Abstract Despite the huge global health burden presented by respiratory viruses, effective broad-spectrum antiviral therapeutic options remain limited. Here we evaluated the antiviral activity of four RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir, ribavirin, favipiravir, and molnupiravir, as monotherapy or dual-drug combinations against respiratory syncytial virus (subtype A, RSVA) and human parainfluenza (serotype 3, hPIV3) using epithelial cell lines and primary human airway culture models. Remdesivir showed the greatest potency across both viruses, while ribavirin and favipiravir also demonstrated inhibition. Molnupiravir was active against RSVA but not hPIV3. Several dual-drug combinations, including remdesivir–favipiravir, remdesivir–molnupiravir and favipiravir–molnupiravir, produced marked synergy against RSVA, and more limited synergy for hPIV3. Antiviral efficacy was validated in primary airway epithelial cultures, where effective concentrations preserved epithelial integrity and attenuated viral disruption of ciliary function. Across both viruses, increasing antiviral exposure was associated with dose-dependent signature mutagenesis. Antivirals induced significantly higher RSVA mutation burden in the primary airway model. These findings highlight the therapeutic potential of RdRp inhibitor combinations for RSVA and hPIV3, provide mechanistic insight through antiviral-related mutational signatures, and demonstrate advantages of the primary human airway culture model for development of effective multi-drug regimens and broad-spectrum antiviral preparedness.

Primary ciliary dyskinesia (PCD) is a genetically and clinically diverse disorder characterised by loss of normal ciliary function leading to chronic oto-sino pulmonary disease, situs abnormalities and subfertility in men and women. There is limited evidence to support robust guidelines on the management of children and adults with PCD; however, there is a clear clinical need to establish a framework of care for the follow-up of these patients. The European Respiratory Society (ERS) has published consensus statements on diagnostic and treatment approaches in children with PCD, and the BEAT-PCD (Better Experimental Approaches to Treat PCD) network provides guidance on infection prevention and control. This is a national consensus statement to outline a set of standards for the provision of specialist care for children and adults with PCD living in England. A national PCD expert panel made up of specialists working in both paediatric and adult UK highly specialist management services, was established to create a consensus statement on the minimum standards of care for PCD. Using a modified Delphi process, consensus to a statement required at least 80% agreement within the PCD expert panel group. Patient organisation representatives were involved in reviewing the statement and have produced an accompanying layperson summary. We present a consensus statement on 15 standards covering provision of pulmonary, ear, nose and throat, and fertility care, screening for situs abnormalities and transition from paediatric to adult care services. It is targeted at clinicians and allied health professionals managing paediatric and adult patients with PCD, patient organisations and patients and their families.

PURPOSE Cetuximab (CET), targeting the epidermal growth factor receptor, is a systemic treatment option for patients with colorectal cancer. One known predictive factor for CET efficacy is the presence of CET-related rash; other putative toxicity factors include fatigue and nausea. Analysis of early CET-associated toxicities may reveal patient subpopulations that clinically benefit from long-term CET treatment. METHODS We analyzed data from CO.20 (ClinicalTrials.gov identifier: NCT00640471 ) trial arms, CET + brivanib alaninate (BRIV) (n = 376) and CET + placebo (n = 374), and CO.17 (ClinicalTrials.gov identifier: NCT00079066 ) trial arms, CET (+best supportive care [BSC]; n = 287) and BSC only (n = 285). Patients were clustered into subpopulations using KmL3D, a machine learning method, to analyze 14 joint longitudinal toxicity trajectories from weeks 0 to 8 of treatment. Landmark survival analyses were performed from 8 weeks after treatment initiation. Regression analyses assessed the relationship between subpopulations and plasma CET concentrations. Three supervised machine learning models were developed to assign patients in the CO.20-CET trial arm into subpopulations, which were then validated using CO.20-CET-BRIV and CO.17-CET trial arm data. RESULTS Joint longitudinal toxicity clustering revealed dichotomous high- and low-toxicity clusters, with all CET-containing arms showing consistent toxicity trajectories and characteristics. High-toxicity clusters were associated with male predilection, fewer metastatic sites, fewer colon-only primaries, and higher body mass indices. In CO.20 trial samples, higher toxicity clusters were associated with improved overall survival and progression-free survival outcomes (adjusted hazard ratios ranging from 2.21 to 4.36) and higher CET concentrations ( P = .003). The random forest predictive model performed the best, with an AUC of 0.981 (0.963-0.999). CONCLUSION We used an innovative machine learning approach to analyze longitudinal joint drug toxicities, demonstrating their role in predicting patient outcomes through a putative pharmacokinetic mechanism.

