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Abstract In natural populations, vital rates such as survival and reproduction are influenced by a complex interplay of abiotic conditions (e.g., environment), density dependence, and individual factors (e.g., phenotypic traits). Studies at the extremes of species distributions, particularly high elevations, offer unique insights due to the intensified effects of abiotic stressors, which can amplify both direct and indirect effects on vital rates. In this study, we focus on a high‐elevation population of the common toad (Bufo bufo) located near the upper limit of its elevational range in the Swiss Alps. This setting provides a critical context for examining how extreme abiotic conditions interact with density dependence and individual factors to influence life history traits. Utilizing 28 years of capture–mark–recapture data and individual body size measurements from nearly 2500 toads, we applied in a Bayesian statistical framework a Cormack–Jolly–Seber model for estimating male survival probabilities, and a multistate model for assessing female survival and breeding probabilities, alongside sex‐specific growth curves. Our analysis indicates that survival probabilities are significantly impacted by interactions between abiotic conditions such as the active season length and temperature at emergence from hibernation, density dependence, and individual phenotypic traits such as body size. The breeding patterns of females showed a biennial cycle, with temperature at hibernation emergence influencing the likelihood of skipping breeding events and density affecting the resumption of breeding. These results highlight the role of abiotic conditions and density in shaping physiological and reproductive strategies in a high‐stress ecological niche. Moreover, we uncovered indications of indirect effects, where both abiotic conditions and density potentially affect asymptotic growth and thus survival, mediated through changes in body size. Our findings illustrate the complex dynamics at play in high‐elevation populations and the importance of long‐term, individual‐based data in studying these processes. This study underscores the value of integrating multiple sources of variation to understand population dynamics comprehensively, particularly in understudied, extreme environments where traditional ecological models may not fully capture the nuanced interdependencies of natural systems.

Introduction Postoperative imaging after neurosurgical interventions is usually performed in the first 72 hours after surgery to provide an accurate assessment of postoperative resection status. Patient frequently report that early postoperative examination after craniotomy for tumour and vascular procedures is associated with distress, exertion, nausea and pain. Delayed postoperative imaging (between 36 and 72 hours postoperatively) may have an advantage regarding psychological and physical stress compared with early imaging. The goal of this study is to evaluate and determine the optimal time frame for postoperative imaging with MRI and CT in terms of medical and neuroradiological implications and patient’s subjective stress level.Methods and analysis Data will be prospectively collected from all patients aged 18–80 years who receive postoperative MRI or CT imaging following a craniotomy for resection of a cerebral tumour (benign and malignant) or vascular surgery. Participants have to complete questionnaires containing visual analogue scores (VAS) for headache and nausea, Body Part Discomfort score and a single question addressing subjective preference of timing of postoperative imaging after craniotomy. The primary endpoint of the study is the difference in subjective stress due to imaging studies after craniotomy, measured just before and after postoperative MRI or CT with the above-mentioned instruments. Subjective stress is defined as a combination of the scores VAS pain, VAS nausea and 0.5* Body Part Discomfort core.This study determines whether proper timing of postoperative imaging can improve patient satisfaction and reduce pain, stress and discomfort caused by postoperative imaging. Factors causing additional postoperative stress are likely responsible for delayed recovery of neurosurgical patients.Ethics and dissemination The institutional review board (Kantonale Ethikkommission Zürich) approved this study on 4 August 2020 under case number BASEC 2020–01590. The authors are planning to publish the data of this study in a peer-reviewed paper. After database closure, the data will be exported to the local data repository (Zurich Open Repository and Archive) of the University of Zurich. The sponsor (LR) and the project leader (MR.G) will make the final decision on the publication of the results. The data that support the findings of this study are available on request from the corresponding author LT. The data are not publicly available due to privacy/ethical restrictions.Trial registration number NCT05112575; ClinicalTrials.gov.

ABSTRACT Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell–cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion.IMPORTANCEHuman cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.

