Around 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species. In addition, devils are affected by two of the three known transmissible cancers in mammals. Immune checkpoint immunotherapy has transformed human medicine, but a lack of species-specific reagents has limited checkpoint immunology in most species. We developed a cut-and-paste reagent development system and used the fluorescent fusion protein system to show that immune checkpoint interactions are conserved across 160,000,000 years of evolution, CD200 is highly expressed on transmissible tumor cells, and coexpression of CD200R1 can block CD200 surface expression. The system’s versatility across species was demonstrated by fusing a fluorescent reporter to a camelid-derived nanobody that binds human programmed death ligand 1. The evolutionarily conserved pathways suggest that naturally occurring cancers in devils and other species can be used to advance our understanding of cancer and immunological tolerance.

This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live coculture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five parts: (A) In silico characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.

Recently, using a deep learning approach, the novel antibiotic halicin was discovered. We compared the antibacterial activities of two novel bactericidal antimicrobial agents, i.e., the synthetic antibacterial and antibiofilm peptide (SAAP)-148 with this antibiotic halicin. Results revealed that SAAP-148 was more effective than halicin in killing planktonic bacteria of antimicrobial-resistant (AMR) Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus, especially in biologically relevant media, such as plasma and urine, and in 3D human infection models. Surprisingly, SAAP-148 and halicin were equally effective against these bacteria residing in immature and mature biofilms. As their modes of action differ, potential favorable interactions between SAAP-148 and halicin were investigated. For some specific strains of AMR E. coli and S. aureus synergism between these agents was observed, whereas for other strains, additive interactions were noted. These favorable interactions were confirmed for AMR E. coli in a 3D human bladder infection model and AMR S. aureus in a 3D human epidermal infection model. Together, combinations of these two novel antimicrobial agents hold promise as an innovative treatment for infections not effectively treatable with current antibiotics.

CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Highlights  Key immune checkpoint receptor-ligand interactions are conserved in marsupials.  Live cell-based assays show Tasmanian devil CD28 and CTLA4 can capture CD80 and CD86 in trans from adjacent cells.  Mutation of the conserved CTLA4MYPPPY ligand binding motif to CTLA4MYPPPA reduces binding to CD80 and intercellular protein transfer.  Removal of conserved CTLA4YVKM protein recycling binding motif in CTLA4 results in bidirectional intercellular protein transfer between CTLA4 and CD80. Highly successful human immune checkpoint immunotherapies have the potential to be translated for veterinary and conservation medicine.

Highlights 33  Key immune checkpoint receptor-ligand interactions are conserved in marsupials. 34  Live cell-based assays show Tasmanian devil CD28 and CTLA4 can capture CD80 35 and CD86 in trans from adjacent cells. 36  Mutation of the conserved CTLA4MYPPPY ligand binding motif to CTLA4MYPPPA 37 reduces binding to CD80 and intercellular protein transfer. 38  Removal of conserved CTLA4YVKM protein recycling binding motif in CTLA4 results 39 in bidirectional intercellular protein transfer between CTLA4 and CD80. 40  Highly successful human immune checkpoint immunotherapies have the potential to 41 be translated for veterinary and conservation medicine. 42 43 Abstract 44 Immune checkpoint immunotherapy is a pillar of human oncology treatment with potential for 45 non-human species. The first checkpoint immunotherapy approved for human cancers targeted 46 the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 47 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 48 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of 49 the CD28-expressing cells. The wild Tasmanian devil (Sarcophilus harrisii) population has 50 declined by 77% due to transmissible cancers that evade immune defenses despite genetic 51 mismatches between the host and tumours. We used a live cell-based assay to demonstrate that 52 devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved 53 motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with 54 patterns observed in other species. These results suggest that checkpoint immunotherapies can 55 be translated to evolutionarily divergent species. 56 57 Keywords 58 immune checkpoint, wild immunology, transmissible cancer, trans-endocytosis, trogocytosis, 59 intercellular protein transfer 60 Abbreviations 61 intercellular protein transfer (IPT), devil facial tumour (DFT), interferon gamma (IFNγ), major 62 histocompatibility complex (MHC), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), 63 blue fluorescent protein (BFP) 64 65 We Amanda Patchett, Camila Espejo, Chrissie Ong, Rob Gasperini, and Ruth Pye for 353 assistance in the laboratory and general advice on this project. 354 355

Aim: A global Train-the-Trainer Program, focused on improving the communication techniques of One Health advocates, is assessed and evaluated in this study. Methods: Between November 2020 and November 2022, a Train-the-Trainer Program engaged 1.583 trainees from 98 countries between the ages of 18 and 74. Trainees practiced public speaking by teaching a lesson about One Health in mostly underserved primary school classrooms. Two surveys of trainees (n = 222 and 203) assessed the impact of the Program’s orientation session on their confidence in science communication to a novice audience. The classroom teacher survey (n = 184 teachers, representing 4.109 students) measured the trainee’s impact at the community level during and following the lesson. Results: The Program significantly improved the trainee’s confidence in communicating science (0.1 scale points) and teaching novice audiences (0.3 scale points) about One Health. Trainees cultivated student interest in One Health [median = 9 (7.3, 10)] and 90.8% of responding classroom teachers reported a desire to continue teaching about One Health after the trainee’s lesson, 99.1% noted a post-lesson increase in student interest in science, and 97.4% recommended this lesson to their colleagues. Conclusion: This study was the first to evaluate an extensive One Health-focused communication program finding the model effective for trainees and communities alike. The trainees, through public speaking, active teaching and engaging mostly primary school students, mainstreamed One Health and inspired action in communities. The classroom teachers benefited by learning about One Health and gained a desire to share this new knowledge with others in their community.

Immune checkpoint immunotherapy has revolutionized medicine, but translational success for new treatments remains low. Around 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species. Additionally, devils are affected by two of the three known transmissible cancers in mammals. Unfortunately, little is known about of immune checkpoints in devils and other non-model species, largely due to a lack of species-specific reagents. We developed a simple cut-and-paste reagent development method applicable to any vertebrate species and show that immune checkpoint interactions are conserved across 160 million years of evolution. The inhibitory checkpoint molecule CD200 is highly expressed on devil facial tumor cells. We are the first to demonstrate that co-expression of CD200R1 can block CD200 expression. The evolutionarily conserved pathways suggest that naturally occurring cancers in devils and other species can serve as models for understanding cancer and immunological tolerance.GRAPHICAL ABSTRACT