Primary ciliary dyskinesia (PCD) is a rare genetic respiratory disorder caused by a reduction in cilia number or cilia dysmotility. Cilia dysmotility leads to breathing difficulties, concurrent infections and severe lung damage if not treated, with no therapies currently available. Improved airway epithelial cell models that mimic the disease phenotype are required for development of new therapeutics, as current models have limited potential of self-renewal in vitro. Here, we describe a human PCD cell model created by lentiviral transduction of airway basal epithelial cells with the BMI1 gene, a regulator of senescence. We report that the cells retain their proliferation and differentiation capacity for at least 19 passages and recapitulate the disease phenotype with immotile cilia lacking DNAH5 and other outer dynein arm proteins. Characterisation of the ion transport properties of these PCD cells grown at an air-liquid interface showed lower activity of the Na+ channel ENaC and enhanced CFTR activity compared to non-PCD cells, which might be linked to ciliary immotility. Our study provides a robust PCD model for therapeutic studies, opening new avenues to investigate the molecular mechanisms of this disease.

Animal-derived components in cell culture, such as foetal bovine serum (FBS) and extracellular matrix proteins (ECM), pose ethical concerns and contribute to variability in experimental outcomes. This study explores the use of animal-free cell culture media and substrates to support the growth and differentiation of primary human bronchial epithelial cells (BECs), as well as their infection by respiratory syncytial virus (RSV). We evaluated the performance of jellyfish collagen 0 and recombinant ECM proteins as alternatives to traditional mammalian substrates. Additionally, we assessed the use of animal-free media and human serum (HS) in viral propagation using HEp2 cells. Results demonstrate that the use of animal-free medium and matrix proteins and human serum can support primary epithelial cell growth and differentiation, with high-levels of ciliation and barrier integrity. RSV propagation in animal-free medium produced an increase in viral titres, indicating the potential of these systems for anti-viral research. Transitioning to include more animal-free medium and substrates for primary cell culture and viral propagation will help improve the ethical standing of research and offer more human-relevant models for studying viral diseases in the future.

Introduction All grade 2/3 gliomas are incurable and at the time of inevitable relapse, patients have significant unmet needs with few effective treatments. This study aims to improve outcomes by molecular profiling of patients at relapse, then matching them with the best available drug based on their molecular profile, maximising the chances of patient benefit while simultaneously testing multiple novel drugs.Methods and analysis Low & Anaplastic Grade Glioma Umbrella Study of MOlecular Guided TherapieS (LUMOS-2) will be an international, phase 2, multicentre, open-label, biomarker-directed, umbrella clinical trial for recurrent isocitrate dehydrogenase mutant, histologically grade 2/3 gliomas. Investigational treatment will be assigned based on molecular profiling of contemporaneous tissue obtained at disease relapse using next-generation sequencing. LUMOS-2 will begin with three therapeutic treatment arms: paxalisib, cadonilimab and selinexor. Patient molecular profiles will be assessed by an expert, multidisciplinary Molecular Tumour Advisory Panel. Patients whose molecular profile is considered suitable for a targeted agent like paxalisib will be allocated to that arm, others will be randomised to the available arms of the trial. The primary endpoint is progression-free survival at 6 months. Secondary objectives include assessment of overall survival, response rate, safety and quality of life measures. Two additional therapeutic arms are currently in development.Ethics and dissemination Central ethics approval was obtained from the Sydney Local Health District Ethics Review Committee, Royal Prince Alfred Hospital Zone, Sydney, Australia (Approval: 2022/ETH02230). Other clinical sites will provide oversight through local governance processes, including obtaining informed consent from suitable participants. A report describing the results of the study will be submitted to international meetings and peer-reviewed journals.Trial registration number ACTRN12623000096651.

