipids, Autophagy physiology, Proteasome Endopeptidase Complex metabolism
Abstract: Autophagosome isolation enables the thorough investigation of structural components and engulfed materials. Recently, we introduced a novel antibody-based FACS-mediated method for isolation of native macroautophagic/autophagic vesicles and confirmed the quality of the preparations. We performed phospholipidomic and proteomic analyses to characterize autophagic vesicle-associated phospholipids and protein cargoes under different autophagy conditions. Lipidomic analyses identified phosphoglycerides and sphingomyelins within autophagic vesicles and revealed that the lipid composition was unaffected by different rates of autophagosome formation. Proteomic analyses identified more than 4500 potential autophagy substrates and showed that in comparison to autophagic vesicles isolated under basal autophagy conditions, starvation only marginally affected the cargo profile. Proteasome inhibition, however, resulted in the enhanced degradation of ubiquitin-proteasome system components. Taken together, the novel isolation method enriched large quantities of autophagic vesicles and enabled detailed analyses of their lipid and cargo composition.
Published: 2023
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41. Comprehensive investigation of lateral dose profile and output factor measurements in small proton fields from different delivery techniques.

Author: Kretschmer J, Brodbek L, Behrends C, Kugel F, Koska B, Bäumer C, Wulff J, Timmermann B, Poppe B, and Looe HK
Subjects: Monte Carlo Method, Particle Accelerators, Algorithms, Photons therapeutic use, Protons, Radiometry methods
Abstract: Background and Purpose: As a part of the commissioning and quality assurance in proton beam therapy, lateral dose profiles and output factors have to be acquired. Such measurements can be performed with point detectors and are especially challenging in small fields or steep lateral penumbra regions as the detector's volume effect may lead to perturbations. To address this issue, this work aims to quantify and correct for such perturbations of six point detectors in small proton fields created via three different delivery techniques., Methods: Lateral dose profile and output measurements of three proton beam delivery techniques (pencil beam scanning, pencil beam scanning combined with collimators, passive scattering with collimators) were performed using high-resolution EBT3 films, a PinPoint 3D 31022 ionization chamber, a microSilicon diode 60023 and a microDiamond detector 60019 (all PTW Freiburg, Germany). Detector specific lateral dose response functions K(x,y) acting as the convolution kernel transforming the undisturbed dose distribution D(x,y) into the measured signal profiles M(x,y) were applied to quantify perturbations of the six investigated detectors in the proton fields and correct the measurements. A signal theoretical analysis in Fourier space of the dose distributions and detector's K(x,y) was performed to aid the understanding of the measurement process with regard to the combination of detector choice and delivery technique., Results: Quantification of the lateral penumbra broadening and signal reduction at the fields center revealed that measurements in the pencil beam scanning fields are only compromised slightly even by large volume ionization chambers with maximum differences in the lateral penumbra of 0.25 mm and 4% signal reduction at the field center. In contrast, radiation techniques with collimation are not accurately represented by the investigated detectors as indicated by a penumbra broadening up to 1.6 mm for passive scattering with collimators and 2.2 mm for pencil beam scanning with collimators. For a 3 mm diameter collimator field, a signal reduction at field center between 7.6% and 60.7% was asserted. Lateral dose profile measurements have been corrected via deconvolution with the corresponding K(x,y) to obtain the undisturbed D(x,y). Corrected output ratios of the passively scattered collimated fields obtained for the microDiamond, microSilicon and PinPoint 3D show agreement better than 0.9% (one standard deviation) for the smallest field size of 3 mm., Conclusion: Point detector perturbations in small proton fields created with three delivery techniques were quantified and found to be especially pronounced for collimated small proton fields with steep dose gradients. Among all investigated detectors, the microSilicon diode showed the smallest perturbations. The correction strategies based on detector's K(x,y) were found suitable for obtaining unperturbed lateral dose profiles and output factors. Approximation of K(x,y) by considering only the geometrical averaging effect has been shown to provide reasonable prediction of the detector's volume effect. The findings of this work may be used to guide the choice of point detectors in various proton fields and to contribute toward the development of a code of practice for small field proton dosimetry., (© 2023 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)
Published: 2023
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42. Membrane Atg8ylation, stress granule formation, and MTOR regulation during lysosomal damage.

