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Abstract Alzheimer’s disease (AD) is a progressive and incurable neurodegenerative disease characterized by the extracellular deposition of amyloid plaques. Investigation into the composition of these plaques revealed a high amount of amyloid-β (Aβ) fibrils and a high concentration of lipids, suggesting that fibril-lipid interactions may also be relevant for the pathogenesis of AD. Therefore, we grew Aβ40 fibrils in the presence of lipid vesicles and determined their structure by cryo-electron microscopy (cryo-EM) to high resolution. The fold of the major polymorph is similar to the structure of brain-seeded fibrils reported previously. The majority of the lipids are bound to the fibrils, as we show by cryo-EM and NMR spectroscopy. This apparent lipid extraction from vesicles observed here in vitro provides structural insights into potentially disease-relevant fibril-lipid interactions.

Abstract If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow—and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis-spliced peptides, whereas trans-spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation.

In this work, we report the discovery of potent anti-epidermal growth factor receptor (EGFR) allosteric heavy-chain antibodies by combining camelid immunization and fluorescence-activated cell sorting (FACS). After immunization and yeast surface display library construction, allosteric clones were obtained by introducing the labeled EGF Fc fusion protein as an additional criterion for FACS. This sorting method enabled the identification of 11 heavy-chain antibodies that did not compete with the orthosteric ligand EGF for the binding to EGFR. These antibodies bind to a triple-negative breast cancer cell line expressing EGFR with affinities in the picomolar to nanomolar range. Those camelid-derived antibodies also exhibit interesting properties by modulating EGFR affinity for EGF. Moreover, they are also able to inhibit EGF-induced downstream signaling pathways. In particular, we identified one clone that is more potent than the approved blocking antibody cetuximab in inhibiting both PI3K/AKT and MAPK/ERK pathways. Our results suggest that allosteric antibodies may be potential new modalities for therapeutics.

Here we report the structure of Opa60 in lipid bilayers using proton-detected magic-angle spinning nuclear magnetic resonance (MAS NMR). Preparations including near-native oligosaccharide lipids reveal a consistent picture of a stable transmembrane beta barrel with a minor increase in the structured region as compared with the previously reported detergent structure. The large variable loops known to interact with host proteins could not be detected, confirming their dynamic nature even in a lipid bilayer environment. The structure provides a starting point for investigation of the functional role of Opa60 in gonococcal infection, which is understood to involve interaction with host proteins. At the same time, it demonstrates the recent advances in proton-detected methodology for membrane protein structure determination at atomic resolution by MAS NMR.

Abstract Alzheimer’s disease begins with mild memory loss and slowly destroys memory and thinking. Cognitive impairment in Alzheimer’s disease has been associated with the localization of the microtubule-associated protein Tau at the postsynapse. However, the correlation between Tau at the postsynapse and synaptic dysfunction remains unclear. Here, we show that Tau arrests liquid-like droplets formed by the four postsynaptic density proteins PSD-95, GKAP, Shank, Homer in solution, as well as NMDA (N-methyl-D-aspartate)-receptor-associated protein clusters on synthetic membranes. Tau-mediated condensate/cluster arrest critically depends on the binding of multiple interaction motifs of Tau to a canonical GMP-binding pocket in the guanylate kinase domain of PSD-95. We further reveal that competitive binding of a high-affinity phosphorylated peptide to PSD-95 rescues the diffusional dynamics of an NMDA truncated construct, which contains the last five amino acids of the NMDA receptor subunit NR2B fused to the C-terminus of the tetrameric GCN4 coiled-coil domain, in postsynaptic density-like condensates/clusters. Taken together, our findings propose a molecular mechanism where Tau modulates the dynamic properties of the postsynaptic density.

