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We report on a study of laser plasma instabilities with 527 nm laser pulses in an intensity range of 0.5 × 10 13 − 1.1 × 10 15 W cm − 2 and plasma parameters entering a regime that is relevant for direct drive inertial confinement fusion. Using the kilojoule high repetition rate L4n laser at the Extreme Light Infrastructure—Beamlines, more than 1300 shots were collected, and the onset and the growth of stimulated Brioullin scattering (SBS) and stimulated Raman scattering (SRS) were studied with a high confidence level. The measured onset intensities are 0.2 × 10 14 W cm − 2 for SBS and 1.4 × 10 14 W cm − 2 for SRS. At the maximum intensity, the total fraction of backscattered energy reaches 2.5% for SBS and 0.1% for SRS. These results are of high relevance for advanced concepts for inertial fusion energy, which rely on the use of 527 nm laser light to drive the implosion of the fuel target, and in particular, they can be used as a benchmark for advanced simulations. [ABSTRACT FROM AUTHOR]

Endotracheal intubation is a medical procedure to secure a patient’s airways, which can be challenging in emergency situations due to an individual’s anatomy. Intubation blades can benefit from an adjustable design with shape memory alloys. We present a novel blade design with two degrees of freedom, continuously transforming a straight into a curved blade. Our demonstrators reached mean angular deflections up to 16∘ without exceeding outer blade temperatures of 40∘C. Mean forces of almost 24N were applicable. This approach has the potential to reduce the complexity of intubation procedures and increase the success rate, especially in time-critical emergency situations.

Type A uncertainty evaluation can significantly benefit from incorporating prior knowledge about the precision of an employed measurement device, which allows for reliable uncertainty assessments with limited observations. The Bayesian framework, employing Bayes theorem and Markov Chain Monte Carlo (MCMC), is recommended to incorporate such prior knowledge in a statistically rigorous way. While MCMC is recommended, metrologists are usually well-familiar with plain Monte Carlo sampling and previous work demonstrated the integration of similar prior knowledge into an uncertainty evaluation framework following the plain Monte Carlo sampling of JCGM 101–the Supplement 1 to the GUM. In this work, we explore the potential and limitations of such an approach, presenting classes of data distributions for informative Type A uncertainty evaluations. Our work justifies an informative extension of the JCGM 101 Type A uncertainty evaluation from a statistical perspective, providing theoretical insight and practical guidance. Explicit distributions are proposed for input quantities in Type A scenarios, aligning with Bayesian uncertainty evaluations. In addition, inherent limitations of the JCGM 101 Monte Carlo approach are discussed concerning general Bayesian inference. Metrological examples support the theoretical findings, significantly expanding the applicability of the JCGM 101 Monte Carlo technique from a Bayesian perspective.

Objectives: This case report describes a digital workflow for designing and 3D printing a guide with the dual purpose of gingivectomy and tooth preparation in the esthetic zone, to be followed by cementation of ceramic veneers under rubber dam isolation., Clinical Considerations: The patient's primary concern was to improve her smile. After clinical evaluation, our recommendation was for a minimal gingivectomy to match the gingival zenith for both central incisors, to be followed by ceramic laminate veneers. A digitally designed and 3D printed guide was used for gingivectomy and tooth preparation for the ceramic veneers. Ceramic veneers were cemented under rubber dam isolation to maximize bonding properties., Conclusions: A single guide can be digitally designed and 3D printed to perform controlled gingivectomy and minimally invasive tooth preparations. The outcome of the gingivectomy improved the gingival architecture for central incisors, and the ceramic veneers fulfilled the patient's esthetic demands., (© Operative Dentistry, 2024.)

Competing Interests: Die Übersicht über die Interessenkonflikte der Autorinnen und Autoren ist im Anhang veröffentlicht.

