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Climate change-driven alterations in Arctic environments can influence habitat availability, species distributions and interactions, and the breeding, foraging, and health of marine mammals. Phocine distemper virus (PDV), which has caused extensive mortality in Atlantic seals, was confirmed in sea otters in the North Pacific Ocean in 2004, raising the question of whether reductions in sea ice could increase contact between Arctic and sub-Arctic marine mammals and lead to viral transmission across the Arctic Ocean. Using data on PDV exposure and infection and animal movement in sympatric seal, sea lion, and sea otter species sampled in the North Pacific Ocean from 2001-2016, we investigated the timing of PDV introduction, risk factors associated with PDV emergence, and patterns of transmission following introduction. We identified widespread exposure to and infection with PDV across the North Pacific Ocean beginning in 2003 with a second peak of PDV exposure and infection in 2009; viral transmission across sympatric marine mammal species; and association of PDV exposure and infection with reductions in Arctic sea ice extent. Peaks of PDV exposure and infection following 2003 may reflect additional viral introductions among the diverse marine mammals in the North Pacific Ocean linked to change in Arctic sea ice extent.

Background: This ORATOR sub-study evaluated swallowing physiology in patients treated with transoral robotic surgery (TORS) versus radiotherapy (RT) for early-stage oropharynx cancer., Methods: Swallowing physiology was evaluated using videofluoroscopy and outcomes were compared across treatment arms and correlated with MDADI scores., Results: Of the 68 patients in the ORATOR trial, 21 participated in this sub-study (30.8%), including 15 RT Arm and six TORS Arm patients. Swallowing profiles were not significantly different between the arms. MBSImP pharyngeal scores for RT Arm versus TORS Arm patients were 4.8 (±2.1) versus 4.3 (±1.5) at baseline, 6.2 (±1.2) versus 9.6 (±4.8) at 6 months and 5.9 (±1.8) versus 8.0 (±4.7) at 12 months. MBSImP pharyngeal scores demonstrated weak associations with several MDADI subscales and PAS scores., Conclusions: To best describe swallowing outcomes in studies of RT and/or surgery, instrumental swallowing assessments should be strongly considered in addition to quality of life measures., (© 2024 The Author(s). Head & Neck published by Wiley Periodicals LLC.)

The IPD-MHC Database project (http://www.ebi.ac.uk/ipd/mhc/) serves as a comprehensive and expertly curated repository for major histocompatibility complex (MHC) sequences from non-human species, providing the necessary infrastructure and tools to study the function and evolution of this highly polymorphic genomic region. In its latest version, the IPD-MHC database has expanded both in content and in the tools for data visualization and comparison. The database now hosts over 18 000 MHC alleles from 125 species, organized into eleven taxonomic groups, all manually curated and named by the Comparative MHC Nomenclature Committee. A cetacean section has recently been included, offering researchers valuable data to study the immune system of whales, dolphins, and porpoises, as well establishing the official nomenclature platform for the Cetacea Leukocyte Antigens (CeLA). In response to user demand and reflecting broader trends in bioinformatics and immunogenetics, IPD-MHC now includes the predicted tertiary structure of over 8000 alleles and allows comparison and visualisation of allele variation within and between species at single residue resolution. These latest developments maintain the critically important link between official nomenclature of curated alleles and the ability to analyse this complex polymorphism using the most up to date methods within a single repository., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)'s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.

