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Background Infectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that represents a large problem for salmonid aquaculture worldwide. Current prevention and treatment methods are only partially effective. Genetic selection and genome engineering have the potential to develop ISAV resistant salmon stocks. Both strategies can benefit from an improved understanding of the genomic regulation of ISAV pathogenesis. Here, we used single-cell RNA sequencing of an Atlantic salmon cell line to provide the first high dimensional insight into the transcriptional landscape that underpins host-virus interaction during early ISAV infection. Results Salmon head kidney (SHK-1) cells were single-cell RNA sequenced at 24, 48 and 96 h post-ISAV challenge. At 24 h post infection, cells showed expression signatures consistent with viral entry, with genes such as PI3K, FAK or JNK being upregulated relative to uninfected cells. At 48 and 96 h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. Uninfected bystander cells at 48 and 96 h also showed clear transcriptional differences, potentially suggesting paracrine signalling from infected cells. These bystander cells expressed pathways such as mRNA sensing, RNA degradation, ubiquitination or proteasome; and up-regulation of mitochondrial ribosome genes also seemed to play a role in the host response to the infection. Correlation between viral and host genes revealed novel genes potentially key for this fish-virus interaction. Conclusions This study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection and revealed host-virus interactions at the cellular level. Our results highlight various potential key genes in this host-virus interaction, which can be manipulated in future functional studies to increase the resistance of Atlantic salmon to ISAV.

Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway, suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however, their role in the hematopoietic development in vivo remains unknown. Here, we identify a subpopulation of NG2 + Runx1 + perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2 + cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs, endothelial cells and hematopoietic cells in the murine AGM. Importantly, this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental, as NG2 + Runx1 + cells were not detected in the adult bone marrow, demonstrating the existence of a specialised pericyte population in the HSC-generating niche, unique to the embryo. Hematopoietic stem cells are supported by niche cells that help balance stem cell self-renewal and differentiation. Here they show that Runx1 deletion in the embryonic perivascular HSC niche impairs hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs, endothelial cells and hematopoietic cells in the murine AGM.

Few quantifiable tissue biomarkers for the diagnosis and prognosis of prostate cancer exist. Using an unbiased, quantitative approach, this study evaluates the potential of three proteins of the 40S ribosomal protein complex as putative biomarkers of malignancy in prostate cancer. Prostate tissue arrays, constructed from 82 patient samples (245 tissue cores, stage pT3a or pT3b), were stained for antibodies against three ribosomal proteins, RPS19, RPS21 and RPS24. Semi-automated Ox-DAB signal quantification using ImageJ software revealed a significant change in expression of RPS19, RPS21 and RPS24 in malignant vs non-malignant tissue (p<0.0001). Receiver operating characteristics curves were calculated to evaluate the potential of each protein as a biomarker of malignancy in prostate cancer. Positive likelihood ratios for RPS19, RPS21 and RPS24 were calculated as 2.99, 4.21, and 2.56 respectively, indicating that the overexpression of the protein is correlated with the presence of disease. Triple-labelled, quantitative, immunofluorescence (with RPS19, RPS21 and RPS24) showed significant changes (p<0.01) in the global intersection coefficient, a measure of how often two fluorophore signals intersect, for RPS19 and RPS24 only. No change was observed in the co-localization of any other permutations of the three proteins. Our results show that RPS19, RPS21 or RPS24 are upregulated in malignant tissue and may serve as putative biomarkers for prostate cancer.

Conventional dendritic cells (cDCs) are antigen-presenting cells (APCs) that play a central role in linking innate and adaptive immunity. cDCs have been well described in a number of different mammalian species, but remain poorly characterised in the chicken. In this study, we use previously described chicken cDC specific reagents, a novel gene-edited chicken line and single-cell RNA sequencing (scRNAseq) to characterise chicken splenic cDCs. In contrast to mammals, scRNAseq analysis indicates that the chicken spleen contains a single, chemokine receptor XCR1 expressing, cDC subset. By sexual maturity the XCR1 + cDC population is the most abundant mononuclear phagocyte cell subset in the chicken spleen. scRNAseq analysis revealed substantial heterogeneity within the chicken splenic XCR1 + cDC population. Immature MHC class II (MHCII) LOW XCR1 + cDCs expressed a range of viral resistance genes. Maturation to MHCII HIGH XCR1 + cDCs was associated with reduced expression of anti-viral gene expression and increased expression of genes related to antigen presentation via the MHCII and cross-presentation pathways. To visualise and transiently ablate chicken XCR1 + cDCs in situ , we generated XCR1 -iCaspase9-RFP chickens using a CRISPR-Cas9 knockin transgenesis approach to precisely edit the XCR1 locus, replacing the XCR1 coding region with genes for a fluorescent protein (TagRFP), and inducible Caspase 9. After inducible ablation, the chicken spleen is initially repopulated by immature CD1.1 + XCR1 + cDCs. XCR1 + cDCs are abundant in the splenic red pulp, in close association with CD8 + T-cells. Knockout of XCR1 prevented this clustering of cDCs with CD8 + T-cells. Taken together these data indicate a conserved role for chicken and mammalian XCR1 + cDCs in driving CD8 + T-cells responses.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease.

