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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.

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.

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.

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.

To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why.

Good pharmacokinetic (PK) behavior is a key prerequisite for sufficient efficacy of therapeutic monoclonal antibodies (mAbs). Fc glycosylation is a critical quality attribute (CQA) of mAbs, due to its impact on stability and effector functions. However, the effects of various IgG Fc glycoforms on antibody PK remain unclear. We used a combination of glycoengineering and glycoform-resolved PK measurements by mass spectrometry (MS) to assess glycoform effects on PK. Four differently glycoengineered mAbs, each still containing multiple glycoforms, were separately injected into rats. Rat models have been shown to be predictive of human PK. At different time points, blood was taken, from which the mAbs were purified and analyzed with a liquid chromatography-MS-based bottom-up glycoproteomics approach. This allowed us to follow changes in the glycosylation profiles of each glycoengineered mAb over time. Enzyme-linked immunosorbent assay measurements provided an absolute concentration in the form of a sum value for all glycoforms. Information from both readouts was then combined to calculate PK parameters per glycoform. Thereby, multiple glycoform kinetics were resolved within one mAb preparation. We confirmed increased clearance of high-mannose (Man5) and hybrid-type (Man5G0) glycoforms. Specifically, Man5 showed a 1.8 to 2.6-fold higher clearance than agalactosylated, complex glycans (G0F). Unexpectedly, clearance was even higher (4.7-fold) for the hybrid-type glycan Man5G0. In contrast, clearance of agalactosylated, monoantennary glycoforms (G0F-N) was only slightly increased over G0F (1.2 to 1.4-fold). Thus, monoantennary, hybrid-type and high-mannose glycoforms should be distinguished in CQA assessments. Strikingly, α2,3-linked sialylation did not affect clearance, contradicting the involvement of the asialoglycoprotein receptor in mAb clearance.

Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.

The manufacturing of advanced high-strength steels with enhanced ductility is a persistent aim of research. The ability of a material to absorb high loads while showing a high deformation behavior is a major task for many industrial fields like the mobility sector. Therefore, the material properties of advanced high-strength steels are one of the most important impact factors on the resulting cyclic fatigue behavior. To adjust advanced material properties, resulting in high tensile strengths as well as an enhanced ductility, the heat treatment process of quenching and partitioning (QP) was developed. The quenching takes place in a field between martensite start and martensite finish temperature and the subsequent partitioning is executed at slightly elevated temperatures. Regarding the sparsely investigated field of fatigue research on quenched and partitioned steels, the present work investigates the influence of a QP heat treatment on the resulting microstructure by light and scanning electron microscopy as well as on the mechanical properties such as tensile strength and resistance against fatigue regarding two different heat treatment conditions (QP1, QP2) in comparison to the cold-rolled base material of 42SiCr steel. Therefore, the microscopic analysis proved the presence of a characteristic quenched and partitioned microstructure consisting of a martensitic matrix and partial areas of retained austenite, whereas carbides were also present. Differences in the amount of retained austenite could be observed by using X-ray diffraction (XRD) for the different QP routes, which influence the mechanical properties resulting in higher tensile strength of about 2000 MPa for QP1 compared to about 1600 MPa for QP2. Furthermore, the transition for the fatigue limit was approximated by using stepwise load increase tests (LIT) and afterwards verified by constant amplitude tests (CAT) in accordance with the staircase method, whereas the QP1 condition reached the highest fatigue strength of 900 MPa. Subsequent light and scanning electron microscopy of selected fractured surfaces and runouts showed a different behavior regarding the size of the fatigue fracture area and also differences in the microstructure of these runouts.

Minor changes in the quality of biologically manufactured monoclonal antibodies (mAbs) can affect their bioactivity and efficacy. One of the most important variations concerns the N-glycosylation pattern, which directly affects an anti-tumor mechanism called antibody-dependent cell-meditated cytotoxicity (ADCC). Thus, careful engineering of mAbs is expected to enhance both protein-receptor binding and ADCC. The specific aim of this study is to evaluate the influence of terminal carbohydrates within the Fc region on the interaction with the FcγRIIIa/CD16a receptor in native and label-free conditions. The single mAb molecule comprises variants with minimal and maximal galactosylation, as well as α2,3 and α2,6-sialic acid isomers. Here, we apply native electrospray ionization mass spectrometry to determine the solution-phase antibody-receptor equilibria and by using temperature-controlled nanoelectrospray, a thermal stability of the complex is examined. Based on these, we prove that the galactosylation of a fucosylated Fc region increases the binding to CD16a 1.5-fold when compared with the non-galactosylated variant. The α2,6-sialylation has no significant effect on the binding, whereas the α2,3-sialylation decreases it 1.72-fold. In line with expectation, the galactoslylated and α2,6-sialylated mAb:CD16a complex exhibit higher thermal stability when measured in the temperature gradient from 20 to 50°C. The similar binding pattern is observed based on surface plasmon resonance analysis and immunofluorescence staining using natural killer cells. The results of our study provide new insight into N-glycosylation-based interaction of the mAb:CD16a complex.