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children. At present, the long-term survival from pediatric ALL is well over 90%. However, the probability of event-free survival is reduced if the lumbar puncture (LP) procedures at the beginning of the patient’s intrathecal therapy cause blood leakage into the spinal canal and blast cells contaminate the cerebrospinal fluid. According to the literature, such traumatic LP procedures concern one out of five pediatric patients with ALL. Recently, a novel medical device measuring the tissue bioimpedance at the tip of a spinal needle was found feasible in pediatric patients with ALL. The LP procedure was successful at the first attempt in 80% of procedures, and the incidence of traumatic LPs was then 11%. The purpose of the present study is to compare the bioimpedance spinal needle system with the standard clinical practice resting on a conventional spinal needle and investigate its efficacy in clinical practice. Methods The study is a multicenter, randomized, two-arm crossover noninferiority trial of pediatric hemato-oncology patients that will be conducted within the usual clinical workflow. Patients’ LP procedures will be performed alternately either with the IQ-Tip system (study arm A) or a conventional Quincke-type 22G spinal needle (study arm B). For each enrolled patient, the order of procedures is randomly assigned either as ABAB or BABA. The total number of LP procedures will be at least 300, and the number of procedures per patient between two and four. After each study LP procedure, the performance will be recorded immediately, and 1-week diary-based and 4-week record-based follow-ups on symptoms, complications, and adverse events will be conducted thereafter. The main outcomes are the incidence of traumatic LP, first puncture success rate, and incidence of post-dural puncture headache. Discussion The present study will provide sound scientific evidence on the clinical benefit, performance, and safety of the novel bioimpedance spinal needle compared with the standard clinical practice of using conventional spinal needles in the LP procedures of pediatric patients with leukemia. Trial registration ISRCTN ISRCTN16161453. Registered on 8 July 2022.

ABSTRACTHere, we generated bispecific antibody (bsAb) derivatives that mimic the function of interleukin (IL)-18 based on single domain antibodies (sdAbs) specific to IL-18 Rα and IL-18 Rβ. For this, camelids were immunized, followed by yeast surface display (YSD)-enabled discovery of VHHs targeting the individual receptor subunits. Upon reformatting into a strictly monovalent (1 + 1) bispecific sdAb architecture, several bsAbs triggered dose-dependent IL-18 R downstream signaling on IL-18 reporter cells, as well as IFN-γ release by peripheral blood mononuclear cells in the presence of low-dose IL-12. However, compared with IL-18, potencies and efficacies were considerably attenuated. By engineering paratope valencies and the spatial orientation of individual paratopes within the overall design architecture, we were able to generate IL-18 mimetics displaying significantly augmented functionalities, resulting in bispecific cytokine mimetics that were more potent than IL-18 in triggering proinflammatory cytokine release. Furthermore, generated IL-18 mimetics were unaffected from inhibition by IL-18 binding protein decoy receptor. Essentially, we demonstrate that this strategy enables the generation of IL-18 mimetics with tailor-made cytokine functionalities.

The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55–100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.

Abstract Background For helicopter emergency service systems (HEMS), the prehospital time consists of response time, on-scene time and transport time. Little is known about the factors that influence on-scene time or about differences between adult and paediatric missions in a physician-staffed HEMS. Methods We analysed the HEMS electronic database of Swiss Air-Rescue from 01-01-2011 to 31-12-2021 (N = 110,331). We included primary missions and excluded missions with National Advisory Committee for Aeronautics score (NACA) score 0 or 7, resulting in 68,333 missions for analysis. The primary endpoint ‘on-scene time’ was defined as first physical contact with the patient until take-off to the hospital. A multivariable linear regression model was computed to examine the association of diagnosis, type and number of interventions and monitoring, and patient's characteristics with the primary endpoint. Results The prehospital time and on-scene time of the missions studied were, respectively, 50.6 [IQR: 41.0–62.0] minutes and 21.0 [IQR: 15.0–28.6] minutes. Helicopter hoist operations, resuscitation, airway management, critical interventions, remote location, night-time, and paediatric patients were associated with longer on-scene times. Conclusions Compared to adult patients, the adjusted on-scene time for paediatric patients was longer. Besides the strong impact of a helicopter hoist operation on on-scene time, the dominant factors contributing to on-scene time are the type and number of interventions and monitoring: improving individual interventions or performing them in parallel may offer great potential for reducing on-scene time. However, multiple clinical interventions and monitoring interact and are not single interventions. Compared to the impact of interventions, non-modifiable factors, such as NACA score, type of diagnosis and age, make only a minor contribution to overall on-scene time.

Interactions between α-synuclein fibrils and lipids have been associated with the development of Parkinson’s disease. This cryo-EM study reveals structural details of these interactions and suggests a mechanism for fibril-induced lipid extraction.

Understanding how small molecules bind to pathological aggregates is of importance for therapeutic and diagnostic development in diseases such as Parkinson’s Disease. Here, the authors reveal a binding site of anle138b to lipid-induced α-synuclein fibrils.