Radiotherapy (RT) and transoral robotic surgery (TORS) are both curative-intent treatment options for oropharyngeal squamous cell carcinoma (OPSCC). Herein, we report the final outcomes of the ORATOR trial comparing these modalities, 5 years after enrollment completion. We randomly assigned 68 patients with T1-2N0-2 OPSCC to RT (with chemotherapy if node-positive) versus TORS plus neck dissection (± adjuvant RT/chemoradiation). The primary end point was swallowing quality of life (QOL) assessed with the MD Anderson Dysphagia Inventory (MDADI). Secondary end points included overall and progression-free survival (OS, PFS), adverse events (AEs), and other QOL metrics. The primary end point has been previously reported (Nichols 2019). In this report, the median follow-up was 5.1 years (IQR, 5.0-5.3 years). MDADI total scores converged by 5 years and were not significantly different across the follow-up period ( P = .11). EORTC QLQ-C30 and H&N35 scores demonstrated differing profiles, including worse dry mouth in the RT arm ( P = .032) and worse pain in the TORS arm ( P = .002). Grade 2-5 AE rates did not differ between arms (91% [n = 31] v 97% [n = 33] respectively, P = .61), with more neutropenia and hearing loss in the RT arm, and more dysphagia and other pain in the TORS arm based on grades 2-5 (all P < .05). There were no differences in OS or PFS. In conclusion, toxicity and QOL profiles differ in some domains between RT and TORS, but oncologic outcomes were excellent in both arms. Choice of treatment should remain a shared decision between the patient and their providers.

Cattle express three subclasses of IgG antibody - IgG1, IgG2 and IgG3. Unlike IgG1 and IgG2, IgG3 was described relatively recently and the role of this subclass in immunity is unknown. Using recombinant bovine IgG1, IgG2 and IgG3 monoclonal antibodies (mAbs), we demonstrated that only one of the commercially available anti-bovine IgG mAbs tested was able to recognize IgG3, and no mAb exclusively bound IgG3. Here, we evaluated a small ankyrin repeat protein, called Ankyron™ AZS40101, that was generated to bind to bovine IgG3. Ankyron™ AZS40101 bound specifically to IgG3 with minimal reactivity to IgG1 and IgG2. Ankyron™ AZS40101 was shown to be useful in ELISA assays as either a capture or detection reagent. Utilisation of Ankyron™ AZS40101 alongside IgG1 and IgG2 specific mAbs to detect antigen-specific IgG subclasses in the serum of cattle sequentially vaccinated with heterologous foot-and-mouth disease virus capsid antigens revealed a low-level antigen-specific IgG3 response, in addition to IgG1 and IgG2 responses. Assessment of the total IgG1, IgG2 and IgG3 levels in healthy cattle plasma samples showed that IgG3 was measurable at a mean concentration of 0.19 mg/mL, although this was significantly lower than those of IgG1 (mean 3.28 mg/mL) and IgG2 (mean 3.41 mg/mL). Thus, Ankyron™ AZS40101 is a new reagent that provides utility for measurement of bovine IgG3 responses to infection and vaccination., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The inbred Babraham pig serves as a valuable biomedical model for research due to its high level of homozygosity, including in the major histocompatibility complex (MHC) loci and likely other important immune-related gene complexes, which are generally highly diverse in outbred populations. As the ability to control for this diversity using inbred organisms is of great utility, we sought to improve this resource by generating a long-read whole genome assembly and transcriptome atlas of a Babraham pig. The genome was de novo assembled using PacBio long reads and error-corrected using Illumina short reads. Assembled contigs were then mapped to the porcine reference assembly, Sscrofa11.1, to generate chromosome-level scaffolds. The resulting TPI&#95;Babraham&#95;pig&#95;v1 assembly is nearly as contiguous as Sscrofa11.1 with a contig N50 of 34.95 Mb and contig L50 of 23. The remaining sequence gaps are generally the result of poor assembly across large and highly repetitive regions such as the centromeres and tandemly duplicated gene families, including immune-related gene complexes, that often vary in gene content between haplotypes. We also further confirm homozygosity across the Babraham MHC and characterize the allele content and tissue expression of several other immune-related gene complexes, including the antibody and T cell receptor loci, the natural killer complex, and the leukocyte receptor complex. The Babraham pig genome assembly provides an alternate highly contiguous porcine genome assembly as a resource for the livestock genomics community. The assembly will also aid biomedical and veterinary research that utilizes this animal model such as when controlling for genetic variation is critical., (© 2024. The Author(s).)

