Understanding host response to infectious salmon anaemia virus in an Atlantic salmon cell line using single-cell RNA sequencing.

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.

scRNA Transcription Profile of Adult Zebrafish Podocytes Using a Novel Reporter Strain.

BACKGROUND/AIMS: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage. METHODS: We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line. RESULTS: We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes. CONCLUSION: We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair.

Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo.

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.

Organized B cell sites in cartilaginous fishes reveal the evolutionary foundation of germinal centers.

The absence of germinal centers (GCs) in cartilaginous fishes lies at odds with data showing that nurse sharks can produce robust antigen-specific responses and affinity mature their B cell repertoires. To investigate this apparent incongruity, we performed RNA sequencing on single nuclei, allowing us to characterize the cell types present in the nurse shark spleen, and RNAscope to provide in situ cellular resolution of key marker gene expression following immunization with R-phycoerythrin (PE). We tracked PE to the splenic follicles where it co-localizes with CXCR5high centrocyte-like B cells and a population of putative T follicular helper (Tfh) cells, surrounded by a peripheral ring of Ki67+ AID+ CXCR4+ centroblast-like B cells. Further, we reveal selection of mutations in B cell clones dissected from these follicles. We propose that the B cell sites identified here represent the evolutionary foundation of GCs, dating back to the jawed vertebrate ancestor.

Development and function of chicken XCR1+ conventional dendritic cells.

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 MHCIIHIGH 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.

Comparative Studies of Renin-Null Zebrafish and Mice Provide New Functional Insights.

BACKGROUND: The renin-angiotensin system is highly conserved across vertebrates, including zebrafish, which possess orthologous genes coding for renin-angiotensin system proteins, and specialized mural cells of the kidney arterioles, capable of synthesising and secreting renin. METHODS: We generated zebrafish with CRISPR-Cas9-targeted knockout of renin (ren-/-) to investigate renin function in a low blood pressure environment. We used single-cell (10×) RNA sequencing analysis to compare the transcriptome profiles of renin lineage cells from mesonephric kidneys of ren-/- with ren+/+ zebrafish and with the metanephric kidneys of Ren1c-/- and Ren1c+/+ mice. RESULTS: The ren-/- larvae exhibited delays in larval growth, glomerular fusion and appearance of a swim bladder, but were viable and withstood low salinity during early larval stages. Optogenetic ablation of renin-expressing cells, located at the anterior mesenteric artery of 3-day-old larvae, caused a loss of tone, due to diminished contractility. The ren-/- mesonephric kidney exhibited vacuolated cells in the proximal tubule, which were also observed in Ren1c-/- mouse kidney. Fluorescent reporters for renin and smooth muscle actin (Tg(ren:LifeAct-RFP; acta2:EGFP)), revealed a dramatic recruitment of renin lineage cells along the renal vasculature of adult ren-/- fish, suggesting a continued requirement for renin, in the absence of detectable angiotensin metabolites, as seen in the Ren1YFP Ren1c-/- mouse. Both phenotypes were rescued by alleles lacking the potential for glycosylation at exon 2, suggesting that glycosylation is not essential for normal physiological function. CONCLUSIONS: Phenotypic similarities and transcriptional variations between mouse and zebrafish renin knockouts suggests evolution of renin cell function with terrestrial survival.

Deciphering Mesenchymal Drivers of Human Dupuytren's Disease at Single-Cell Level.

Dupuytren's disease (DD) is a common, progressive fibroproliferative disease affecting the palmar fascia of the hands, causing fingers to irreversibly flex toward the palm with significant loss of function. Surgical treatments are limited; therefore, effective new therapies for DD are urgently required. To identify the key cellular and molecular pathways driving DD, we employed single-cell RNA sequencing, profiling the transcriptomes of 35,250 human single cells from DD, nonpathogenic fascia, and healthy dermis. We identify a DD-specific population of pathogenic PDPN+/FAP+ mesenchymal cells displaying an elevated expression of fibrillar collagens and profibrogenic genes. In silico trajectory analysis reveals resident fibroblasts to be the source of this pathogenic population. To resolve the processes governing DD progression, genes differentially expressed during fibroblast differentiation were identified, including upregulated TNFRSF12A and transcription factor SCX. Knockdown of SCX and blockade of TNFRSF12A inhibited the proliferation and altered the profibrotic gene expression of cultured human FAP+ mesenchymal cells, demonstrating a functional role for these genes in DD. The power of single-cell RNA sequencing is utilized to identify the major pathogenic mesenchymal subpopulations driving DD and the key molecular pathways regulating the DD-specific myofibroblast phenotype. Using this precision medicine approach, inhibition of TNFRSF12A has shown potential clinical utility in the treatment of DD.