High levels of pre-existing antibodies are a major challenge for the application of viral vectors since they can severely limit their efficacy. To identify promising candidates among adeno-associated virus (AAV) based vectors for future gene therapies for the treatment of hereditary neuromuscular disorders (NMDs), we investigated the antibody levels in sera from patients with NMDs against 18 AAV types, including 11 AAVs with wild-type capsids, 5 AAVs with peptide-modified capsids and 2 AAVs with shuffled capsids. With regard to the wild-type capsid AAVs, the lowest binding antibody levels were detected against AAV6, AAV5, AAV12 and AAV9, whereas the highest binding antibody levels were detected against AAV10, AAV8, AAV1, and AAV2. The lowest neutralizing antibody levels against wild-type AAVs were detected against AAV12, AAV5, AAV9, AAV7, AAV8 and AAV10, and the highest neutralizing antibody levels were detected against AAV13, AAV2 and AAV3. Interestingly, the influence of peptide modifications or shuffling of AAV capsids on antibody binding and AAV neutralization seemed to depend on the parental AAV. While the sex of the serum donors had no significant impact on binding or neutralizing antibody levels, we observed a trend to higher binding antibodies in older serum donors against some AAV types and a clear positive correlation of neutralizing antibody titers with the age of the serum donors. The disease status on the other hand did not have a meaningful impact on antibody levels, with no changes in AAV neutralization. Our data indicate that several wild-type or peptide-modified AAV may be good candidates for therapeutic application due to low pre-existing antibody levels, and that the age of potential recipients rather than their health status with regard to NMDs has the biggest impact on vector applicability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Wang, Klann, Wiedtke, Sano, Fischer, Schiller, Elfert, Güttsches, Weyen, Grimm, Vorgerd and Bayer.)

The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome., (© 2024. The Author(s).)

Background and Objective: Follow-up for patients with testicular cancer should ensure early detection of relapses. Optimal schedules and minimum requirements for cross-sectional imaging are not clearly defined, and guideline recommendations differ. Our aim was to analyse the clinical impact of different imaging modalities for detection of relapse in a large prospective cohort (Swiss Austrian German Testicular Cancer Cohort Study, SAG TCCS)., Methods: Patients with seminoma or nonseminoma were prospectively enrolled between January 2014 and February 2023 after initial treatment ( n  = 1175). Follow-up according to the study schedule was individualised for histology and disease stage. Only patients who had received primary treatment were considered. We analysed the total number of imaging modalities and scans identifying relapse and the timing of relapse., Key Findings and Limitations: We analysed data for 1006 patients (64% seminoma, 36% nonseminoma); 76% had stage I disease. Active surveillance was the most frequent management strategy (65%). Recurrence occurred in 82 patients, corresponding to a 5-yr relapse-free survival rate of 90.1% (95% confidence interval 87.7-92.1%). Median follow-up for patients without relapse was 38.4 mo (interquartile range 21.6-61.0). Cross-sectional imaging of the abdomen was the most important indicator of relapse 57%, abdominal CT accounting for 46% and MRI for 11%. Marker elevation indicated relapse in 24% of cases. Chest X-ray was the least useful modality, indicating relapse in just 2% of cases., Conclusions and Clinical Implications: On the basis of findings from our prospective register, we have adapted a follow-up schedules with an emphasis on abdominal imaging and a reduction in chest X-rays. This schedule might provide additional guidance for clinicians and will be prospectively evaluated as SAG TCCS continues to enrol patients., Patient Summary: We analysed the value of different types of imaging scans for detection of relapse of testicular cancer. We used our findings to propose an optimum follow-up schedule for patients with testicular cancer., (© 2024 The Author(s).)