Objectives: The study aimed to i) understand the experiences of newly qualified physiotherapists from racially minoritised backgrounds in a large hospital physiotherapy department and ii) co-create and implement inclusive strategies to affect work culture., Design: The project used an action research design with co-creation principles and was in 3 phases. Phase 1 explored the experiences of inclusion for newly qualified racially minoritised staff, using focus groups and transcripts analysed thematically. The themes identified were used to co-create strategies for inclusion that were implemented in Phase 2 and evaluated Phase 3., Setting: A large urban teaching hospital physiotherapy department in the UK., Participants: Participants were invited to join focus groups for newly qualified racially minoritised staff, newly qualified white staff, or senior staff., Results: Phase 1 findings are reported here, while phases 2 and 3 are reported elsewhere. Seventeen newly qualified physiotherapists (eight racially minoritised, nine white colleagues) and ten senior physiotherapists participated in seven focus groups. Thematic analysis identified four themes; 1) Fear spectrum: all colleagues struggled to find a common ground for discussing race, 2) Race as an additional burden for racially minoritised staff in belonging in the department, 3) Contradiction between the organisation culture and impact on racially minoritised staff, and 4) Consistent work practices give an illusion of inclusion., Conclusions: The findings reflect previous studies that racially minoritised physiotherapists experience additional burdens not fully understood by their white peers. There are implications for the department which are currently being enacted and evaluated and will be reported elsewhere. CONTRIBUTION OF THE PAPER., Competing Interests: Conflict of Interest Jacky Jones, Gareth Jones and Trupti Bhandari are all senior leaders in the physiotherapy department where the research takes place. Charmaine Turner and Trupti Bhandari identify from a racially minoritised group while all other researchers are white. John Hammond is also Associate Editor of the journal. There are no other conflicts of interest., (Copyright © 2024 Chartered Society of Physiotherapy. Published by Elsevier Ltd. All rights reserved.)

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form β-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Å resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing., (open access.)

Background: The advent and continual improvement of high-throughput sequencing technologies has made immunoglobulin repertoire sequencing accessible and informative regardless of study species. However, to fully map dynamic changes in polyclonal responses precise framework and complementarity determining region annotation of rearranging genes is pivotal. Most sequence annotation tools are designed primarily for use with human and mouse antibody sequences which use databases with fixed species lists, applying very specific assumptions which select against unique structural characteristics. For this reason, data agnostic tools able to learn from presented data can be very useful with new species or with novel datasets., Results: We have developed IgMAT, which utilises a reduced amino acid alphabet, that incorporates multiple HMM alignments into a single consensus to automatically annotate immunoglobulin sequences from most organisms. Additionally, the software allows the incorporation of user defined databases to better represent the species and/or antibody class of interest. To demonstrate the accuracy and utility of IgMAT, we present analysis of sequences extracted from structural data and immunoglobulin sequence datasets from several different species., Conclusions: IgMAT is fully open-sourced and freely available on GitHub ( https://github.com/TPI-Immunogenetics/igmat ) for download under GPLv3 license. It can be used as a CLI application or as a python module to be integrated in custom scripts., (© 2023. The Author(s).)