PDGFRß+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny.

Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRß signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification in vivo and whether PDGF-B/PDGFRß is involved. Here, we show that PDGFRß is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRß+ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRß+ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of HSPCs in vitro.

Quantitative analysis of RNAscope staining for c-fos expression in mouse brain tissue as a measure of Neuronal Activation.

The expression of c-fos mRNA is an indirect marker of neuronal activity. RNAscope ACD Bio RNAscope (now Biotechne) is a proprietary in-situ mRNA detection technology using branched DNA amplification and z paired probes to deliver a robust and specific assay designed primarily for use on formalin fixed paraffin sections [1]. In the present study we adapted this technology to be used in frozen sections to allow quantitative analysis of c-fos gene expression in different mouse brain regions during neuropharmacology studies. The method was applied by Cosi et al. 2021 [2] and the image analysis is described here in details. •The patented RNAscope (ACD Bio) flourescent in-situ hybridisation technology designed primarily for use on formalin fixed paraffin sections was adapted to be used on frozen section from mouse brain.•We carefully controlled sample preparation and handling to maximise mRNA preservation and used the fluorescent properties of the fast Red substrate combined with fluorescent whole slide scanning and image analysis.•A customized algorithm was set up for image analysis•The method developed permitted the quantitative analysis of c-fos expression in specific brain regions from whole sections.

Quantitative Analysis of Seven New Prostate Cancer Biomarkers and the Potential Future of the 'Biomarker Laboratory'.

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.

Estrés e Inteligencia Emocional en Adultos Mayores de Ciudad Chihuahua, Chihuahua, México / Stress and Emotional Intelligence in Older Adults in Chihuahua, Chihuahua, Mexico

Resumen Objetivo: Conocer la relación que existe entre la Inteligencia Emocional y los Eventos Estresantes de la Vida en los Adultos Mayores. Materiales y Métodos: Estudio descriptivo correlacional realizado en 95 participantes de Chihuahua, Chihuahua, México. Se aplicó una Cedula de Datos Personales, la Escala de Meta-Estado de Ánimo y el Inventario de Estrés en la Vida de Adultos Mayores. Resultados: El 64.2% de los participantes pertenecieron al sexo femenino, los eventos que más experimentaron los AM en el último año fueron la presencia de una lesión o enfermedad en el Adulto Mayor (67.4%), el no poder realizar actividades que disfrutaba mucho (65.3%) y la muerte de un miembro cercano a la familia (63.8%). No se encontró relación significativa entre la Inteligencia Emocional y los Eventos Estresantes de la Vida en los Adultos Mayores (rs = -.06, p = .56), sin embargo, se realizó en análisis con cada uno de los eventos y los resultados mostraron que la Inteligencia Emocional se relacionó de forma positiva con la muerte de un miembro cercano a la familia (rs = .65, p = .05), la alteración en la salud de algún miembro en la familia (rs = .54, p = .05), el no poder realizar actividades que disfrutaba (rs = .54, p = .05). Conclusiones: La experiencia repetida de los Eventos Estresantes de la Vida pueden impactar de forma negativa la salud del Adulto Mayor, se requiere de más estudios que permitan identificar como la IE puede ayudar al Adulto Mayor a enfrentar estos eventos.

Abstract Objective: To know the relationship that exists between Emotional Intelligence and Stressful Life Events in Older Adults. Materials and Methods: Correlational descriptive study, in 95 participants from Chihuahua, Chihuahua, Mexico. A Personal Data Record, the MetaMood Scale and the Stress Inventory in the Life of Older Adults were applied. Results: 64.2% of the participants were females, the events that the elderly experienced the most in the last year were the presence of an injury or illness in the Elderly (67.4%), not being able to carry out activities that they enjoyed a lot (65.3%) and the death of a close family member (63.8%). No significant relationship was found between Emotional Intelligence and Stressful Life Events in Older Adults (rs = -.06, p = .56), however, an analysis was carried out with each one of the events and the results showed that Emotional Intelligence was positively related to the death of a close family member (rs = .65, p = .05), the alteration in the health of a family member (rs = .54, p = .05), not being able to do activities they enjoyed (rs = .54, p = .05). Conclusions: The repeated experience of Stressful Life Events can negatively impact the health of the Elderly, more studies are required to identify how Emotional Intelligence can help the Elderly to face these events.

Expression of ribosomal proteins in normal and cancerous human prostate tissue.

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.

Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

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.