Author: Jia J, Wang F, Bhujabal Z, Peters R, Mudd M, Duque T, Allers L, Javed R, Salemi M, Behrends C, Phinney B, Johansen T, and Deretic V
Subjects: Animals, Humans, Stress Granules, RNA Helicases metabolism, Poly-ADP-Ribose Binding Proteins metabolism, Proteomics, RNA Recognition Motif Proteins metabolism, Autophagy, SARS-CoV-2, TOR Serine-Threonine Kinases metabolism, Lysosomes metabolism, Cytoplasmic Granules metabolism, Mammals metabolism, Galectins metabolism, DNA Helicases metabolism, COVID-19
Abstract: The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, Mycobacterium tuberculosis infection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage. Abbreviations: Atg8: autophagy related 8; ATG: autophagy related; ATF4: activating transcription factor 4; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; GABARAP: GABA type A receptor-associated protein; G3BP1: G3BP stress granule assembly factor 1; LLOMe: L-leucyl-L-leucine methyl ester; LysoIP: lysosome immunopurification; mRNA: messenger ribonucleic acid; MTOR: mechanistic target of rapamycin kinase; NUFIP2: nuclear FMR1 interacting protein 2; ORF3a: open reading frame 3a protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SG: stress granule; TIA1: TIA1 cytotoxic granule associated RNA binding protein.
Published: 2023
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43. Optimization of proton pencil beam positioning in collimated fields.

Author: Behrends C, Bäumer C, Verbeek NG, Wulff J, and Timmermann B
Subjects: Protons, Phantoms, Imaging, Radiotherapy Dosage, Monte Carlo Method, Water, Radiotherapy Planning, Computer-Assisted methods, Proton Therapy methods
Abstract: Background: The addition of static or dynamic collimator systems to the pencil beam scanning delivery technique increases the number of options for lateral field shaping. The collimator shape needs to be optimized together with the intensity modulation of spots., Purpose: To minimize the proton field's lateral penumbra by investigating the fundamental relations between spot and collimating aperture edge position., Methods: Analytical approaches describing the effect of spot position on the resulting spot profile are presented. The theoretical description is then compared with Monte Carlo simulations in TOPAS and in the RayStation treatment planning system, as well as with radiochromic film measurements at a clinical proton therapy facility. In the model, one single spot profile is analyzed for various spot positions in air. Further, irradiation setups in water with different energies, the combination with a range shifter, and two-dimensional proton fields were investigated in silico., Results: The further the single spot is placed beyond the collimating aperture edge ('overscanning'), the sharper the relative lateral dose fall-off and thus the lateral penumbra. Overscanning up to 5 mm $5\,\text{mm}$ reduced the lateral penumbra by about 20% on average after a propagation of 13 cm $13\,\text{cm}$ in air. This benefit from overscanning is first predicted by the analytical proofs and later verified by simulations and measurements. Corresponding analyses in water confirm the benefit in lateral penumbra with spot position optimization as observed theoretically and in air. The combination of spot overscanning with fluence modulation facilitated an additional improvement., Conclusions: The lateral penumbra of single spots in collimated scanned proton fields can be improved by the method of spot overscanning. This suggests a better sparing of proximal organs at risk in smaller water depths at higher energies, especially in the plateau of the depth dose distribution. All in all, spot overscanning in collimated scanned proton fields offers particular potential in combination with techniques such as fluence modulation or dynamic collimation for optimizing the lateral penumbra to spare normal tissue., (© 2023 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)
Published: 2023
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44. Atg8 family proteins, LIR/AIM motifs and other interaction modes.

Author: Rogov VV, Nezis IP, Tsapras P, Zhang H, Dagdas Y, Noda NN, Nakatogawa H, Wirth M, Mouilleron S, McEwan DG, Behrends C, Deretic V, Elazar Z, Tooze SA, Dikic I, Lamark T, and Johansen T
Abstract: The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies., Competing Interests: Disclosure statement No potential conflict of interest was reported by the author(s).
Published: 2023
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45. Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2.