The radula is the anatomical structure used for feeding in most species of Mollusca. Previous studies have revealed that radulae can be adapted to the food or the substrate the food lies on, but the real, in vivo forces exerted by this organ on substrates and the stresses that are transmitted by the teeth are unknown. Here, we relate physical properties of the radular teeth of Cornu aspersum (Müller. 1774 Vermium terrestrium et fluviatilium, seu animalium infusoriorum, helminthicorum, et testaceorum, non marinorum, succincta historia. Volumen alterum. Heineck & Faber, Havniæ & Lipsiæ.), a large land snail, with experiments revealing their radula scratching force. The radula motion was recorded with high-speed video, and the contact area between tooth cusps and the substrate was calculated. Forces were measured in all directions; highest forces (106.91 mN) were exerted while scratching, second highest forces while pulling the radula upwards and pressing the food against its counter bearing, the jaw, because the main ingesta disaggregation takes place during those two processes. Nanoindentation revealed that the tooth hardness and elasticity in this species are comparable to wood. The teeth are softer than some of their ingesta, but since the small contact area of the tooth cusps (227 µm2) transmits high local pressure (4698.7 bar) on the ingesta surface, harder material can still be cut or pierced with abrasion. This method measuring the forces produced by the radula during feeding could be used in further experiments on gastropods for better understanding functions and adaptations of radulae to ingesta or substrate, and hence, gastropods speciation and evolution.

Friction stir welding (FSW) is an innovative solid-state joining process, which is suitable for joining dissimilar materials with strongly differing physical and chemical properties such as aluminum and steel. Where other joining methods such as fusion welding struggle to achieve appropriate joint strengths due to the excessive formation of brittle aluminum-rich intermetallic phases (IMP), FSW joints of aluminum and steel only show small layers of IMP, thus, sufficient tensile strengths in proximity to the maximum tensile strength of the weaker aluminum base material can be reached. With the aim to optimize the mechanical and microstructural properties of such dissimilar joints for widening the field for possible industrial applications, several hybrid friction stir welding methods have been developed which include an additional energy input, whereas the ultrasound enhancement (USE-FSW) is one of the most promising. The current work was carried out on AA6061/DC04 joints which were successfully friction stir welded with and without ultrasound support, in respect to the influence of varying the ultrasound transmission side. The functionality of the USE-FSW setup could be verified by multi point laser vibrometer measurements. Additionally, a higher proportion of transversal oscillation for the transmission of power ultrasound into aluminum could be detected using a scanning vibrometer. In comparison to the conventionally friction stir welded joints the ultrasound enhancement led to an avoidance of weld defects and an increase of the steel particle volume in the stir zone. The joint produced with power ultrasound transmission via aluminum resulted in a more uniform interface.

El presente artículo analiza varios textos indigenistas de Gamaliel Churata ("El gamonal", "Tojjras" y El pez de oro) comparándolos con los tópicos de la narrativa indigenista de autores como Alcides Arguedas, Jorge Icaza, Ciro Alegría y José María Arguedas. Se observa que la narrativa de Churata plasma una especie de metaindigenismo que trasciende los indigenismos ortodoxos antes de que estos se hubieran establecido.The present article analyses several indigenist texts by Gamaliel Churata ("El gamonal", "Tojjras" y El pez de oro), comparing these with themes in indigenist fiction written by authors such as Alcides Arguedas, Jorge Icaza, Ciro Alegría and José María Arguedas. It observes that Churata’s writing gives expression to a type of meta-indigenism that transcends orthodox indigenisms before these had been established.