Background: Patients with advanced cancer often experience immense cancer pain that negatively impacts their quality of life. Interventions to address cancer-related pain are limited., Methods: We conducted a randomized trial of a digital therapeutic app (ePAL) for patients with advanced cancer receiving care in a specialty palliative care clinic at a tertiary care hospital. Patients were randomized to ePAL or usual care. ePAL included 1) active pain monitoring; 2) artificial intelligence algorithm to triage patient symptoms; and 3) patient education to address barriers to pain management. Participants were instructed to use ePAL over eight weeks. Patient-reported pain symptoms were assessed at baseline, Week-4, and Week-8 (primary endpoint) using the Brief Pain Inventory. Secondary outcomes include pain-related hospitalizations by Week-8., Results: We enrolled 112 patients who were randomly assigned to ePAL (N = 56) or usual care (N = 56). Patients utilized ePAL on average 2.1 times per week to report pain symptoms, and 47.6% reported their pain at least once per week over eight weeks. Patients randomized to ePAL reported lower pain scores at Week-4 (mean: 3.16 vs. 4.28, P = 0.010) and week-8 (mean:2.99 vs. 4.05, P = 0.017), compared to those receiving usual care. Participants randomized to ePAL were less likely to experience a pain-related hospitalization compared to those in the usual care group (7.1% vs. 23.2% P = 0.018) CONCLUSIONS: ePAL was associated with lower patient-reported pain and fewer pain-related hospitalizations compared to usual care in patients with advanced cancer. This study demonstrates the promise of digital therapeutics for improving patients' symptoms while reducing burdensome hospitalizations., (Copyright © 2024 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ohnezeit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 10 4 copies/ml (corresponding to C T values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of C T values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings., Competing Interests: Declaration of competing interest The authors declare to have no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform., Methods: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays., Results: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %., Discussion: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations., Competing Interests: Declaration of competing interest ML received speaker honoraria and related travel expenses from Roche Diagnostics and Qiagen GmbH. DN received speaker honoraria and related travel expenses from Roche Diagnostics. All other authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Cochlear implantation is a prevalent remedy for severe-to-profound hearing loss. Optimising outcomes and hearing preservation, and minimising insertion trauma, require precise electrode placement. Objective monitoring during the insertion process can provide valuable insights and enhance surgical precision. This study assesses the feasibility and performance of an impedance-based method for monitoring electrode insertion, compared to the surgeon's feedback., Methods: The study utilised the Insertion Monitoring Tool (IMT) research software, allowing for real-time measurement of impedance and evoked compound action potential (eCAP) during electrode insertion in 20 patient implantations. This enabled an impedance-based method to continuously assess the status of each electrode during the insertion process. The feasibility and performance was evaluated and compared to the surgeon's feedback approach. eCAP measurements focused merely on feasibility without searching specific responses., Results: The IMT demonstrated feasibility in measuring real-time impedances and eCAP during the insertion of the electrode array. The impedance-based method exhibited potential for accurately monitoring the insertion depth with a high success rate. However, further development is needed to improve the number of usable contacts., Conclusions: Objective monitoring with the impedance-based method shows promise as a valuable tool to enhance the precision of cochlear implant electrode insertion respecting insertion distance estimation. The IMT research software proved feasible in recording real-time impedances and eCAP during electrode insertion. While this impedance-based method exhibits high success rates, further improvements are required to optimise the number of usable contacts. This study highlights the potential of objective monitoring techniques to enhance cochlear implantation outcomes., (© 2024. The Author(s).)

Background: The recent global pandemic due to severe acute respiratory syndrome coronavirus-2 resulted in a high rate of multi-organ failure and mortality in a large patient population across the world. As such, a possible correlation between acute kidney injury (AKI) and increased mortality rate in these patients has been suggested in literature., Methods: This is a two-year retrospective study of critically ill adult patients infected with COVID-19 that were admitted to the intensive care unit (ICU) on ventilatory support. Two groups of patients were identified in this study, those who were directly admitted to the ICU or those who were initially admitted to the Medical Floor and were later transferred to the ICU due to either worsening respiratory status or change in their hemodynamic conditions. Within each group, three subgroups were created based on the status of AKI, namely, those who did not develop AKI, those who developed AKI, and those who with previous history of dialysis dependent AKI., Results: The AKI subgroup had the highest mortality rate in the ICU and Floor patients. Of note, those patients who were directly admitted to the Floor and were later transferred to the ICU for worsening conditions also experienced a higher mortality rate if they had developed AKI during their course of hospital stay., Conclusions: This study identified a statistically significant higher mortality in patients who developed AKI than those who did not develop AKI among critically ill patients., Trial Registration: Clinicaltrials.gov registration number NCT05964088. Date of registration: July 24 2023., (© 2024. The Author(s).)