Author: Nalbach K, Schifferer M, Bhattacharya D, Ho-Xuan H, Tseng WC, Williams LA, Stolz A, Lichtenthaler SF, Elazar Z, and Behrends C
Subjects: Autophagosomes, Autophagy genetics, Golgi Apparatus, Protein Transport, Carrier Proteins metabolism, Nerve Tissue Proteins metabolism, Proteomics, Secretory Pathway
Abstract: Hereditary sensory and autonomic neuropathy 9 (HSAN9) is a rare fatal neurological disease caused by mis- and nonsense mutations in the gene encoding for Tectonin β-propeller repeat containing protein 2 (TECPR2). While TECPR2 is required for lysosomal consumption of autophagosomes and ER-to-Golgi transport, it remains elusive how exactly TECPR2 is involved in autophagy and secretion and what downstream sequels arise from defective TECPR2 due to its involvement in these processes. To address these questions, we determine molecular consequences of TECPR2 deficiency along the secretory pathway. By employing spatial proteomics, we describe pronounced changes with numerous proteins important for neuronal function being affected in their intracellular transport. Moreover, we provide evidence that TECPR2's interaction with the early secretory pathway is not restricted to COPII carriers. Collectively, our systematic profiling of a HSAN9 cell model points to specific trafficking and sorting defects which might precede autophagy dysfunction upon TECPR2 deficiency., (© 2023. The Author(s).)
Published: 2023
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46. Commissioning and validation of a novel commercial TPS for ocular proton therapy.

Author: Wulff J, Koska B, Heufelder J, Janson M, Bäcker CM, Siregar H, Behrends C, Bäumer C, Foerster A, Bechrakis NE, and Timmermann B
Subjects: Humans, Radiotherapy Planning, Computer-Assisted methods, Radiotherapy Dosage, Protons, Algorithms, Phantoms, Imaging, Monte Carlo Method, Proton Therapy methods
Abstract: Background: Until today, the majority of ocular proton treatments worldwide were planned with the EYEPLAN treatment planning system (TPS). Recently, the commercial, computed tomography (CT)-based TPS for ocular proton therapy RayOcular was released, which follows the general concepts of model-based treatment planning approach in conjunction with a pencil-beam-type dose algorithm (PBA)., Purpose: To validate RayOcular with respect to two main features: accurate geometrical representation of the eye model and accuracy of its dose calculation algorithm in combination with an Ion Beam Applications (IBA) eye treatment delivery system., Methods: Different 3D-printed eye-ball-phantoms were fabricated to test the geometrical representation of the corresponding CT-based model, both in orthogonal 2D images for X-ray image overlay and in fundus view overlaid with a funduscopy. For the latter, the phantom was equipped with a lens matching refraction of the human eye. Funduscopy was acquired in a Zeiss Claus 500 camera. Tantalum clips and fiducials attached to the phantoms were localized in the TPS model, and residual deviations to the actual position in X-ray images for various orientations of the phantom were determined, after the nominal eye orientation was corrected in RayOcular to obtain a best overall fit. In the fundus view, deviations between known and displayed distances were measured. Dose calculation accuracy of the PBA on a 0.2 mm grid was investigated by comparing between measured lateral and depth-dose profiles in water for various combinations of range, modulation, and field-size. Ultimately, the modeling of dose distributions behind wedges was tested. A 1D gamma-test was applied, and the lateral and distal penumbra were further compared., Results: Average residuals between model clips and visible clips/fiducials in orthogonal X-ray images were within 0.3 mm, including different orientations of the phantom. The differences between measured distances on the registered funduscopy image in the RayOcular fundus view and the known ground-truth were within 1 mm up to 10.5 mm distance from the posterior pole. No clear benefit projection of either polar mode or camera mode could be identified, the latter mimicking camera properties. Measured dose distributions were reproduced with gamma-test pass-rates of >95% with 2%/0.3 mm for depth and lateral profiles in the middle of spread-out Bragg-peaks. Distal falloff and lateral penumbra were within 0.2 mm for fields without a wedge. For shallow depths, the agreement was worse, reaching pass-rates down to 80% with 5%/0.3 mm when comparing lateral profiles in air. This is caused by low-energy protons from a scatter source in the IBA system not modeled by RayOcular. Dose distributions modified by wedges were reproduced, matching the wedge-induced broadening of the lateral penumbra to within 0.4 mm for the investigated cases and showing the excess dose within the field due to wedge scatter., Conclusion: RayOcular was validated for its use with an IBA single scattering delivery nozzle. Geometric modeling of the eye and representation of 2D projections fulfill clinical requirements. The PBA dose calculation reproduces measured distributions and allows explicit handling of wedges, overcoming approximations of simpler dose calculation algorithms used in other systems., (© 2022 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)
Published: 2023
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47. Vps37a regulates hepatic glucose production by controlling glucagon receptor localization to endosomes.