Background:Animal-derived components in cell culture, such as fetal bovine serum and extracellular matrix proteins, pose ethical concerns and contribute to variability in experimental outcomes. This study explores the use of animal-free cell culture media and substrates to support the growth and differentiation of primary human bronchial epithelial cells, as well as their infection by respiratory syncytial virus (RSV). Methods:We evaluated the performance of jellyfish collagen 0 and recombinant extracellular matrix proteins as alternatives to traditional mammalian substrates. Additionally, we assessed the use of animal-free medium and human serum (HS) in viral propagation using HEp2 cells. Results:The use of animal-free medium, matrix proteins and HS can support primary epithelial cell growth and differentiation with high levels of ciliation and barrier integrity. RSV propagation in animal-free medium produced an increase in viral titres, indicating the potential of these systems for antiviral research. Conclusion:Transitioning to include more animal-free medium and substrates for primary cell culture and viral propagation will help improve the ethical standing of research and offer more human-relevant models for studying viral diseases in the future.

BACKGROUND:Immune checkpoint inhibitors (ICIs) benefit patients across various tumor types. ICIs block cancer and T-cell interactions whereas cannabinoids may inhibit T-cell activation, reducing lysis of tumor cells. Interactions between cannabinoid use and dual ICI treatment remain unknown. METHODS:Individual patient data from 4 Canadian Cancer Trials Group (CCTG) trials of patients treated with dual ICI ± chemotherapy (n = 684) were pooled. Cochran - Mantel - Haenszel and log-rank tests (stratified by trial/treatment arms) correlated cannabinoid use with clinicopathologic characteristics, Best Overall Response (BOR)/iBOR per RECIST 1.1/iRECIST, Progression-Free Survival (PFS)/iPFS, Overall Survival (OS) and immune-related adverse events (irAEs). RESULTS:Sixty-five (9.5%) patients took cannabinoids at any time on trial, 32 (4.7%) of which were using cannabinoids at baseline. By multivariate analysis, cannabinoid use at baseline was significantly associated with improved iPFS (0.05), but not iBOR (p = 0.15), PFS (p = 0.12), OS (p = 0.35) or incidence of grade 1/2 or 3/4 irAEs (p = 0.96 and 0.65 respectively). Results were not significantly different with cannabinoid use at any time on trial. CONCLUSION:Improved iPFS with cannabinoid use in patients treated with durvalumab plus tremelimumab ± chemotherapy did not translate into OS benefits. This study supports the safe use of cannabinoids in the context of combination ICI therapy.

Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro . Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as ‘conditional reprogramming’. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium (‘EpMED’; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation. ### Competing Interest Statement SMJ has received fees for advisory board membership from BARD1 Life Sciences. He has received grant income from GRAIL Inc. and is an unpaid member of a GRAIL advisory board. SMJ has received lecture fees for academic meetings from Chiesi and AstraZeneca and his wife works for AstraZeneca. REH has received speaker fees from AstraZeneca and REH and DRP have received royalties as inventors on licensed intellectual property licensed to AstraZeneca that is unrelated to the work in this manuscript. The remaining authors declare no conflicts of interest. Medical Research Council, https://ror.org/03×94j517, MR/R015635/1, MR/W025051/1 Debra, GR000070 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982 Rosetrees Trust NIHR Great Ormond Street Hospital Biomedical Research Centre Royal Society, RG\R1\241421 Great Ormond Street Hospital Children's Charity Cancer Research UK