In the human phonation process, acoustic standing waves in the vocal tract can influence the fluid flow through the glottis as well as vocal fold oscillation. To investigate the amount of acoustic back-coupling, the supraglottal flow field has been recorded via high-speed particle image velocimetry (PIV) in a synthetic larynx model for several configurations with different vocal tract lengths. Based on the obtained velocity fields, acoustic source terms were computed. Additionally, the sound radiation into the far field was recorded via microphone measurements and the vocal fold oscillation via high-speed camera recordings. The PIV measurements revealed that near a vocal tract resonance frequency fR, the vocal fold oscillation frequency fo (and therefore also the flow field’s fundamental frequency) jumps onto fR. This is accompanied by a substantial relative increase in aeroacoustic sound generation efficiency. Furthermore, the measurements show that fo-fR-coupling increases vocal efficiency, signal-to-noise ratio, harmonics-to-noise ratio and cepstral peak prominence. At the same time, the glottal volume flow needed for stable vocal fold oscillation decreases strongly. All of this results in an improved voice quality and phonation efficiency so that a person phonating with fo-fR-coupling can phonate longer and with better voice quality.

Sound generation in human phonation and the underlying fluid–structure–acoustic interaction that describes the sound production mechanism are not fully understood. A previous experimental study, with a silicone made vocal fold model connected to a straight vocal tract pipe of fixed length, showed that vibroacoustic coupling can cause a deviation in the vocal fold vibration frequency. This occurred when the fundamental frequency of the vocal fold motion was close to the lowest acoustic resonance frequency of the pipe. What is not fully understood is how the vibroacoustic coupling is influenced by a varying vocal tract length. Presuming that this effect is a pure coupling of the acoustical effects, a numerical simulation model is established based on the computation of the mechanical-acoustic eigenvalue. With varying pipe lengths, the lowest acoustic resonance frequency was adjusted in the experiments and so in the simulation setup. In doing so, the evolution of the vocal folds’ coupled eigenvalues and eigenmodes is investigated, which confirms the experimental findings. Finally, it was shown that for normal phonation conditions, the mechanical mode is the most efficient vibration pattern whenever the acoustic resonance of the pipe (lowest formant) is far away from the vocal folds’ vibration frequency. Whenever the lowest formant is slightly lower than the mechanical vocal fold eigenfrequency, the coupled vocal fold motion pattern at the formant frequency dominates.

The authors provide a litmus test for the recognition mechanism of transiently binding proteins based on nuclear magnetic resonance and find a conformational selection binding mechanism through concentration-dependent kinetics of ubiquitin and SH3.

Toft-Bertelsen et al. describe repurposing of anti-influenza drug amantadine and its derivatives for the treatment of SARS-CoV-2. They show that Amantadine, Emodin and Xanthene show significant blockage of ionchannels formed by SARS-CoV-2 which are crucial for its assembly and pathophysiology.

Abstract Background The goal of improving quality through centralisation of specialised medical services must be balanced against potential harm caused by delayed access to emergency treatments in rural areas. This study aims to assess the duration of transfers of critically ill patients with cardiovascular emergencies from smaller hospitals to major medical centres by a helicopter emergency medical service (HEMS) in Switzerland. Methods This retrospective observational cohort study includes all consecutive emergency interfacility transfers (IFTs) conducted by Switzerland’s largest HEMS provider between July 3rd, 2019, and March 31st, 2021. All patients with acute myocardial infarction, non-traumatic strokes, ruptured aortic aneurysms, and other acute vascular emergencies were included. The duration and distance of each HEMS IFT were compared to calculated distances and duration of travel for the same missions using ground-based transportation (GEMS). The ground-based mission distance beyond which the total mission duration of HEMS is expected to be faster than GEMS was calculated. Findings A total of 645 patients were transferred for stroke (n = 364), myocardial infarction (n = 252) and other acute vascular emergencies (n = 29). The median total mission duration from emergency call to landing at the destination was 59.9 (IQR 51.5 to 70.5) minutes. The median road distance for the same missions was 60 (IQR 43 to 72) km. Regression analysis revealed that HEMS is expected to be faster if the road distance is more than 51.3 km. Interpretation Centralisation of specialised medical services should be accompanied by a comprehensive and specialised rescue chain. HEMS in Switzerland ensures time-sensitive IFT in medical emergencies, even in topographically challenging terrain. Graphical Abstract

Abstract Objective We aimed to investigate the medical characteristics of helicopter hoist operations (HHO) in HEMS missions. Methods We designed a retrospective study evaluating all HHO and other human external cargo (HEC) missions performed by Swiss Air-Rescue (Rega) between January 1, 2010, and December 31, 2019. Results During the study period, 9,963 (88.7 %) HEMS missions with HHO and HEC were conducted during the day, and 1,265 (11.3 %) at night. Of the victims with time-critical injuries (NACA ≥ 4), 21.1 % (n = 400) reached the hospital within 60 min during the day, and 9.1 % (n = 18) at night. Nighttime missions, a trauma diagnosis, intubation on-site, and NACA Score ≥ 4 were independently and highly significantly associated with longer mission times (p

