Angiotensin-Converting Enzyme 2 Expression Is Detectable in Keratinocytes, Cutaneous Appendages, and Blood Vessels by Multiplex RNA In Situ Hybridization.

OBJECTIVE: The angiotensin-converting enzyme 2 (ACE2) receptor mediates uptake of SARS-CoV-2, the virus responsible for COVID-19. Previous work analyzing publicly available bulk RNA-sequencing data sets has shown the expression of ACE2 in human keratinocytes. This finding is potentially relevant for the etiology of COVID-19-associated rashes and might also suggest a possible entry mechanism for the SARS-CoV-2 virus. In this study, the authors examined the spatial localization of ACE2 mRNA in vivo. METHODS AND RESULTS: The authors analyzed several publicly available single-cell RNA-sequencing data sets. They determined spatial localization of ACE2 mRNA using multiplex RNA in situ hybridization in human skin. CONCLUSIONS: Both analyses supported ACE2 expression in keratinocytes and skin vasculature, which could reflect a potential cutaneous entry point for SARS-CoV-2, particularly in damaged or broken skin. Moreover, ACE2 expression in vascular endothelial cells may support direct, virally mediated mechanisms in the etiology of the chilblain-like acral eruption that is seen in patients with COVID-19.

QuArray: an application for tissue array whole slide image export and signal analysis.

MOTIVATION: Tissue array (TA) staining, combined with whole slide imaging (WSI) methods facilitate discovery of biomarkers for diagnosis, prognostication and disease stratification. A key impediment in TA WSI analysis is handling missing tissue and artefacts when identifying tissue cores before quantitative, standardized downstream analysis. There is a need for an open access, user friendly, integrated analysis of the WSI generated using TAs in clinical and scientific research laboratories. RESULTS: We have developed QuArray (Quantitative Array Application) for image export and signal analysis of TAs using WSI. The application input is a WSI and a corresponding TA configuration file. QuArray identifies and exports core images and analyses chromogen staining in a simple graphical user interface. Output data is saved to file for further analysis including indexed data. AVAILABILITYAND IMPLEMENTATION: Available for download from https://github.com/c-arthurs/QuArray under an MIT licence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Contractile function of detrusor smooth muscle from children with posterior urethral valves - The role of fibrosis.

INTRODUCTION: Posterior urethral valves (PUV) is the most common cause of congenital bladder outflow obstruction with persistent lower urinary tract and renal morbidities. There is a spectrum of functional bladder disorders ranging from hypertonia to bladder underactivity, but the aetiology of these clinical conditions remains unclear. AIMS AND OBJECTIVES: We tested the hypothesis that replacement of detrusor muscle with non-muscle cells and excessive deposition of connective tissue is an important factor in bladder dysfunction with PUV. We used isolated detrusor samples from children with PUV and undergoing primary or secondary procedures in comparison to age-matched data from children with functionally normal bladders. In vitro contractile properties, as well as passive stiffness, were measured and matched to histological assessment of muscle and connective tissue. We examined if a major pathway for fibrosis was altered in PUV tissue samples. METHODS: Isometric contractions were measured in vitro in response to either stimulation of motor nerves to detrusor or exposure to cholinergic and purinergic receptor agonists. Passive mechanical stiffness was measured by rapid stretching of the tissue and recording changes to muscle tension. Histology measured the relative amounts of detrusor muscle and connective tissue. Multiplex quantitative immunofluorescence labelling using five epitope markers was designed to determine cellular pathways, in particular the Wnt-signalling pathway, responsible for any changes to excessive deposition of connective tissue. RESULTS AND DISCUSSION: PUV tissue showed equally reduced contractile function to efferent nerve stimulation or exposure to contractile agonists. Passive muscle stiffness was increased in PUV tissue samples. The smooth muscle:connective tissue ratio was also diminished and mirrored the reduction of contractile function and the increase of passive stiffness. Immunofluorescence labelling showed in PUV samples increased expression of the matrix metalloproteinase, MMP-7; as well as cyclin-D1 expression suggesting cellular remodelling. However, elements of a fibrosis pathway associated with Wnt-signalling were either reduced (ß-catenin) or unchanged (c-Myc). The accumulation of extracellular matrix, containing collagen, will contribute to the reduced contractile performance of the bladder wall. It will also increase tissue stiffness that in vivo would lead to reduced filling compliance. CONCLUSIONS: Replacement of smooth muscle with fibrosis is a major contributory factor in contractile dysfunction in the hypertonic PUV bladder. This suggests that a potential strategy to restore normal contractile and filling properties is development of the effective use of antifibrotic agents.

Equine penile squamous cell carcinoma: expression of biomarker proteins and EcPV2.

Equine penile squamous cell carcinoma (EpSCC) is a relatively common cutaneous neoplasm with a poor prognosis. In this study, we aimed to determine the protein expression and colocalisation of FRA1, c-Myc, Cyclin D1, and MMP7 in normal (NT), tumour (T), hyperplastic epidermis and/or squamous papilloma (Hyp/Pap), poorly-differentiated (PDSCC), or well-differentiated (WDSCC) EpSCC using a tissue array approach. Further objectives were to correlate protein expression to (i) levels of inflammation, using a convolutional neural network (ii) equine papillomavirus 2 (EcPV2) infection, detected using PCR amplification. We found an increase in expression of FRA1 in EpSCC compared to NT samples. c-Myc expression was higher in Hyp/Pap and WDSCC but not PDSCC whereas MMP7 was reduced in WDSCC compared with NT. There was a significant increase in the global intersection coefficient (GIC) of FRA1 with MMP7, c-Myc, and Cyclin D1 in EpSCC. Conversely, GIC for MMP7 with c-Myc was reduced in EpSCC tissue. Inflammation was positively associated with EcPV2 infection in both NT and EpSCC but not Hyp/Pap. Changes in protein expression could be correlated with EcPV2 for Cyclin D1 and c-Myc. Our results evaluate novel biomarkers of EpSCC and a putative correlation between the expression of biomarkers, EcPV2 infection and inflammation.

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.

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.