The increase in metabolic dysfunction-associated steatotic liver disease (MASLD) and its progression to metabolic dysfunction-associated steatohepatitis (MASH) is a worldwide healthcare challenge. Heterogeneity between men and women in the prevalence and mechanisms of MASLD and MASH is related to differential sex hormone signalling within the liver, and declining hormone levels during aging. In this study we used biochemically characterised pluripotent stem cell derived 3D liver spheres to model the protective effects of testosterone and estrogen signalling on metabolic liver disease ‘in the dish’. We identified sex steroid-dependent changes in gene expression which were protective against metabolic dysfunction, fibrosis, and advanced cirrhosis patterns of gene expression, providing new insight into the pathogenesis of MASLD and MASH, and highlighting new druggable targets. Additionally, we highlight gene targets for which drugs already exist for future translational studies.

Tomato brown rugose fruit virus (ToBRFV), being a mechanically transmitted disease, is usually difficult to control; therefore, an effective alternative to reduce transmission and replication in the crop is by spraying with chlorine dioxide (ClO2) during routine crop management. In this research, the efficacy of chlorine dioxide (ClO2) for ToBRFV management in a greenhouse and open field was determined. The phytotoxicity of ClO2 and its effective concentration against ToBRFV in Nicotiana longiflora plants were evaluated. Subsequently, the effect of ClO2 on ToBRFV was evaluated in tomato plants grown in an open field. Finally, the effectiveness of ClO2 on plants inoculated with ToBRFV under greenhouse conditions was evaluated and the number of necrotic local lesions (NLLs) was quantified. The results revealed that ClO2 at 760 mg L−1 did not show phytotoxicity and reduced the number of NLLs in N. longiflora plants. It also decreased ToBRFV transmission and replication in field- and greenhouse-grown tomato plants, improving agronomic parameters. ClO2 reduced replication in plants inoculated with different amounts of ToBRFV inoculum in a greenhouse. N. longiflora leaves expressed lower numbers of NLLs when inoculated with ClO2-treated tomato plant extracts. Finally, the results demonstrate that ClO2 represents an effective management alternative when used by direct application to plants. To our knowledge, this is the first study where the use of an antiviral compound is carried out under field and greenhouse conditions.

Prostate cancer is the third highest cause of male mortality in the developed world, with the burden of the disease increasing dramatically with demographic change. There are significant limitations to the current diagnostic regimens and no established effective screening modality. To this end, research has discovered hundreds of potential ‘biomarkers’ that may one day be of use in screening, diagnosis or prognostication. However, the barriers to bringing biomarkers to clinical evaluation and eventually into clinical usage have yet to be realised. This is an operational challenge that requires some new thinking and development of paradigms to increase the efficiency of the laboratory process and add ‘value’ to the clinician. Value comes in various forms, whether it be a process that is seamlessly integrated into the hospital laboratory environment or one that can provide additional ‘information’ for the clinical pathologist in terms of risk profiling. We describe, herein, an efficient and tissue-conserving pipeline that uses Tissue Microarrays in a semi-automated process that could, one day, be integrated into the hospital laboratory domain, using seven putative prostate cancer biomarkers for illustration.

Phytotoxicity caused by secondary metabolites of botanical extracts is a drawback in agriculture. The objective of this study was to evaluate the phytotoxic effects of methanolic extracts of Crotalaria longirostrata and Argemone mexicana on the germination and physiological variables of tomato seedlings. The results indicated that high doses of both extracts (Clong500 and Amex500) inhibited tomato seed germination, while their mixture (Cl50 + Am50) promoted germination by 100%. At 30 days after transplanting (dat), the plant height increased by 15.4% with a high dose of C. longirostrata (Clong500) compared to the control. At 30 dat, the vigor index displayed a notable increase with Cl50 + Am50, reaching 29.5%. The root length increased with the mean dose of A. mexicana (Amex95) at 10, 20, and 30 dat (59.7%, 15.1%, and 22.4%, respectively). The chlorophyll content increased with Amex95 by 66.1% in 10 dat, 22.6% at 20 dat, and 19.6% at 30 dat. On the other hand, Amex95 had a higher nitrogen content throughout the trial. Amex95 produced the greatest increase in root dry weight by 731.5% and 209.4% at 10 and 20 dat. The foliage dry weight increased by 85.7% at 10 dat with Amex95 and up to 209.7% with Amex50 at 30 dat. The present investigation reveals the ability of the extracts to stimulate tomato growth at low and medium doses, though at high doses they exhibit allelopathic effects.