The most frequent cause of familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are hexanucleotide repeat expansions in the non-coding region of the C9ORF72 gene that are translated into five dipeptide repeat (DPR) proteins. Here, the authors show that proline/arginine (PR) DPRs inhibit the prolyl isomerase PPIA and reveal the molecular mechanism of the impaired protein folding activity of PPIA by performing NMR measurements and determining a PR DPR bound PPIA crystal structure.

Methods to quantify the complexity of trajectory datasets are still a missing piece in benchmarking human trajectory prediction models. In order to gain a better understanding of the complexity of trajectory prediction tasks and following the intuition, that more complex datasets contain more information, an approach for quantifying the amount of information contained in a dataset from a prototype-based dataset representation is proposed. The dataset representation is obtained by first employing a non-trivial spatial sequence alignment, which enables a subsequent learning vector quantization (LVQ) stage. A large-scale complexity analysis is conducted on several human trajectory prediction benchmarking datasets, followed by a brief discussion on indications for human trajectory prediction and benchmarking.

Cyclophilin A (CypA) is a peptidylprolyl isomerase that also has chaperone activity and interacts with the intrinsically disordered protein α-Synuclein (aSyn). Here, the authors combine NMR measurements and biochemical experiments to characterise the interplay between the catalysis of proline isomerization and molecular chaperone activity of CypA and find that both activities have opposing effects on aSyn and further show that the that cis/trans isomerization outpowers the holding activity of CypA.

α-Syn in CSF is a biomarker of neurodegenerative diseases; however, the detection of clinically relevant species is difficult. Here, the authors create a nanobody biosensor that reveals the presence of α-Syn in cells, which allow the detection of transmittable forms of α-Syn present in human CSF.

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and SLP65 is a key scaffold protein mediating BCR signaling. Here authors show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles.

A novel active turbulence grid of the Institute of Fluid Mechanics at FAU Erlangen-Nuremberg is introduced. The focus of this grid is not on basic investigations of fluid mechanics, as is usually the case with active turbulence grids, but the generation of defined inflow conditions for axial fans. Thus, by means of the active turbulence grid, individual turbulence characteristics in the flow to the fan can be changed; therefore, fundamental interactions between the flow mechanics at the axial fan and the sound radiation can be analyzed. In addition, the replication of the flow fields of heat exchangers by the active turbulence grid is the focus of the investigations. The investigations showed that it is possible to use the active turbulence grid to generate defined inflow conditions for axial fans. It was also possible to reproduce the heat exchanger flow fields both for the mean turbulence values and for the spatial distributions. It was found that the grid induces tonal components due to the drive motors, but also that the inherent noise has no significant influence on the spectrum of the fans under investigation. Based on selected turbulence characteristics, direct correlations were found between the spatial distribution of the turbulence level and sound radiation at the first blade passing frequency of the axial fan. As the variance of the turbulence level increases, the sound radiation of the tonal components becomes more pronounced. The total sound pressure level, however, is mainly determined by the low-frequency broadband sound. A linear relationship between the spatial mean value of the turbulence level and the total sound pressure level was found for the investigated axial fan.

Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are characterized by the pathological accumulation of α-synuclein. Here the authors employ fluorescent probes, electron microscopy and NMR spectroscopy to study the properties of α-synuclein aggregates that were amplified from patient brain extracts and observe a greater structural diversity among PD patients compared to MSA patients.

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.

α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson’s disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked hαSynTP or hαSynΔ119 (h stands for “human”) into the murine αSyn locus. hαSynTP is a structure-based mutant with triple alanine to proline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster. hαSynΔ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of hαSynTP or hαSynΔ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that hαSynTP and hαSynΔ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSynTP and hαSynΔ119 mice, despite the lower levels of hαSynTP and hαSynΔ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSynΔ119 mice, while a compulsive behavior was exclusively detected in hαSynTP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches.