Cattle possess three IgG subclasses. However, the key immune functions, including complement and NK cell activation, and enhancement of phagocytosis, are not fully described for bovine IgG1, 2 and 3. We produced chimeric monoclonal antibodies (mAbs) consisting of a defined variable region linked to the constant regions of bovine IgG1, 2 and 3, and expressed His-tagged soluble recombinant bovine Fc gamma receptors (FcγRs) IA (CD64), IIA (CD32A), III (CD16) and Fcγ2R. Functional assays using bovinized mAbs were developed. IgG1 and IgG3, but not IgG2, activated complement-dependent cytotoxicity. Only IgG1 could activate cattle NK cells to mobilize CD107a after antigen crosslinking, a surrogate assay for antibody-dependent cell cytotoxicity. Both IgG1 and IgG2 could trigger monocyte-derived macrophages to phagocytose fluorescently labelled antigen-expressing target cells. IgG3 induced only weak antibody-dependent cellular phagocytosis (ADCP). By contrast, monocytes only exhibited strong ADCP when triggered by IgG2. IgG1 bound most strongly to recombinant FcγRs IA, IIA and III, with weaker binding by IgG3 and none by IgG2, which bound exclusively to Fcγ2R. Immune complexes containing IgG1, 2 and 3 bound differentially to leukocyte subsets, with IgG2 binding strongly to neutrophils and monocytes and all subclasses binding platelets. Differential expression of the FcγRs on leukocyte subsets was demonstrated by surface staining and/or RT-qPCR of sorted cells, e.g., Fcγ2R mRNA was expressed in monocytes/macrophages, neutrophils, and platelets, potentially explaining their strong interactions with IgG2, and FcγRIII was expressed on NK cells, presumably mediating IgG1-dependent NK cell activation. These data reveal differences in bovine IgG subclass functionality, which do not correspond to those described in humans, mice or pigs, which is relevant to the study of these IgG subclasses in vaccine and therapeutic antibody development., 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 © 2023 Noble, Paudyal, Schwartz, Mwangi, Munir, Tchilian, Hammond and Graham.)

Background: There is a paucity of evidence on the risk of donor-recipient transmission of the SARS-CoV-2 in solid organ transplant recipients. Initial impressions suggest non-lung solid organs may be safely transplanted from SARS-CoV-2-positive donors without risk of viral transmission., Methods: We reviewed clinical results of transplants in which SARS-CoV-2-negative recipients received non-lung solid organs from SARS-CoV-2-positive donors at a single transplant center. No prisoners were used in this study, and participants were neither coerced nor paid. The manuscript was created in compliance with the Helsinki Congress and the Declaration of Istanbul., Results: Between June 2021 and January 2023, we transplanted 26 solid organs, including 13 kidneys, 8 livers, 3 hearts, and 1 simultaneous heart and kidney, from 23 SARS-CoV-2-positive donors into 25 SARS-CoV-2 negative recipients. Two of the recipients had a positive SARS-CoV-2 real-time polymerase chain reaction after transplantation, but otherwise, patients had no SARS-CoV-2-related complications, and all patients to date are alive with excellent allograft function., Conclusion: Transplantation of non-lung solid organs from SARS-CoV-2-positive donors into uninfected recipients can be safely performed without adverse effects from SARS-CoV-2., Competing Interests: Declaration of Competing Interest All the authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cattle, sheep, and goats are the only species outside primates known to have an expanded and diversified family of killer immunoglobulin-like receptors (KIR). Primate KIR are expressed on the surface of NK and T cells and bind MHC-I to control activation. However, the surface expression, ligands and function of bovid KIR remain unknown. Cattle botaKIR2DL1 is the only functional KIR of the same DL-lineage as the expanded KIR in primates and we examined if leukocyte expression patterns were consistent with human. We raised a specific mouse anti-botaKIR2DL1 monoclonal antibody and assessed its utility in flow cytometry, ELISA, and western blot. Unlike primates, cattle DL-lineage KIR (botaKIR2DL1) is present on B cells and monocytes in addition to T cells and low-level expression on NK cells. Expression decreases after in vitro PBMC stimulation with IL-2. This suggests that botaKIR2DL1 has different functions, and potentially ligands, compared to primate KIR., 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.)

Mutations in the proline-rich domain (PRD) of annexin A11 are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, and generate abundant neuronal A11 inclusions by an unknown mechanism. Here, we demonstrate that recombinant A11-PRD and its ALS-associated variants form liquidlike condensates that transform into β-sheet-rich amyloid fibrils. Surprisingly, these fibrils dissolved in the presence of S100A6, an A11 binding partner overexpressed in ALS. The ALS variants of A11-PRD showed longer fibrillization half-times and slower dissolution, even though their binding affinities for S100A6 were not significantly affected. These findings indicate a slower fibril-to-monomer exchange for these ALS variants, resulting in a decreased level of S100A6-mediated fibril dissolution. These ALS-A11 variants are thus more likely to remain aggregated despite their slower fibrillization.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (T Naïve ), central memory (T CM ), effector memory (T EM ) and terminal effector (T TE ) T cells. Activated cattle T cells (T AV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle T Naïve , T AV , T CM , T EM , T TE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