Author: Sekar R, Motzler K, Kwon Y, Novikoff A, Jülg J, Najafi B, Wang S, Warnke AL, Seitz S, Hass D, Gancheva S, Kahl S, Yang B, Finan B, Schwarz K, Okun JG, Roden M, Blüher M, Müller TD, Krahmer N, Behrends C, Plettenburg O, Miaczynska M, Herzig S, and Zeigerer A
Published: 2022
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48. Lipid and protein content profiling of isolated native autophagic vesicles.

Author: Schmitt D, Bozkurt S, Henning-Domres P, Huesmann H, Eimer S, Bindila L, Behrends C, Boyle E, Wilfling F, Tascher G, Münch C, Behl C, and Kern A
Subjects: Humans, Autophagy, Phospholipids, Proteasome Endopeptidase Complex, Proteomics
Abstract: Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)
Published: 2022
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49. Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage.

Author: Jia J, Wang F, Bhujabal Z, Peters R, Mudd M, Duque T, Allers L, Javed R, Salemi M, Behrends C, Phinney B, Johansen T, and Deretic V
Subjects: Animals, Cytoplasmic Granules metabolism, Lysosomes metabolism, Mammals metabolism, Poly-ADP-Ribose Binding Proteins metabolism, RNA Recognition Motif Proteins metabolism, Stress Granules, TOR Serine-Threonine Kinases genetics, TOR Serine-Threonine Kinases metabolism, Autophagy-Related Protein 8 Family metabolism, DNA Helicases metabolism, RNA Helicases metabolism
Abstract: We report that lysosomal damage is a hitherto unknown inducer of stress granule (SG) formation and that the process termed membrane atg8ylation coordinates SG formation with mTOR inactivation during lysosomal stress. SGs were induced by lysosome-damaging agents including SARS-CoV-2ORF3a, Mycobacterium tuberculosis, and proteopathic tau. During damage, mammalian ATG8s directly interacted with the core SG proteins NUFIP2 and G3BP1. Atg8ylation was needed for their recruitment to damaged lysosomes independently of SG condensates whereupon NUFIP2 contributed to mTOR inactivation via the Ragulator-RagA/B complex. Thus, cells employ membrane atg8ylation to control and coordinate SG and mTOR responses to lysosomal damage., (© 2022 Jia et al.)
Published: 2022
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50. Impact of Sterilization on the Colloidal Stability of Ligand-Free Gold Nanoparticles for Biomedical Applications.

Author: Johny J, van Halteren CER, Zwiehoff S, Behrends C, Bäumer C, Timmermann B, Rehbock C, and Barcikowski S
Subjects: Reactive Oxygen Species, Particle Size, Ligands, Sterilization, Gold, Metal Nanoparticles
Abstract: Sterilization is a major prerequisite for the utilization of nanoparticle colloids in biomedicine, a process well examined for particles derived from chemical synthesis although highly underexplored for electrostatically stabilized ligand-free gold nanoparticles (AuNPs). Hence, in this work, we comprehensively examined and compared the physicochemical characteristics of laser-generated ligand-free colloidal AuNPs exposed to steam sterilization and sterile filtration as a function of particle size and mass concentration and obtained physicochemical insight into particle growth processes. These particles exhibit long-term colloidal stability (up to 3 months) derived from electrostatic stabilization without using any ligands or surfactants. We show that particle growth attributed to cluster-based ripening occurs in smaller AuNPs (∼5 nm) following autoclaving, while larger particles (∼10 and ∼30 nm) remain stable. Sterile filtration, as an alternative effective sterilizing approach, has no substantial impact on the colloidal stability of AuNPs, regardless of particle size, although a mass loss of 5-10% is observed. Finally, we evaluated the impact of the sterilization procedures on potential particle functionality in proton therapy, using the formation of reactive oxygen species (ROS) as a readout. In particular, 5 nm AuNPs exhibit a significant loss in activity upon autoclaving, probably dedicated to specific surface area reduction and surface restructuring during particle growth. The filtered analog enhanced the ROS release by up to a factor of ∼2.0, at 30 ppm gold concentration. Our findings highlight the need for carefully adapting the sterilization procedure of ligand-free NPs to the desired biomedical application with special emphasis on particle size and concentration.
Published: 2022
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