Wetting is strongly influenced by adsorbate layers, which are omnipresent on surfaces. The influence of the composition and thickness of adsorbate layers on the water contact angle of sessile drops on different substrates was systematically investigated in the present work. Measurements were carried out for gold-sputtered substrates. These new results are compared to results from a previous study, in which corresponding measurements were carried out for technical steel and titanium substrates. In all experiments, different pretreatments of the samples were used to obtain variations of the adsorbate layer. The samples were either exposed to an oil bath or not, and different cleaning agents were used. The analysis of the adsorbate layer was carried out with X-ray photoelectron spectroscopy (XPS). The results for the different substrates reveal that the water contact angle depends mainly on the composition of the adsorbate layer. The substrate has only an indirect influence, as it influences the composition of the adsorbate layer. The thickness of the adsorbate layers was between 1.4 and 14 nm and was large enough to prevent a direct influence of the substrate on the water contact angle. It is shown that using the information on the adsorbate layer composition from XPS and the results for the water contact angle obtained for the gold samples alone, the water contact angles on the steel and titanium samples can be predicted.

Bispecific antibodies comprise extremely diverse architectures enabling complex modes of action, such as effector cell recruitment or conditional target modulation via dual targeting, not conveyed by monospecific antibodies. In recent years, research on bispecific therapeutics has substantially grown. However, evaluation of binding moiety combinations often leads to undesired prolonged development times. While high throughput screening for small molecules and classical antibodies has evolved into a mature discipline in the pharmaceutical industry, dual-targeting antibody screening methodologies lack the ability to fully evaluate the tremendous number of possible combinations and cover only a limited portion of the combinatorial screening space. Here, we propose a novel combinatorial screening approach for bispecific IgG-like antibodies to extenuate screening limitations in industrial scale, expanding the limiting screening space. Harnessing the ability of a protein trans-splicing reaction by the split intein Npu DnaE, antibody fragments were reconstituted within the hinge region in vitro. This method allows for fully automated, rapid one-pot antibody reconstitution, providing biological activity in several biochemical and functional assays. The technology presented here is suitable for automated functional and combinatorial high throughput screening of bispecific antibodies.

Abstract Background Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Results Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. Conclusion We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.

Abstract Crucial for immune and anti-inflammatory cellular responses, signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 and -13 -induced tyrosine phosphorylation by direct interaction with coactivators. The interaction of STAT6 with nuclear coactivator 1 (NCoA1) is mediated by a short region of the STAT6 transactivation domain that includes the motif LXXLL and interacts with the PAS-B domain of NCoA1. Despite the availability of an X-ray structure of the PAS-B domain/ Leu794-Gly814-STAT6 complex, the mechanistic details of this interaction are still poorly understood. Here, we determine the structure of the NCoA1257–385/STAT6783–814 complex using Nuclear Magnetic Resonance (NMR) and X-ray crystallography. The STAT6783–814 peptide binds with additional N-terminal amino acids to NCoA1257–385, compared to the STAT6794–814 peptide, explaining its higher affinity. Secondary and tertiary structures existing in the free peptide are more highly populated in the complex, suggesting binding by conformational selection.

The available magnetic field strength for high resolution NMR in persistent superconducting magnets has recently improved from 23.5 to 28 Tesla, increasing the proton resonance frequency from 1 to 1.2 GHz. For magic-angle spinning (MAS) NMR, this is expected to improve resolution, provided the sample preparation results in homogeneous broadening. We compare two-dimensional (2D) proton detected MAS NMR spectra of four membrane proteins at 950 and 1200 MHz. We find a consistent improvement in resolution that scales superlinearly with the increase in magnetic field for three of the four examples. In 3D and 4D spectra, which are now routinely acquired, this improvement indicates the ability to resolve at least 2 and 2.5 times as many signals, respectively.

Abstract Background Urinary tract infection is the most common complication after kidney transplantation. It can cause severe sepsis and transplant loss. Emergence of drug resistance among gram-negative urinary pathogens is the current challenge for urinary tract infection treatment after kidney transplantation. Methods This study analyzes the antimicrobial susceptibility of gram-negative urinary pathogens after kidney transplantation from 2009 to 2012 at the Transplant Outpatient Clinic of the University Hospital Essen, Germany. Kidney transplant patients at the University Hospital Essen receive regular follow up examinations after transplantation. Midstream urines were examined for bacteriuria at each follow up visit. Results From 2009 to 2012 15.741 urine samples were obtained from 859 patients. In 2985 (19%) samples bacterial growth was detected. The most frequently detected gram-negative bacteria were E.coli 1109 (37%), Klebsiella spp. 242 (8%) and Pseudomonas aeruginosa 136 (4.5%). Klebsiella spp. showed a significant increase of resistance to trimethoprim-sulfamethoxazole by 19% (p = 0.02), ciprofloxacin by 15% (p = 0.01) and ceftazidime by 17% (p = 0.004). E.coli and P. aeruginosa isolates presented no significant differences of antimicrobial susceptibility to the analyzed antibiotics. Conclusions Antimicrobial resistance of Klebsiella spp. increased significant to trimethoprim-sulfamethoxazole, ciprofloxacin and ceftazidime from 2009 to 2012.

