On the importance of integrating comparative anatomy and One Health perspectives in anatomy education.

Funder: UCD ResearchAs a result of many factors, including climate change, unrestricted population growth, widespread deforestation and intensive agriculture, a new pattern of diseases in humans is emerging. With increasing encroachment by human societies into wild domains, the interfaces between human and animal ecosystems are gradually eroding. Such changes have led to zoonoses, vector-borne diseases, infectious diseases and, most importantly, the emergence of antimicrobial-resistant microbial strains as challenges for human health. Now would seem to be an opportune time to revisit old concepts of health and redefine some of these in the light of emerging challenges. The One Health concept addresses some of the demands of modern medical education by providing a holistic approach to explaining diseases that result from a complex set of interactions between humans, environment and animals, rather than just an amalgamation of isolated signs and symptoms. An added advantage is that the scope of One Health concepts has now expanded to include genetic diseases due to advancements in omics technology. Inspired by such ideas, a symposium was organised as part of the 19th International Federation of Associations of Anatomists (IFAA) Congress (August 2019) to investigate the scope of One Health concepts and comparative anatomy in contemporary medical education. Speakers with expertise in both human and veterinary anatomy participated in the symposium and provided examples where these two disciplines, which have so far evolved largely independent of each other, can collaborate for mutual benefit. Finally, the speakers identified some key concepts of One Health that should be prioritised and discussed the diverse opportunities available to integrate these priorities into a broader perspective that would attempt to explain and manage diseases within the scopes of human and veterinary medicine

Cavalier King Charles Spaniels with Chiari-like malformation and Syringomyelia have increased variability of spatio-temporal gait characteristics

Abstract Background Chiari-like malformation in the Cavalier King Charles Spaniel is a herniation of the cerebellum and brainstem into or through the foramen magnum. This condition predisposes to Syringomyelia; fluid filled syrinxes within the spinal cord. The resulting pathology in spinal cord and cerebellum create neuropathic pain and changes in gait. This study aims to quantify the changes in gait for Cavalier King Charles Spaniel with Chiari-like malformation and Syringomyelia. Methods We compared Cavalier King Charles Spaniel with Chiari-like malformation with (n = 9) and without (n = 8) Syringomyelia to Border Terriers (n = 8). Two video cameras and manual tracking was used to quantify gait parameters. Results and conclusions We found a significant increase in coefficient of variation for the spatio-temporal characteristics and ipsilateral distance between paws and a wider base of support in the thoracic limbs but not in the pelvic limbs for Cavalier King Charles Spaniels compared with the border terrier

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.This research was funded by the Prostate Cancer Research Centre charity (registered UK charity no. 1156027), Grant Number AA1. A small financial contribution was also made through intra-mural funds from the Royal Veterinary College.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.016110

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders

Sensitivity of the Natriuretic Peptide/cGMP System to Hyperammonaemia in Rat C6 Glioma Cells and GPNT Brain Endothelial Cells.

C-type natriuretic peptide (CNP) is the major natriuretic peptide of the central nervous system and acts via its selective guanylyl cyclase-B (GC-B) receptor to regulate cGMP production in neurons, astrocytes and endothelial cells. CNP is implicated in the regulation of neurogenesis, axonal bifurcation, as well as learning and memory. Several neurological disorders result in toxic concentrations of ammonia (hyperammonaemia), which can adversely affect astrocyte function. However, the relationship between CNP and hyperammonaemia is poorly understood. Here, we examine the molecular and pharmacological control of CNP in rat C6 glioma cells and rat GPNT brain endothelial cells, under conditions of hyperammonaemia. Concentration-dependent inhibition of C6 glioma cell proliferation by hyperammonaemia was unaffected by CNP co-treatment. Furthermore, hyperammonaemia pre-treatment (for 1 h and 24 h) caused a significant inhibition in subsequent CNP-stimulated cGMP accumulation in both C6 and GPNT cells, whereas nitric-oxide-dependent cGMP accumulation was not affected. CNP-stimulated cGMP efflux from C6 glioma cells was significantly reduced under conditions of hyperammonaemia, potentially via a mechanism involving changed in phosphodiesterase expression. Hyperammonaemia-stimulated ROS production was unaffected by CNP but enhanced by a nitric oxide donor in C6 cells. Extracellular vesicle production from C6 cells was enhanced by hyperammonaemia, and these vesicles caused impaired CNP-stimulated cGMP signalling in GPNT cells. Collectively, these data demonstrate functional interaction between CNP signalling and hyperammonaemia in C6 glioma and GPNT cells, but the exact mechanisms remain to be established

Expression of Snail2 in long bone osteosarcomas correlates with tumour malignancy

Snail2 is a marker of malignancy in epithelial tumours; however, in sarcomas, it is not known if this protein is present. Here we examine the expression of Snail2 in one type of sarcoma, osteosarcoma, and explore its relationship to tumour grade, subtype and anatomical location in cases of long bone and cranial bone osteosarcoma. Long bone osteosarcomas typically have a much greater metastatic capability and a poorer prognosis. We find that Snail2 is expressed in the three main subtypes of long bone osteosarcoma—osteoblastic, chondroblastic and fibroblastic. Regression analysis showed that Snail 2 expression was statistically correlated with tumour grade (p = 0.014) in all of these subtypes. Snail2 was only expressed in high-grade cranial bone osteosarcomas, suggesting a link between Snail2 expression and metastasis. This is the first time Snail2 has been associated with any sarcoma, and this study shows that Snail2 may be a useful prognostic marker for this disease

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues