Defining the molecular signatures of Achilles tendinopathy and anterior cruciate ligament ruptures: A whole-exome sequencing approach.

Musculoskeletal soft tissue injuries are complex phenotypes with genetics being one of many proposed risk factors. Case-control association studies using the candidate gene approach have predominately been used to identify risk loci for these injuries. However, the ability to identify all risk conferring variants using this approach alone is unlikely. Therefore, this study aimed to further define the genetic profile of these injuries using an integrated omics approach involving whole exome sequencing and a customised analyses pipeline. The exomes of ten exemplar asymptomatic controls and ten exemplar cases with Achilles tendinopathy were individually sequenced using a platform that included the coverage of the untranslated regions and miRBase miRNA genes. Approximately 200 000 variants were identified in the sequenced samples. Previous research was used to guide a targeted analysis of the genes encoding the tenascin-C (TNC) glycoprotein and the α1 chain of type XXVII collagen (COL27A1) located on chromosome 9. Selection of variants within these genes were; however, not predetermined but based on a tiered filtering strategy. Four variants in TNC (rs1061494, rs1138545, rs2104772 and rs1061495) and three variants in the upstream COL27A1 gene (rs2567706, rs2241671 and rs2567705) were genotyped in larger Achilles tendinopathy and anterior cruciate ligament (ACL) rupture sample groups. The CC genotype of TNC rs1061494 (C/T) was associated with the risk of Achilles tendinopathy (p = 0.018, OR: 2.5 95% CI: 1.2-5.1). Furthermore, the AA genotype of the TNC rs2104772 (A/T) variant was significantly associated with ACL ruptures in the female subgroup (p = 0.035, OR: 2.3 95% CI: 1.1-5.5). An inferred haplotype in the TNC gene was also associated with the risk of Achilles tendinopathy. These results provide a proof of concept for the use of a customised pipeline for the exploration of a larger genomic dataset. This approach, using previous research to guide a targeted analysis of the data has generated new genetic signatures in the biology of musculoskeletal soft tissue injuries.

Concentration of lipocalin region of collagen XXVII alpha 1 in the serum of dogs with hemangiosarcoma.

BACKGROUND: Hemangiosarcoma (HSA) is a common malignancy of dogs with characteristic early, aggressive metastasis. Diagnosis of HSA is challenging because of lack of sensitive and specific diagnostic tests. HYPOTHESIS: Specific proteins that are increased in serum of dogs with HSA might represent useful biomarkers of the disease. ANIMALS: Thirty-four dogs with HSA and 42 healthy dogs from the Ontario Veterinary College Teaching Hospital. METHODS: This case-control study compared serum proteins in dogs with HSA and healthy dogs. Proteins were separated by 2-dimensional difference gel electrophoresis and identified by liquid chromatography and tandem mass spectrometry. RESULTS: Western blot analysis showed that serum collagen XXVII peptide concentration in serum of dogs with large metastatic HSA burdens (1,488, 231-3,754 DU; median, minimum-maximum); was, on average, 9.5-fold higher than in healthy dogs (156; 46-2,101 DU). While concentrations for dogs with osteosarcomas (678; 124-3,251 DU), lymphomas (423; 92-2,777 DU), carcinomas (1,022; 177-3,448 DU), and inflammatory disease were also increased, values were consistently lower than those for HSA. Receiver operating characteristic curves revealed an estimated area under the curve of 83% for HSA cases whereas areas for other neoplastic and nonneoplastic diseases were nondiscriminatory. Serum collagen XXVII peptide concentration before splenectomy (1,350; 1,156-1,929 DU) was reduced after tumor removal (529; 452-562 DU) and chemotherapy but increased in 2 dogs with tumor recurrence (511-945 DU; 493-650 DU). CONCLUSIONS AND CLINICAL IMPORTANCE: Collagen XXVII peptide might be useful for diagnosis and monitoring of advanced HSA.

Antifibrotic effects of chronic baicalein administration in a CCl4 liver fibrosis model in rats.

Baicalein was a major bioactive flavonoid derived from Radix Scutellariae in Xiao-Chai-Hu-Tang which was commonly used to treat chronic hepatitis and liver fibrosis in China. The aim of this study was to assess whether chronic baicalein administration could prevent liver fibrosis induced by carbon tetrachloride (CCl(4)) in rats and investigate its possible protective mechanism. The antifibrotic effects of baicalein were assessed directly by hepatic histology and indirectly by measuring levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hepatic hyaluronic acid, laminin and procollagen type III (PCIII) in serum, as well as hydroxyproline and matrix metalloproteinases (MMPs) in liver. In addition, we further investigated protein synthesis of platelet derived growth factor (PDGF) beta receptor which has been identified as attractive target for therapeutic intervention. CCl(4) treatment increased levels of AST, ALT, hyaluronic acid, laminin, and PCIII in serum, as well as hydroxyproline and MMPs in liver. Baicalein treatment (20, 40, or 80 mg/kg for 10 weeks) dose-dependently decreased levels of these markers. Baicalein also reduced inflammation, destruction of liver architecture, and collagen accumulation and significantly inhibited protein synthesis of PDGF-beta receptor. Together, our results suggest that chronic baicalein administration inhibits stellate cell activation and proliferation by the down-regulation of PDGF-beta receptor and prevents the development of CCl(4) induced liver fibrosis in rats.

Immunodetection of collagen types I, II, III, and IV for differentiation of liver fibrosis stages in patients with chronic HCV.

The current study is aimed at evaluating serum collagens and other serum biochemical markers as useful, non-invasive markers of hepatic fibrosis associated with chronic hepatitis C virus (HCV). Collagen types I, II, III, and IV were detected in serum using ELISA and Western blot techniques. The ELISA levels of collagen I, II, III, and IV increased significantly with the progression of fibrosis staging. Based on receiver-operating characteristic (ROC) curve analysis, the collagen type III (70 kDa) and type IV (200 kDa) were more useful than other serum bio-markers for differentiating severe fibrosis from mild fibrosis. Multivariate discriminant analysis (MDA) selected a fibrosis discriminant score (FDS) = [2.345 + Collagen III (microg/mL) x 1.923 + Collagen IV (microg/mL) x 1.544 + ALT (U/mL) x 0.005] - [albumin(g/L) x 0.046]. The FDS correctly classified 87% of the severe fibrosis patients at a cut-off score = 2.2 (i.e., more than 2.2 indicated severe fibrotic liver and less than 2.2 indicated mild fibrotic liver) with specificity of 97%. In a validation study, the FDS was applied to the second cohort of patients and the results were reproduced without significant difference. In conclusion, the developed four-parameter based FDS is useful for identifying severe liver fibrosis in patients with chronic HCV infection.