This 8-color panel has been optimized to distinguish between functionally distinct subsets of cattle B cells in both fresh and cryopreserved peripheral blood mononuclear cells (PBMCs). Existing characterized antibodies against cell surface molecules (immunoglobulin light chain (S-Ig[L]), CD20, CD21, CD40, CD71, and CD138) enabled the discrimination of 24 unique populations within the B-cell population. This allows the identification of five putative functionally distinct B-cell subsets critical to infection and vaccination responses: (1) naïve B cells (B Naïve ), (2) regulatory B cells (B Reg ), (3) memory B cells (B Mem ), (4) plasmablasts (PB), and (5) plasma cells (PC). Although CD3 and CD8α can be included as an additional dump channel, it does not significantly improve the panel's ability to separate "classical" B cells. This panel will promote better characterization and tracking of B-cell responses in cattle as well as other bovid species as the reagents are likely to cross react., (© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Background: In the United Kingdom (UK), whilst nurse educators are responsible for developing compassion through providing high quality education, there is limited literature exploring how their lived experience of compassion is interpreted in nurse education., Objectives: To explore how nurse educators make meaning of compassion through their lived experiences in the UK., Design: Hermeneutic phenomenology., Setting: A UK school of nursing., Participants: Purposeful sampling was used to recruit twelve nurse educators., Method: Semi-structured interviews were used to explore participant experiences of compassion. Data analysis involved crafting stories and was interpreted by applying Heideggerian and Gadamerian philosophical notions to surface meanings of everyday experiences., Findings: The phenomenological themes identified nurse educators interpreted compassion through Being-with is Care; settling their colliding worlds of nursing practice and nurse education, and balancing Kairos or 'felt' time with negotiated time for compassion in nurse education., Conclusion: This research demonstrates that nurse educators share genuine concern for Being-with others that is interpreted as compassion. However, the emotional aspect of compassion is avoided in their professional practice as a means of protecting students and their own feelings of vulnerability. There are colliding views in understanding compassion. Emotional intelligence is identified as necessary to grasp 'felt' moments or negotiate a time to Be-with that is interpreted as compassion. Training and support is necessary for nurse educators to understand and develop compassion in their professional practices., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies., 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., (Copyright © 2022 Paudyal, Mwangi, Rijal, Schwartz, Noble, Shaw, Sealy, Bonnet-Di Placido, Graham, Townsend, Hammond and Tchilian.)

Importance: The optimal approach for treatment deescalation in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas (OPSCCs) is unknown., Objective: To assess a primary radiotherapy (RT) approach vs a primary transoral surgical (TOS) approach in treatment deescalation for HPV-related OPSCC., Design, Setting, and Participants: This international, multicenter, open-label parallel-group phase 2 randomized clinical trial was conducted at 9 tertiary academic cancer centers in Canada and Australia and enrolled patients with T1-T2N0-2 p16-positive OPSCC between February 13, 2018, and November 17, 2020. Patients had up to 3 years of follow-up., Interventions: Primary RT (consisting of 60 Gy of RT with concurrent weekly cisplatin in node-positive patients) vs TOS and neck dissection (ND) (with adjuvant reduced-dose RT depending on pathologic findings)., Main Outcomes and Measures: The primary end point was overall survival (OS) compared with a historical control. Secondary end points included progression-free survival (PFS), quality of life, and toxic effects., Results: Overall, 61 patients were randomized (30 [49.2%] in the RT arm and 31 [50.8%] in the TOS and ND arm; median [IQR] age, 61.9 [57.2-67.9] years; 8 women [13.6%] and 51 men [86.4%]; 31 [50.8%] never smoked). The trial began in February 2018, and accrual was halted in November 2020 because of excessive toxic effects in the TOS and ND arm. Median follow-up was 17 months (IQR, 15-20 months). For the OS end point, there were 3 death events, all in the TOS and ND arm, including the 2 treatment-related deaths (0.7 and 4.3 months after randomization, respectively) and 1 of myocardial infarction at 8.5 months. There were 4 events for the PFS end point, also all in the TOS and ND arm, which included the 3 mortality events and 1 local recurrence. Thus, the OS and PFS data remained immature. Grade 2 to 5 toxic effects occurred in 20 patients (67%) in the RT arm and 22 (71%) in the TOS and ND arm. Mean (SD) MD Anderson Dysphagia Inventory scores at 1 year were similar between arms (85.7 [15.6] and 84.7 [14.5], respectively)., Conclusions and Relevance: In this randomized clinical trial, TOS was associated with an unacceptable risk of grade 5 toxic effects, but patients in both trial arms achieved good swallowing outcomes at 1 year. Long-term follow-up is required to assess OS and PFS outcomes., Trial Registration: Clinicaltrials.gov Identifier: NCT03210103.