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Bacterial DNA methylases are a diverse group of enzymes which have been pivotal in the development of technologies with applications including genetic engineering, bacteriology, biotechnology and agriculture. This review describes bacterial DNA methylase types, the main technologies for targeted methylation or demethylation and the recent roles of these enzymes in molecular and synthetic biology. Bacterial methylases can be exocyclic or endocyclic and can exist as orphan enzymes or as a part of the restriction-modifications (R-M) systems. As a group, they display a rich diversity of sequence-specificity. Additional technologies for targeting methylation involve using fusion proteins combining a methylase and a DNA-binding protein (DNBP) such as a zinc-finger (ZF), transcription activator-like effector (TALE) or CRISPR/dCas9. Bacterial methylases have contributed significantly to the creation of novel DNA assembly techniques, to the improvement of bacterial transformation and to crop plant engineering. Future studies to define the characteristics of more bacterial methylases have potential to identify new tools of value in synthetic and molecular biology and with widespread applications. • Bacterial methylases can be used to direct methylation to specific sequences in target DNA • DNA methylation using bacterial methylases has been applied to improve DNA assembly and to increase the efficiency of bacterial transformation • Site-selective methylation using bacterial methylases can alter plant gene expression and phenotype

Background. The current landscape of organ donation and transplantation (ODT) registries is not well established. This narrative review sought to identify and characterize the coverage, structure, and data capture of ODT registries globally. Methods. We conducted a literature search using Ovid Medline and web searches to identify ODT registries from 2000 to 2023. A list of ODT registries was compiled based on publications of registry design, studies, and reports. Extracted data elements included operational features of registries and the types of donor and recipient data captured. Results. We identified 129 registries encompassing patients from all continents except Antarctica. Most registries were active, received funding from government or professional societies, were national in scope, included both adult and pediatric patients, and reported patient-level data. Registries included kidney (n = 99), pancreas (n = 32), liver (n = 44), heart (n = 35), lung (n = 30), intestine (n = 15), and islet cell (n = 5) transplants. Most registries captured donor data (including living versus deceased) and recipient features (including demographics, cause of organ failure, and posttransplant outcomes) but there was underreporting of other domains (eg, donor comorbidities, deceased donor referral rates, waitlist statistics). Conclusions. This review highlights existing ODT registries globally and serves as a call for increased visibility and transparency in data management and reporting practices. We propose that standards for ODT registries, a common data model, and technical platforms for collaboration, will enable a high-functioning global ODT system responsive to the needs of transplant candidates, recipients, and donors.