Short peptide sequences consisting of two cysteine residues separated by three other amino acids display complete change from random coil to α-helical secondary structure in response to addition of Ag + ions. The folded CXXXC/Ag + complex involves formation of multinuclear Ag + species and is stable in a wide pH range from below 3 to above 8. The complex is stable through reversed-phase HPLC separation as well as towards a physiological level of chloride ions, based on far-UV circular dichroism spectroscopy. In electrospray MS under acidic conditions a peptide dimer with four Ag + ions bound was observed, and modelling based on potentiometric experiments supported this to be the dominating complex at neutral pH together with a peptide dimer with 3 Ag + and one proton at lower pH. The complex was demonstrated to work as a N-terminal nucleation site for inducing α-helicity into longer peptides. This type of silver-mediated peptide assembly and folding may be of more general use for stabilizing not only peptide folding but also for controlling oligomerization even under acidic conditions., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Purpose: To evaluate whether cooled anaesthetic eyedrops and antiseptics alleviate pain and minimise subconjunctival haemorrhage following intravitreal injection., Methods: A prospective, double-masked, randomised controlled trial of 100 participants receiving either cooled (n = 50) or room temperature (n = 50) topical anaesthetic eyedrops and antiseptics before receiving an injection of bevacizumab. Baseline tolerability was estimated using a self-reported pain sensitivity questionnaire., Results: Overall tolerability was comparable between the study group and the control group (0.75 ± 0.13 vs. 0.74 ± 0.14, respectively, p = 0.99). Subconjunctival haemorrhage incidence was similar in both groups (80% vs. 86%, respectively, p = 0.113), as was subconjunctival haemorrhage size (2.75 ± 5.51 mm 2 vs. 5.53 ± 10.72 mm 2 , respectively, p = 0.11). Sub-group analysis demonstrated that the participants taking daily ocular eyedrops who received cooled anaesthetic eyedrops and antiseptics reported less pain at 10 min and less burning sensation at 24 h compared with matched controls (0.67 ± 1.50 vs. 2.50 ± 3.03, respectively, p = 0.040 and 0.00 ± 0.00 vs. 1.44 ± 2.96, respectively, p = 0.045). Participants who received cooled eyedrops and did not use antithrombotic therapy had smaller-sized subconjunctival haemorrhages compared with matched controls (1.55 ± 1.87 mm 2 vs. 8.29 ± 14.61 mm 2 , respectively, p = 0.038). Participants with hypertension who received cooled eyedrops had smaller-sized subconjunctival haemorrhage compared with matched controls (2.33 ± 4.99 mm 2 vs. 6.89 ± 12.41 mm 2 , respectively, p = 0.045)., Conclusion: The benefit of using cooled anaesthetic eyedrops and antiseptics to alleviate pain and minimise subconjunctival haemorrhage following intravitreal injection was not proven in the general population. It may be beneficial in part for some patients, such as those who regularly use eyedrops, patients with hypertension or those not on antithrombotic therapy., (© 2023 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Malignant catarrhal fever (MCF), caused by ovine herpesvirus 2 (OvHV2; Orthoherpesviridae , Macavirus ovinegamma2 ), has sheep as natural hosts. OvHV2 is an important macavirus globally that induces fatal disease in dead-end hosts. Goats, which can be infected subclinically with OvHV2, rarely develop MCF. A 28-wk-old female goat was presented with fever and multifocal crusty skin lesions. Histologic examination of a skin biopsy suggested erythema multiforme (EM), with pyoderma and dermal vasculitis. The doe was euthanized and subjected to postmortem and histologic examination. MCF was suspected and PCR assays for macaviruses were performed, followed by immunohistochemistry (IHC) for OvHV2 latency-associated nuclear antigen (oLANA), RNA in situ hybridization for Ov2.5 mRNA, and IHC to characterize infiltrating leukocytes. The main postmortem finding was severe multifocal ulcerative dermatitis with macrophage- and T cell-mediated arteritis. The latter was also detected in kidney, spleen, heart, and intestinal wall. The PCR assay detected high loads of OvHV2 in tissues. OvHV2 oLANA and Ov2.5 mRNA were expressed within the lesions in leukocytes, endothelial cells, fibroblasts, and/or keratinocytes. Our case confirms that MCF can initially manifest clinically as a skin disease in goats and as EM with confirmed viral etiology., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Bumble bees are an important group of insects that provide essential pollination services as a consequence of their foraging behaviors. These pollination services are driven, in part, by energetic exchanges between flowering plants and individual bees. Thus, it is important to examine bumble bee energy metabolism and explore how it might be influenced by external stressors contributing to declines in global pollinator populations. Two stressors that are commonly encountered by bees are insecticides, such as the neonicotinoids, and nutritional stress, resulting from deficits in pollen and nectar availability. Our study uses a metabolomic approach to examine the effects of neonicotinoid insecticide exposure on bumble bee metabolism, both alone and in combination with nutritional stress. We hypothesized that exposure to imidacloprid disrupts bumble bee energy metabolism, leading to changes in key metabolites involved in central carbon metabolism. We tested this by exposing Bombus impatiens workers to imidacloprid according to one of three exposure paradigms designed to explore how chronic versus more acute (early or late) imidacloprid exposure influences energy metabolite levels, then also subjecting them to artificial nectar starvation. The strongest effects of imidacloprid were observed when bees also experienced nectar starvation, suggesting a combinatorial effect of neonicotinoids and nutritional stress on bumble bee energy metabolism. Overall, this study provides important insights into the mechanisms underlying the impact of neonicotinoid insecticides on pollinators, and underscores the need for further investigation into the complex interactions between environmental stressors and energy metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC 50 : 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor., (© 2024. The Author(s).)

In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.

Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.