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo . Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo . Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.

Purpose: The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has risen rapidly, because of an epidemic of human papillomavirus infection. The optimal management of early-stage OPSCC with surgery or radiation continues to be a clinical controversy. Long-term randomized data comparing these paradigms are lacking., Methods: We randomly assigned patients with T1-T2, N0-2 (≤ 4 cm) OPSCC to radiotherapy (RT) (with chemotherapy if N1-2) versus transoral robotic surgery plus neck dissection (TORS + ND) (with or without adjuvant therapy). The primary end point was swallowing quality of life (QOL) at 1-year using the MD Anderson Dysphagia Inventory. Secondary end points included adverse events, other QOL outcomes, overall survival, and progression-free survival. All analyses were intention-to-treat. Herein, we present long-term outcomes from the trial., Results: Sixty-eight patients were randomly assigned (n = 34 per arm) between August 10, 2012, and June 9, 2017. Median follow-up was 45 months. Longitudinal MD Anderson Dysphagia Inventory analyses demonstrated statistical superiority of RT arm over time ( P = .049), although the differences beyond 1 year were of smaller magnitude than at the 1-year timepoint (year 2: 86.0 ± 13.5 in the RT arm v 84.8 ± 12.5 in the TORS + ND arm, P = .74; year 3: 88.9 ± 11.3 v 83.3 ± 13.9, P = .12). These differences did not meet the threshold to qualify as a clinically meaningful change at any timepoint. Certain differences in QOL concerns including more pain and dental concerns in the TORS + ND arm seen at 1 year resolved at 2 and 3 years; however, TORS patients started to use more nutritional supplements at 3 years ( P = .015). Dry mouth scores were higher in RT patients over time ( P = .041)., Conclusion: On longitudinal analysis, the swallowing QOL difference between primary RT and TORS + ND approaches persists but decreases over time. Patients with OPSCC should be informed about the pros and cons of both treatment options (ClinicalTrials.gov identifier: NCT01590355)., Competing Interests: Anthony C. NicholsResearch Funding: Novartis Canada Pharmaceuticals Inc (Inst) Eric BertheletResearch Funding: Eisai Eric WinquistConsulting or Advisory Role: Merck, Bayer, Eisai, Amgen, Roche, IpsenResearch Funding: Roche/Genentech (Inst), Merck (Inst), Pfizer (Inst), Eisai (Inst), Ayala Pharmaceuticals (Inst) David A. PalmaThis author is a member of the Journal of Clinical Oncology Editorial Board. Journal policy recused the author from having any role in the peer review of this manuscript.Employment: London Health Sciences Centre (LHSC)Patents, Royalties, Other Intellectual Property: US patent: a method for analyzing a three-dimensional computed tomography image, US patent application no. 61/896349 (not licensed, no commercial or financial impact)No other potential conflicts of interest were reported.