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the Ear, Nose and Throat department of a tertiary pediatric hospital linked to the United Kingdom’s National EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wildtype LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof-of-concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Aim Primary ciliary dyskinesia (PCD) is a genetic disorder affecting motile cilia. Most cases are inherited recessively, due to variants in more than 50 genes that result in abnormal or absent motile cilia. This leads to chronic upper and lower airway disease, sub-fertility and laterality defects in some cases. Given overlapping clinical features and genetic heterogeneity, diagnosis can be difficult and often occurs late. Of those tested, an estimated 30% of genetically screened PCD patients still lack a molecular diagnosis. Here, we aimed to identify how readily a genetic diagnosis could be made in a clinically diagnosed population using whole genome sequencing (WGS) to facilitate identification of pathogenic variants in known genes as well as identify novel PCD candidate genes.Maethods WGS was used to screen for variants causing PCD in 8 clinically diagnosed PCD patients, sequenced as trios where parental samples were available.Results Seven of the eight cases (87.5%) had homozygous or biallelic variants in DNAH5 , DNAAF4 or DNAH11 that were classified as pathogenic or likely pathogenic. Three of the variants were deletions, ranging from 3kb to 13kb, for which WGS identified precise breakpoints, permitting confirmation by Sanger sequencing. WGS yielded a high genetic diagnostic rate from this clinically diagnosed population, in part through detection of structural variants as well as identification of a de novo variant in a novel PCD gene TUBB4B .Conclusion A molecular diagnosis allows for appropriate clinical management for cases and their families, including prediction of phenotypic features correlated to genotype. Here, WGS uplifted genetic diagnosis in cases of clinically diagnosed PCD by identifying structural variants and novel modes of inheritance in new candidate genes. Our study suggests that WGS could be a powerful part of the PCD diagnostic toolkit to increase the current molecular diagnostic yield from 70%. It provides important new insight into our understanding of fundamental biology of motile cilia as well as of variation in the non-coding genome in PCD.Summary Whole genome sequencing (WGS) yielded a high genetic diagnostic rate (100%) in eight Scottish patients with clinically diagnosed primary ciliary dyskinesia (PCD) by detection of large structural variants, homology modelling and identification of a novel disease gene with a dominant mode of inheritance. Prioritised WGS may facilitate early genetic diagnosis in PCD.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe Scottish Genomes Partnership is funded by the Chief Scientist Office of the Scottish Government Health Directorates [SGP\_1] and the MRC Whole Genome Sequencing for Health and Wealth Initiative (MC\_PC\_15080). We acknowledge support from the MRC (PM: MC\_UU\_00007\_14, MR\_Y015002\_1); an MRC Career Development Award (MR\_M02122X\_1) and Lister Prize Fellowship to JAM; an NHS Research Scotland fellowship to SU; and an NRS/R+D fellowship from the NHS Lothian R&D office to DU. ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Signed and informed consent was obtained from the affected individual as well as relatives through approved protocols. Sample IDs were assigned and the key known only those within the research group. The study was approved by the London-West London and Gene Therapy Advisory Committee Research Ethics Committee (REC number 11/LO/0883).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors.

PURPOSE Treatment options for refractory advanced gastric and esophagogastric junction cancer (AGOC) are limited. Regorafenib, an oral multikinase inhibitor, prolonged progression-free survival (PFS) versus placebo in the INTEGRATE I phase II trial. INTEGRATE IIa was designed to examine whether regorafenib improved overall survival (OS). METHODS A double-blind placebo-controlled phase III trial compared regorafenib and best supportive care (BSC) versus placebo and BSC for participants with confirmed evaluable metastatic/advanced AGOC who failed ≥two prior therapies on a 2:1 random assignment, stratified by tumor location, geographic region (Asia v rest of world), and prior vascular endothelial growth factor inhibitors. The primary end point was OS. Treatment efficacy on OS was first tested in the pooled INTEGRATE I + INTEGRATE IIa cohort and, if significant, then in the INTEGRATE IIa cohort. Secondary end points were PFS, objective response rate, safety, and quality of life (QoL). RESULTS INTEGRATE IIa enrolled 251 participants: 157 from Asia and 94 from rest of world and 169 received regorafenib and 82 received placebo. No significant heterogeneity was observed between INTEGRATE I and INTEGRATE IIa studies on OS. Pooled OS analysis hazard ratio (HR) was 0.70 (95% CI, 0.56 to 0.87; P = .001; 361 events). INTEGRATE IIa alone OS HR was 0.68 (95% CI, 0.52 to 0.90; P = .006; 238 events), the median OS was 4.5 months versus 4.0 months, and 12-month survival rates were 19% and 6%, for regorafenib versus placebo, respectively. After a preplanned adjustment for multiplicity, there were no statistically significant differences across regions or other prespecified subgroups. Regorafenib improved PFS (HR, 0.53 [95% CI, 0.40 to 0.70]; P < .0001) and delayed deterioration in global QoL (HR, 0.68 [95% CI, 0.52 to 0.89]; P = .0043). The toxicity profile was consistent with that of previous reports. CONCLUSION Regorafenib improves survival compared with placebo in refractory AGOC.