The outer mitochondrial membrane translocator protein (TSPO) mediates several mitochondrial functions and binds cholesterol with a high affinity. Here the authors use solid-state NMR to show that cholesterol binding to TSPO results in allosteric changes that modulate TSPO oligomerization.

The operating limits of oscillating heat pipes (OHP) are crucial for the optimal design of cooling systems. In particular, the dryout limit is a key factor in optimizing the functionality of an OHP. As shown in previous studies, experimental approaches to determine the dryout limit lead to contradictory results. This work proposes a compact theory to predict a dryout threshold that unifies the experimental and analytical data. The theory is based on the influence of vapor quality on the flow pattern. When the vapor quality exceeds a certain limit (x = 0.006), the flow pattern changes from slug flow to annular flow and the heat transfer decreases abruptly. The results indicate a uniform threshold value, which has been validated experimentally and by the literature. With that approach, it becomes possible to design an OHP with an optimized filling ratio and, hence, substantially improve its cooling abilities.

Hydrometeorological data are commonly serially dependent and thereby deviate from the assumption of independence that underlies the Spearman rho trend test. The presence of autocorrelation will influence the significance of observed trends. Specifically, the positive autocorrelation inflates Type І errors, while it deflates the power of trend detection in some cases. To address this issue, we derive a theoretical formula and recommend an appropriate empirical formula to calculate the rho variance of dependent series. The proposed procedure of the variance correction for the Spearman rho method is capable of mitigating the effect of autocorrelation on both, Type І error and power of the test. Similar to the Block Bootstrap method, it has the advantage that it does not require an initial knowledge of the autocorrelation structure or modification of the series. In comparison, the capability of the Pre-Whitening method is sensitive to model misspecification if the series are whitened by a parametric autocorrelation model. The Trend-Free Pre-Whitening method tends to lead to an overestimation of the statistical significance of trends, similar to the original Spearman rho test. The results of the study emphasize the importance of selecting a reliable method for trend detection in serially dependent data.

Single visual object tracking from an unmanned aerial vehicle (UAV) poses fundamental challenges such as object occlusion, small-scale objects, background clutter, and abrupt camera motion. To tackle these difficulties, we propose to integrate the 3D structure of the observed scene into a detection-by-tracking algorithm. We introduce a pipeline that combines a model-free visual object tracker, a sparse 3D reconstruction, and a state estimator. The 3D reconstruction of the scene is computed with an image-based Structure-from-Motion (SfM) component that enables us to leverage a state estimator in the corresponding 3D scene during tracking. By representing the position of the target in 3D space rather than in image space, we stabilize the tracking during ego-motion and improve the handling of occlusions, background clutter, and small-scale objects. We evaluated our approach on prototypical image sequences, captured from a UAV with low-altitude oblique views. For this purpose, we adapted an existing dataset for visual object tracking and reconstructed the observed scene in 3D. The experimental results demonstrate that the proposed approach outperforms methods using plain visual cues as well as approaches leveraging image-space-based state estimations. We believe that our approach can be beneficial for trafficmonitoring, video surveillance, and navigation.

The marine beta-glucan laminarin is an abundant storage polysaccharide in microalgae. High production rates and rapid digestion by heterotrophic bacteria turn laminarin into an ideal carbon and energy source, and it is therefore a key player in the marine carbon cycle. As a main storage glucan laminarin also plays a central role in the energy metabolism of the microalgae (Percival and Ross, 1951; Myklestad, 1974; Painter, 1983). We take advantage of enzymes that digest laminarin selectively and can thereby quantify only this polysaccharide in environmental samples. These enzymes hydrolyze laminarin into glucose and oligosaccharides, which are measured with a standard reducing sugar assay to obtain the laminarin concentration. Prior to this assay, the three enzymes need to be produced via heterologous expression and purification. The assay can be used to monitor laminarin concentrations in environmental microalgae, which were concentrated from seawater by filtering, or in samples derived from algal lab cultures.