The major histocompatibility complex (MHC) class I region of cattle is both highly polymorphic and, unlike many species, highly variable in gene content between haplotypes. Cattle MHC class I alleles were historically grouped by sequence similarity in the more conserved 3' end of the coding sequence to form phylogenetic allele groups. This has formed the basis of current cattle MHC class I nomenclature. We presently describe and compare five fully assembled MHC class I haplotypes using the latest cattle and yak genome assemblies. Of the five previously described "pseudogenes" in the cattle MHC class I region, Pseudogene 3 is putatively functional in all haplotypes and Pseudogene 6 and Pseudogene 7 are putatively functional in some haplotypes. This was reinforced by evidence of transcription. Based on full gene sequences as well as 3' coding sequence, we identified distinct subgroups of BoLA-3 and BoLA-6 that represent distinct genetic loci. We further examined allele-specific expression using transcriptomic data revealing that certain alleles are consistently weakly expressed compared to others. These observations will help to inform further studies into how MHC class I region variability influences T cell and natural killer cell functions in cattle., (© 2021 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies (mAbs). In this study we demonstrated that prophylactic intravenous administration of 15 mg/kg of porcine mAb pb18, against the K160-163 site of the hemagglutinin, significantly reduced lung pathology and nasal virus shedding and eliminated virus from the lung of pigs following H1N1pdm09 challenge. When given at 1 mg/kg, pb18 significantly reduced lung pathology and lung and BAL virus loads, but not nasal shedding. Similarly, when pb18 was given in combination with pb27, which recognized the K130 site, at 1 mg/kg each, lung virus load and pathology were reduced, although without an apparent additive or synergistic effect. No evidence for mAb driven virus evolution was detected. These data indicate that intravenous administration of high doses was required to reduce nasal virus shedding, although this was inconsistent and seldom complete. In contrast, the effect on lung pathology and lung virus load is consistent and is also seen at a one log lower dose, strongly indicating that a lower dose might be sufficient to reduce severity of disease, but for prevention of transmission other measures would be needed., 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., (Copyright © 2021 Paudyal, McNee, Rijal, Carr, Nunez, McCauley, Daniels, Townsend, Hammond and Tchilian.)

γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2 + /SWC5 - , CD2 - /SWC5 + , and CD2 - /SWC5 - ). Both CD2 - subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2 + cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2 + and CD2 - γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The addition of cattle health and immunity traits to genomic selection indices holds promise to increase individual animal longevity and productivity, and decrease economic losses from disease. However, highly variable genomic loci that contain multiple immune-related genes were poorly assembled in the first iterations of the cattle reference genome assembly and underrepresented during the development of most commercial genotyping platforms. As a consequence, there is a paucity of genetic markers within these loci that may track haplotypes related to disease susceptibility. By using hierarchical assembly of bacterial artificial chromosome inserts spanning 3 of these immune-related gene regions, we were able to assemble multiple full-length haplotypes of the major histocompatibility complex, the leukocyte receptor complex, and the natural killer cell complex. Using these new assemblies and the recently released ARS-UCD1.2 reference, we aligned whole-genome shotgun reads from 125 sequenced Holstein bulls to discover candidate variants for genetic marker development. We selected 124 SNPs, using heuristic and statistical models to develop a custom genotyping panel. In a proof-of-principle study, we used this custom panel to genotype 1,797 Holstein cows exposed to bovine tuberculosis (bTB) that were the subject of a previous GWAS study using the Illumina BovineHD array. Although we did not identify any significant association of bTB phenotypes with these new genetic markers, 2 markers exhibited substantial effects on bTB phenotypic prediction. The models and parameters trained in this study serve as a guide for future marker discovery surveys particularly in previously unassembled regions of the cattle genome., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly., 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 © 2021 Elsevier Ltd. All rights reserved.)