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure−function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

El presente artículo aclarará las múltiples referencias del autor al simbolismo, al modernismo y al vanguardismo e igualmente ubicará a Rosales y Rosales ante el trasfondo de aquellos discursos antropológicos y estéticos que a partir de 1900 empezaron a resaltar el ritmo como factor decisivo para poder definir la raza. También mediante el análisis de los poemas contenidos en Euterpologio politonal destaca la combinación de lo rítmico con lo racial-indígena, hecho notable, dado que el autor reelaboró el poemario después de que los indígenas salvadoreños fueron aniquilados casi por completo como consecuencia de La Matanza de 1932. Así, el aparente esteticismo apolítico de Rosales y Rosales asume aspectos inesperados de compromiso social que requieren una revalorización de su escritura.This article will discuss the multiple references of Vicente Rosales y Rosales, to the poetics of French symbolism, American modernisms and the international avant-garde in order to locate his position within the ethnographic and aesthetic discourses on the relation of rhythm and race that appeared since the end of the 19th century. This seems to be especially important, though rhythmic and racial elements are both present in Rosales y Rosales’ poems analyzed here. Considering the fact that Salvadorean natives were almost completely killed during the so-called La Matanza in 1932 and that Rosales y Rosales started to rewrite his Euterpologio politonal after this incident, the apparently apolitical frame of Rosales y Rosales’ aestheticism unexpectedly turns to social compromiso —a fact that requires a revalorization of his writing.

This paper applies literary criticism to a cultural phenomenon of the 1930s and re-projects such criticism to the extra-literary present in order to provide alternative routes for the understanding of a possible “nuova Bolivian culture.” Against the sociological, historical, ethnological, and/or pedagogical analysis that until now have framed the study of Warisata’s Escuela- Ayllu, this work is, first of all, an invitation to discuss Bolivian politics from the specific angle of a discipline different from that of the social sciences. This is nothing more than a first outline of possible discursive bonds between some artistic works of Warisata and Evo Morales’ education reform program. Este trabajo aplica las pautas metodológicas de la crítica literaria a un fenómeno cultural de los años 1930 y las re-proyecta al presente extraliterario con el propósito de abrir una vía alternativa para el entendimiento de las bases de una posible “nuova cultura” boliviana. Frente a los análisis sociológicos, históricos, etnológicos, pedagógicos, etc., que hasta ahora se han hecho sobre la Escuela-Ayllu de Warisata, este trabajo es ante todo una invitación a discutir la política boliviana desde el ángulo específico de una disciplina distinta a las ciencias sociales. El texto presente no es más que un primer esbozo de los posibles vínculos discursivos entre algunas obras artísticas de Warisata y el proyecto educativo-estatal de Evo Morales.

Der vorliegende Artikel untersucht die literarischen Repräsentationen von nationalsozialistischen Kriegsverbrechen in den Romanen La ofensa und Medusa des spanischen Schriftstellers Ricardo Menéndez Salmón. Ausgehend von einer Kontextualisierung der Romane im Gesamtschaffen des Autors werden intertextuelle Bezüge nicht nur zu anderen literarischen Texten, sondern auch zur Theorie herausgearbeitet. Gegenüber dem im Zusammenhang mit Holocaust-Darstellungen oft postulierten ‚Bilderverbot‘ realisiert Menéndez Salmón in Anlehnung an Didi-Huberman fiktionalisierte images malgré tout im Medium der Sprache, deren Funktion über die Darstellung von Nazi-Verbrechen hinausgeht und eine metamediale Reflexion über den Zusammenhang von Bild, Sprache und Schrecken anstößt.

Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies FcγRs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual FcγRs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different FcγRs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fcγ-receptors, with the exception of high affinity FcγRI. The FcγRII and FcγRIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the FcγR binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to FcγRIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the decreased affinity of target bound antibody towards FcRs, showing the importance of the IC-formation for the FcR- mediated effector functions.

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.

Cellular DExD/H-box RNA-helicases perform essential functions during mRNA biogenesis. The closely related human proteins UAP56 and URH49 are members of this protein family and play an essential role for cellular mRNA export by recruiting the adaptor protein REF to spliced and unspliced mRNAs. In order to gain insight into their mode of action, we aimed to characterize these RNA-helicases in more detail. Here, we demonstrate that UAP56 and URH49 exhibit an intrinsic CRM1-independent nucleocytoplasmic shuttling activity. Extensive mapping studies identified distinct regions within UAP56 or URH49 required for (i) intranuclear localization (UAP56 aa81-381) and (ii) interaction with REF (UAP56 aa51-428). Moreover, the region conferring nucleocytoplasmic shuttling activity was mapped to the C-terminus of UAP56, comprising the amino acids 195-428. Interestingly, this region coincides with a domain within Uap56p of S. pombe that has been reported to be required for both Rae1p-interaction and nucleocytoplasmic shuttling. However, in contrast to this finding we report that human UAP56 shuttles independently from Rae1. In summary, our results reveal nucleocytoplasmic shuttling as a conserved feature of yeast and human UAP56, while their export receptor seems to have diverged during evolution.