The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications.

Liver transplantation is the only curative option for patients with end-stage liver disease. Despite improvements in surgical techniques, nonanastomotic strictures (characterized by the progressive loss of biliary tract architecture) continue to occur after liver transplantation, negatively affecting liver function and frequently leading to graft loss and retransplantation. To study the biological effects of organ preservation before liver transplantation, we generated murine models that recapitulate liver procurement and static cold storage. In these models, we explored the response of cholangiocytes and hepatocytes to cold storage, focusing on responses that affect liver regeneration, including DNA damage, apoptosis, and cellular senescence. We show that biliary senescence was induced during organ retrieval and exacerbated during static cold storage, resulting in impaired biliary regeneration. We identified decoy receptor 2 (DCR2)–dependent responses in cholangiocytes and hepatocytes, which differentially affected the outcome of those populations during cold storage. Moreover, CRISPR-mediated DCR2 knockdown in vitro increased cholangiocyte proliferation and decreased cellular senescence but had the opposite effect in hepatocytes. Using the p21 ^KO model to inhibit senescence onset, we showed that biliary tract architecture was better preserved during cold storage. Similar results were achieved by administering senolytic ABT737 to mice before procurement. Last, we perfused senolytics into discarded human donor livers and showed that biliary architecture and regenerative capacities were better preserved. Our results indicate that cholangiocytes are susceptible to senescence and identify the use of senolytics and the combination of senotherapies and machine-perfusion preservation to prevent this phenotype and reduce the incidence of biliary injury after transplantation.

Background: Skeletal damage is a challenge for laying hens because the physiological adaptations required for egg laying make them susceptible to osteoporosis. Previously, we showed that genetic factors explain 40% of the variation in end of lay bone quality and we detected a quantitative trait locus (QTL) of large effect on chicken chromosome 1. The aim of this study was to combine data from the commercial founder White Leghorn population and the F2 mapping population to fine-map this QTL and understand its function in terms of gene expression and physiology. Results: Several single nucleotide polymorphisms on chromosome 1 between 104 and 110 Mb (galGal6) had highly significant associations with tibial breaking strength. The alternative genotypes of markers of large effect that flanked the region had tibial breaking strengths of 200.4 vs. 218.1 Newton (P < 0.002) and, in a subsequent founder generation, the higher breaking strength genotype was again associated with higher breaking strength. In a subsequent generation, cortical bone density and volume were increased in individuals with the better bone genotype but with significantly reduced medullary bone quality. The effects on cortical bone density were confirmed in a further generation and was accompanied by increased mineral maturity of the cortical bone as measured by infrared spectrometry and there was evidence of better collagen cross-linking in the cortical bone. Comparing the transcriptome of the tibia from individuals with good or poor bone quality genotypes indicated four differentially-expressed genes at the locus, one gene, cystathionine beta synthase ( CBS ), having a nine-fold higher expression in the genotype for low bone quality. The mechanism was cis -acting and although there was an amino-acid difference in the CBS protein between the genotypes, there was no difference in the activity of the enzyme. Plasma homocysteine concentration, the substrate of CBS, was higher in the poor bone quality genotype. Conclusions: Validated markers that predict bone strength have been defined for selective breeding and a gene was identified that may suggest alternative ways to improve bone health in addition to genetic selection. The identification of how genetic variants affect different aspects of bone turnover shows potential for translational medicine.