Blended learning has received much interest in higher education as a way to increase learning efficiency and effectiveness. By combining face-to-face teaching with technology-enhanced learning through online resources, students can manage their own learning. Blended methods are of particular interest in professional degree programs such as veterinary medicine in which students need the flexibility to undertake intra- and extramural activities to develop the range of competencies required to achieve professional qualification. Yet how veterinary students engage with blended learning activities and whether they perceive the approach as beneficial is unclear. We evaluated blended learning through review of student feedback on a 4-week clinical module in a veterinary degree program. The module combined face-to-face sessions with online resources. Feedback was collected by means of a structured online questionnaire at the end of the module and log data collected as part of a routine teaching audit. The features of blended learning that support and detract from students' learning experience were explored using quantitative and qualitative methods. Students perceived a benefit from aspects of face-to-face teaching and technology-enhanced learning resources. Face-to-face teaching was appreciated for practical activities, whereas online resources were considered effective for facilitating module organization and allowing flexible access to learning materials. The blended approach was particularly appreciated for clinical skills in which students valued a combination of visual resources and practical activities. Although we identified several limitations with online resources that need to be addressed when constructing blended courses, blended learning shows potential to enhance student-led learning in clinical courses.

As hospitals across the world realized their surge capacity would not be enough to care for patients with coronavirus disease-2019 (COVID-19) infection, an urgent need to open field hospitals prevailed. In this article the authors describe the implementation process of opening a Boston field hospital including the development of a culture unique to this crisis and the local community needs. Through first-person accounts, readers will learn (1) about Boston Hope, (2) how leaders managed and collaborated, (3) how the close proximity of the care environment impacted decision-making and management style, and (4) the characteristics of leaders under pressure as observed by the team., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations., Competing Interests: The authors have declared that no competing interests exist.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike-ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife., Competing Interests: The authors have declared that no competing interests exist.

The principle of monoclonality with regard to bacterial infections was considered immutable prior to 30 years ago. This view, espoused by Koch for acute infections, has proven inadequate regarding chronic infections as persistence requires multiple forms of heterogeneity among the bacterial population. This understanding of bacterial plurality emerged from a synthesis of what-were-then novel technologies in molecular biology and imaging science. These technologies demonstrated that bacteria have complex life cycles, polymicrobial ecologies, and evolve in situ via the horizontal exchange of genic characters. Thus, there is an ongoing generation of diversity during infection that results in far more highly complex microbial communities than previously envisioned. This perspective is based on the fundamental tenet that the bacteria within an infecting population display genotypic diversity, including gene possession differences, which result from horizontal gene transfer mechanisms including transformation, conjugation, and transduction. This understanding is embodied in the concepts of the supragenome/pan-genome and the distributed genome hypothesis (DGH). These paradigms have fostered multiple researches in diverse areas of bacterial ecology including host-bacterial interactions covering the gamut of symbiotic relationships including mutualism, commensalism, and parasitism. With regard to the human host, within each of these symbiotic relationships all bacterial species possess attributes that contribute to colonization and persistence; those species/strains that are pathogenic also encode traits for invasion and metastases. Herein we provide an update on our understanding of bacterial plurality and discuss potential applications in diagnostics, therapeutics, and vaccinology based on perspectives provided by the DGH with regard to the evolution of pathogenicity., (© 2020 The Author(s).)

Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.

Using the best animal models to study immune responses against specific pathogens or vaccines can dramatically accelerate our understanding. Veterinary species are well studied, particularly livestock, to reduce their disease burden. They have also proven to be powerful models, especially for zoonotic pathogens and novel vaccination strategies. A prerequisite for any model selection is having the right quality and range of species-specific immunological reagents. To help promote the widest possible use of veterinary species, an open access website (https://www.immunologicaltoolbox.co.uk) has been created as a central community annotated hub for veterinary immunological reagents. The website is also the portal into services offered by the UK Immunological Toolbox project that includes antibody generation, sequencing and recombinant expression. The funding for this effort is linked into sustainable sources, but ultimate success relies on community engagement to continually increase the quality and quantity of information. It is hoped that as more users and reagent owners engage, it will become an essential resource for researchers, veterinarians and clinicians alike by removing barriers that prevent the use of the most informative animal models., (© 2020 The Authors. Immunology published by John Wiley & Sons Ltd.)

It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16-19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives., (Copyright © 2020 Entrican, Lunney, Wattegedera, Mwangi, Hope and Hammond.)