Adeno-associated viruses (AAVs) are useful vehicles for gene therapy because of their stability, low immunogenicity. and non-pathogenicity. However, disparity in AAV sample preparations (e.g., in capsid composition, DNA packaging, and impurities) gives rise to product heterogeneity, with possibly undesired effects on gene delivery. Ideally, AAV production should be with full control of AAV structure and genetic payload. Therefore, robust, efficient, and low material consuming methods are essential to characterize AAVs. Here, we use two emerging single-molecule techniques, mass photometry and Orbitrap-based charge-detection mass spectrometry, and show how they may efficiently and accurately characterize AAVs. We were able to resolve heterogeneous pools of particles, evaluating AAVs from two different serotypes (AAV8 and AAV2), produced by three independent production platforms, either lacking a genome or packed with a transgene. Together our data confirm that the different AAV production methods result in rather different and diverse AAV particle distributions. Especially for the packed AAVs, frequently additional subspecies were observed, next to the expected packed genome, mostly resulting from under- or overpackaging of genome material and/or residual empty particles. This work further establishes that both these single-particle techniques may become valuable tools in characterizing AAVs before they are used in gene therapy.

Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups.

A relatively low clearance is one of the prominent favorable features of immunoglobulin G1-based therapeutic monoclonal antibodies (mAbs). Various studies have observed differential clearance of mAb glycoforms, including oligomannose glycoforms, which are considered a critical quality attribute because they show higher clearance than complex type glycoforms. Glycoform clearance, however, has not previously been studied after subcutaneous injection or in a porcine model system. Here, we performed glycoform-resolved pharmacokinetic (PK) analysis of two mAbs in Göttingen minipigs. We found glycoform effects on clearance to be largely the same for subcutaneous and intravenous injection and in line with observations in other species. Oligomannose glycoforms were cleared up to 25% faster and monoantennary glycoforms up to 8% faster than agalactosylated complex glycoforms. Sialylated glycoforms were cleared at approximately the same rate as fully galactosylated glycoforms. Importantly, we report here an impact of galactosylation on the PK of a mAb for the first time. Whether increased galactosylation led to slower or faster clearance seemed to depend on the overall glycosylation profile. When clearance of galactosylated glycoforms was slower, the mAb showed higher galactosylation in serum at maximum concentration after subcutaneous injection compared to both intravenous injection and the injected material. Whether this higher galactosylation after subcutaneous injection has consequences for therapeutic efficacy remains to be investigated. In conclusion, preferential clearance of antibody glycoforms can be simulated in the minipig model with intravenous as well as subcutaneous injections. Furthermore, we observed a glycoform bias in the absorption from skin into circulation after subcutaneous injection based on galactosylation.Abbreviations: AUC - area under the curve; CL/F - apparent clearance as a function of bioavailability following SC administration; Cmax - maximum serum concentration; CQA critical quality attribute; FcγR - Fc gamma receptor; IgG - immunoglobulin G; IV - intravenous; LC-MS - liquid chromatography - mass spectrometry; mAb - therapeutic monoclonal antibody; PK - pharmacokinetics; SC - subcutaneous; TMDD - target-mediated drug disposition

Informatische Bildung wird auch im Primarbereich immer relevanter. Um in diesem Bereich Unterricht adäquat planen und durchführen zu können, bedarf es qualifizierter Lehrkräfte, die über fachinhaltliche und fachdidaktische informatische Kompetenzen verfügen. Allerdings sind sowohl die Ausbildung angehender Sachunterrichtslehrkräfte als auch das Fortbildungsangebot in allen Phasen der Lehrer*innenbildung noch unzureichend. Dieses Ausbildungsdesiderats nimmt sich das im Folgenden beschriebene Forschungsseminar an, das von Fachdidaktiker*innen aus der Didaktik der Informatik und der Didaktik des Sachunterrichts gemeinsam entwickelt wurde. Zentrales Prinzip des Forschungsseminars war das Forschende Lernen, das zur Professionalisierung der (angehenden) Sachunterrichtslehrkräfte zum Themengebiet „Informatische Bildung“ durchgehend Anwendung fand. In diesem Beitrag werden die Ziele, das Konzept und dessen Ausgestaltung sowie die Durchführung und die Evaluation des Forschungsseminars mit 14 Seminarteilnehmenden dargestellt.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell–cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike’s fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike’s fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2.