α-Synuclein (α-Syn) is intimately linked to the etiology of Parkinson's Disease, as mutations and even subtle increases in gene dosage result in early onset of the disease. However, how this protein causes neuronal dysfunction and neurodegeneration is incompletely understood. We thus examined a comprehensive range of physiological parameters in cultured rat primary neurons overexpressing α-Syn at levels causing a slowly progressive neurodegeneration. In contradiction to earlier reports from non-neuronal assay systems we demonstrate that α-Syn does not interfere with essential ion handling capacities, mitochondrial capability of ATP production or basic electro-physiological properties like resting membrane potential or the general ability to generate action potentials. α-Syn also does not activate canonical stress kinase Signaling converging on SAPK/Jun, p38 MAPK or Erk kinases. Causative for α-Syn-induced neurodegeneration are mitochondrial thiol oxidation and activation of caspases downstream of mitochondrial outer membrane permeabilization, leading to apoptosis-like cell death execution with some unusual aspects. We also aimed to elucidate neuroprotective strategies counteracting the pathophysiological processes caused by α-Syn. Neurotrophic factors, calpain inhibition and increased lysosomal protease capacity showed no protective effects against α-Syn overexpression. In contrast, the major watchdog of outer mitochondrial membrane integrity, Bcl-Xl, was capable of almost completely preventing neuron death, but did not prevent mitochondrial thiol oxidation. Importantly, independent from the quite mono-causal induction of neurotoxicity, α-Syn causes diminished excitability of neurons by external stimuli and robust impairments in endogenous neuronal network activity by decreasing the frequency of action potentials generated without external stimulation. This latter finding suggests that α-Syn can induce neuronal dysfunction independent from its induction of neurotoxicity and might serve as an explanation for functional deficits that precede neuronal cell loss in synucleopathies like Parkinson's disease or dementia with Lewy bodies.

We have designed a complete antibody-like construct where the CH1 and Cκ domains are exchanged for a pair of the CH3 domains and efficient pairing of the heavy and light variable domain is achieved using "Knobs-into-Holes" strategy. This construct, composed of only naturally occurring immunoglobulin sequences without artificial linkers, expressed at a high level in mammalian cells, however exhibited low solubility. Rational mutagenesis aimed at the amino acid residues located at the interface of the variable domains and the exchanged CH3 domains was applied to improve the biophysical properties of the molecule. The domain-exchanged construct, including variable domains of the HER2/neu specific antibody trastuzumab, was able to bind to the surface of the strongly HER2/neu positive cell line SK-BR3 4-fold weaker than trastuzumab, but could nevertheless incite a more potent response in an antibody-dependent cell cytotoxicity (ADCC) reporter assay with FcγRIIIa-overexpressing T-cells. This could be explained with a stronger binding to the FcγRIIIa. Importantly, the novel construct could mediate a specific ADCC effect with natural killer cells similar to the parental antibody.

In their application to air-cooled condensers, axial fans are often subject to the detrimental influence of ambient flow fields at their inlet or outlet. While effects have been investigated mostly under perpendicular cross-flow conditions on fans operating as part of an array in their target design point, this study aims at examining the integral influence of uniform ambient flow fields on a single axial fan over a wide operating range. For this purpose, a wind tunnel fan test rig has been designed and assessed. Multiple angles between uniform ambient flow field and fan axis are examined in their integral influence on the characteristic curve of two distinct industrial axial fans with varying inlet modifications. Increasingly with the fan flow rate, perpendicular inlet cross-flow was found to always have a detrimental influence on fan performance. The straight bladed fan reacted less sensitively than the forward skewed fan, and the adverse cross-flow influence could be reduced with an inlet guard grille and with short conical shroud extensions. Cross-flow at the fan outlet showed potential static fan pressure increases at low flow rates.

In side channel blowers, the pressure field is the result of complex, inner flow mechanisms. While there are already experimental investigations on the wall pressure distributions, little is known about the pressure in the rotating system, i.e., on the blade surface. In this work, we present an experimental setup for measuring the unsteady blade surface pressure in several positions. The acquired data will be complemented by and compared to the additionally measured wall pressure on the side channel housing. Miniature pressure sensors are integrated into the impeller. It is modified to ensure flush mounted membranes of the sensors and to avoid impacting on the flow field. A telemetric system is used for a wireless transfer of the data from the rotating system to the data recorder. As a result, we show the time-resolved pressure distribution as well as its phase-locked ensemble average. The variations of the pressure field are related to the integral pressure difference across the turbomachine and to its rotational speed. Due to the high temporal resolution of the measurement data, an exact spatial localization of crucial flow phenomena is achieved. Low integral pressure differences show a nearly linear increase of the pressure in circumferential direction, while greater integral pressure differences evolve exponentially over the azimuth. The results confirm the circulatory flow theory. Different rotational speeds elicit a comparable behavior. The stripper is a dominant source for pressure fluctuations. Its individual geometric discontinuities are correlated to the flow field. Our results provide a deeper understanding of the flow phenomena in side channel blowers and the theory of pressure generation. Although the measurements were performed for only one type of side channel blower with a double-flow configuration and open blades, the energy transfer mechanism is the same for other modifications like single flow or closed blade versions.