Hepatocyte transplantation has shown promise for genetic diseases of the hepatocytes but to date has shown limited efficacy for non-genetic forms of severe liver injury. Limited cell engraftment and poor function of donor hepatocytes in recipient livers impacts the clinical utility of hepatocyte cell therapy. The mechanisms underpinning this are poorly understood. We explored this in a liver injury model, where predictable levels of injury and hepatocyte senescence were induced in AhCreMdm2^fl/fl mice through genetic excision of hepatocyte Mdm2. Freshly isolated mouse or human cryopreserved hepatocytes were delivered via intrasplenic injection into AhCreMdm2^fl/fl (immune competent and deficient strains) mice. Engraftment kinetics, donor cell engraftment and host liver function were assessed. Paired transcriptomic and proteomic analyses were performed on healthy vs. senescent mouse hepatocytes. We found inhibition of host hepatocyte proliferation and liver injury is a requirement for donor hepatocyte engraftment and long-term repopulation, improving liver repair and function, but excessive senescence inhibited this process, causing a decline in graft function due to transmission of senescence from host to donor cells. Paired proteomic and transcriptomic analyses of healthy vs. senescent hepatocytes reveal a unique senescent signature associated with paracrine senescence. Modification of the host niche prior to transplantation with the senotherapeutic drug ABT737 improved donor cell proliferative capacity. The host niche impacts the initial engraftment and long-term function of transplanted hepatocytes. Targeting paracrine senescence may be a way to improve donor hepatocyte function, optimise therapy and guide translation into the clinic. Hepatocyte transplantation has shown promise for genetic diseases but has limited efficacy for acute and severe liver injury. Poor engraftment and functionality have prevented large-scale clinical application. We show that host senescence provides the required non-competitive niche for donor hepatocytes to repopulate the recipient liver, but can, paradoxically, negatively impact donor function. These findings demonstrate a requirement for a clear understanding of the host niche prior to cell transfusion. This has significant implications not only for hepatocellular therapies, but also when developing and optimising any preclinical and clinical cell therapies.

The multifunctional NS1 protein of influenza A virus has roles in antagonising cellular innate immune responses and promoting viral gene expression. To better understand the interplay between these functions, we tested the effects of NS1 effector domain mutations known to affect homo-dimerisation or interactions with cellular PI3 kinase or Trim25 on NS1 ability to promote nuclear export of viral mRNAs. The NS1 dimerisation mutant W187R retained the functions of binding cellular NXF1 as well as stabilising NXF1 interaction with viral segment 7 mRNAs and promoting their nuclear export. Two PI3K-binding mutants, NS1 Y89F and Y89A still bound NXF1 but no longer promoted NXF1 interactions with segment 7 mRNA or its nuclear export. The Trim25-binding mutant NS1 E96A/E97A bound NXF1 and supported NXF1 interactions with segment 7 mRNA but no longer supported mRNA nuclear export. Analysis of WT and mutant NS1 interaction partners identified hsp70 as specifically binding to NS1 E96A/E97A. Whilst these data suggest the possibility of functional links between NS1’s effects on intracellular signalling and its role in viral mRNA nuclear export, they also indicate potential pleiotropic effects of the NS1 mutations; in the case of E96A/E97A possibly via disrupted protein folding leading to chaperone recruitment.

A state-of-the-art multi-omics approach was applied to improve our understanding of the aetio-pathogenesis of a highly prevalent, performance-limiting disorder of racehorses: mild-to-moderate equine asthma (MMEA). This is a prerequisite to improving prophylactic, management, and therapeutic options for this condition. Although a number of risk factors have been identified, options for intervention are limited. This study applied a multi-omic approach to reveal key inflammatory pathways involved in inflammatory cell recruitment to the lower airways and highlight distinct MMEA inflammatory profiles. We compared bronchoalveolar lavage fluid (BALF) cell gene and protein expression data from horses with non-inflammatory BALF cytology with those isolated from horses with neutrophilic, mastocytic, mixed neutrophilic/mastocytic, and eosinophilic/mastocytic inflammation. The analyses on transcriptomic/proteomic data derived from BALF from horses with neutrophilic cytology showed enrichment in classical inflammatory pathways, and horses with mastocytic inflammation showed enrichment in pathways involved in hypersensitivity reactions related to nonclassical inflammation potentially mimicking a Th2-immune response. The mixed eosinophilic/mastocytic group also presented with a nonclassical inflammatory profile, whereas the mixed neutrophilic/mastocytic group revealed profiles consistent with both neutrophilic inflammation and hypersensitivity. Our adopted multi-omics approach provided a holistic assessment of the immunological status of the lower airways associated with the different cytological profiles of equine asthma.