Self-measurement and documentation of blood-glucose are critical elements of diabetes management, particularly in regimes including insulin. In this study, we analyze the usability of iBG-STAR, the first blood glucose meter connectable to a smartphone. This technology records glucometer measurements, removing the burden of documentation from diabetic patients. This study assesses the potential for implementation of iBG-STAR in routine care. Twelve long-term diabetic patients (4 males; median age of 66.5 years) were enrolled in the study. N = 4/12 reported diabetic polyneuropathy. Reported subjective mental workload for all tasks related to iBG-STAR was on average lower than 12 points, corresponding to the verbal code 'nearly no effort needed'. A "Post Study System Usability Questionnaire", evaluated the glucometer at an average value of 2.06 (SD = 1.02) on a 7-Likert-scale (1 = 'I fully agree' to 7 = 'I completely disagree') for usability. These results represent a positive user-experience. Patients with polyneuropathy may experience physical difficulties in completing the tasks, thereby affecting usability. Technologically savvy patients (n = 6) with a positive outlook on diabetes assessed the product as a suitable tool for themselves and would recommend to other diabetic patients. The main barrier to regular use was treating physicians' inability to retrieve digitally recorded data. This barrier was due to a shortcoming in interoperability of mobile devices and medical information systems.

The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.

[This corrects the article DOI: 10.1371/journal.pbio.2000374.].

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.

Objective The study’s objective was to assess factors contributing to the use of smart devices by general practitioners (GPs) and patients in the health domain, while specifically addressing the situation in Germany, and to determine whether, and if so, how both groups differ in their perceptions of these technologies. Methods GPs and patients of resident practices in the Hannover region, Germany, were surveyed between April and June 2014. A total of 412 GPs in this region were invited by email to participate via an electronic survey, with 50 GPs actually doing so (response rate 12.1%). For surveying the patients, eight regional resident practices were visited by study personnel (once each). Every second patient arriving there (inclusion criteria: of age, fluent in German) was asked to take part (paper-based questionnaire). One hundred and seventy patients participated; 15 patients who did not give consent were excluded. Results The majority of the participating patients (68.2%, 116/170) and GPs (76%, 38/50) owned mobile devices. Of the patients, 49.9% (57/116) already made health-related use of mobile devices; 95% (36/38) of the participating GPs used them in a professional context. For patients, age ( P 0.99). For doctors, neither age ( P = 0.73), professional experience ( P > 0.99) nor gender ( P = 0.19) influenced usage rates. For patients, the primary use case was obtaining health (service)-related information. For GPs, interprofessional communication and retrieving information were in the foreground. There was little app-related interaction between both groups. Conclusions GPs and patients use smart mobile devices to serve their specific interests. However, the full potentials of mobile technologies for health purposes are not yet being taken advantage of. Doctors as well as other care providers and the patients should work together on exploring and realising the potential benefits of the technology.

Alpha-synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 – the Drosophila ortholog of Rab8a – ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS–Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.

Voiced speech is the result of a fluid-structure-acoustic interaction in larynx and vocal tract (VT). Previous studies show a strong influence of the VT on this interaction process, but are limited to individually obtained VT geometries. In order to overcome this restriction and to provide a more general VT replica, we computed a simplified, averaged VT geometry for the vowel /a/. The basis for that were MRI-derived cross-sections along the straightened VT centerline of six professional tenors. The resulting mean VT replica, as well as realistic and simplified VT replicas of each tenor were 3D-printed for experiments with silicone vocal folds that show flow-induced oscillations. Our results reveal that all replicas, including the mean VT, reproduce the characteristic formants with mean deviations of 12% when compared with the subjects’ audio recordings. The overall formant structure neither is impaired by the averaging process, nor by the simplified geometry. Nonetheless, alterations in the broadband, non-harmonic portions of the sound spectrum indicate changed aerodynamic characteristics within the simplified VT. In conclusion, our mean VT replica shows similar formant properties as found in vivo. This indicates that the mean VT geometry is suitable for further investigations of the fluid-structure-acoustic interaction during phonation.