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Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

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Introduction: The anterior cruciate ligament (ACL) is susceptible to degeneration, resulting in joint pain, reduced mobility, and osteoarthritis development. There is currently a paucity of knowledge on how anterior cruciate ligament degeneration and disease leads to osteoarthritis. Small non-coding RNAs (sncRNAs), such as microRNAs and small nucleolar RNA (snoRNA), have diverse roles, including regulation of gene expression. Methods: We profiled the sncRNAs of diseased osteoarthritic ACLs to provide novel insights into osteoarthritis development. Small RNA sequencing from the ACLs of non- or end-stage human osteoarthritic knee joints was performed. Significantly differentially expressed sncRNAs were defined, and bioinformatics analysis was undertaken. Results and Discussion: A total of 184 sncRNAs were differentially expressed: 68 small nucleolar RNAs, 26 small nuclear RNAs (snRNAs), and 90 microRNAs. We identified both novel and recognized (miR-206, -365, and -29b and -29c) osteoarthritis-related microRNAs and other sncRNAs (including SNORD72, SNORD113, and SNORD114). Significant pathway enrichment of differentially expressed miRNAs includes differentiation of the muscle, inflammation, proliferation of chondrocytes, and fibrosis. Putative mRNAs of the microRNA target genes were associated with the canonical pathways "hepatic fibrosis signaling" and "osteoarthritis." The establishing sncRNA signatures of ACL disease during osteoarthritis could serve as novel biomarkers and potential therapeutic targets in ACL degeneration and osteoarthritis development.

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Proteoglycans (PGs) are minor extracellular matrix proteins, and their contributions to the mechanobiology of complex ligaments such as the cranial cruciate ligament (CCL) have not been determined to date. The CCLs are highly susceptible to injuries, and their extracellular matrix comprises higher PGs content than the other major knee ligaments. Hence these characteristics make CCLs an ideal specimen to use as a model in this study. This study addressed the hypothesis that PGs play a vital role in CCL mechanobiology by determining the biomechanical behaviour at low strain rates before and after altering PGs content. For the first time, this study qualitatively investigated the contribution of PGs to key viscoelastic characteristics, including strain rate dependency, hysteresis, creep and stress relaxation, in canine CCLs. Femur-CCL-tibia specimens (n = 6 pairs) were harvested from canine knee joints and categorised into a control group, where PGs were not depleted, and a treated group, where PGs were depleted. Specimens were preconditioned and cyclically loaded to 9.9 N at 0.1, 1 and 10%/min strain rates, followed by creep and stress relaxation tests. Low tensile loads were applied to focus on the toe-region of the stress-strain curves where the non-collagenous extracellular matrix components take significant effect. Biochemical assays were performed on the CCLs to determine PGs and water content. The PG content was ∼19% less in the treated group than in the control group. The qualitative study showed that the stress-strain curves in the treated group were strain rate dependent, similar to the control group. The CCLs in the treated group showed stiffer characteristics than the control group. Hysteresis, creep characteristics (creep strain, creep rate and creep compliance), and stress relaxation values were reduced in the treated group compared to the control group. This study suggests that altering PGs content changes the microstructural organisation of the CCLs, including water molecule contents which can lead to changes in CCL viscoelasticity. The change in mechanical properties of the CCLs may predispose to injury and lead to knee joint osteoarthritis. Future studies should focus on quantitatively identifying the effect of PG on the mechanics of intact knee ligaments across broader demography.

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Cruciate ligaments (CL) of the knee joint are injured following trauma or aging. MicroRNAs (miRs) are potential therapeutic targets in musculoskeletal disorders, but there is little known about the role of miRs and their expression ligaments during aging. This study aimed to (1) identify if mice with normal physical activity, wild-stock house mice are an appropriate model to study age-related changes in the knee joint and (2) investigate the expression of miRs in aging murine cruciate ligaments. Knee joints were collected from 6 and 24 months old C57BL/6 and wild-stock house mice (Mus musculus domesticus) for ligament and cartilage (OARSI) histological analysis. Expression of miR targets in CLs was determined in 6-, 12-, 24-, and 30-month-old wild-stock house mice, followed by the analysis of predicted mRNA target genes and Ingenuity Pathway Analysis. Higher CL and knee OARSI histological scores were found in 24-month-old wild-stock house mice compared with 6- and 24-month-old C57BL/6 and 6-month-old wild-stock house mice (p < 0.05). miR-29a and miR-34a were upregulated in 30-month-old wild-stock house mice in comparison with 6-, 12-, and 24-month-old wild-stock house mice (p < 0.05). Ingenuity Pathway Analysis on miR-29a and 34a targets was associated with inflammation through interleukins, TGFß and Notch genes, and p53 signaling. Collagen type I alpha 1 chain (COL1A1) correlated negatively with both miR-29a (r = -0.35) and miR-34a (r = -0.33). The findings of this study support wild-stock house mice as an appropriate aging model for the murine knee joint. This study also indicated that miR-29a and miR-34a may be potential regulators of COL1A1 gene expression in murine CLs.

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Injuries to the intra-articular anterior cruciate ligament (ACL) and the extra-articular medial collateral ligament (MCL) result in significant knee joint instability, pain, and immobility. Moderate endurance-type exercise can increase ligament strength but little is known on the effect of short-term regular bouts of high-intensity exercise on the extracellular matrix (ECM) structure of knee ligaments. Therefore, this study aimed to identify the effect of short-term regular bouts high exercise on the proteome of the rat ACL and MCL using mass spectrometry. Sprague-Dawley male rats (n = 6) were split into control and exercise groups, and subjected to high-intensity training for four 4 weeks followed by proteomic analyses of the ACL and MCL. Knee joint health status was assessed using OARSI and a validated histological scoring system. Histopathological analyses demonstrated no significant changes in either in cruciate, collateral ligaments, or cartilage between the control and exercised knee joints. However, significant proteins were found to be more abundant in the exercised ACL compared to ACL control group but not between the exercised MCL and control MCL groups. The significant abundant proteins in ACL exercise groups were mostly cytoskeletal, ribosomal and enzymes with several abundant matrisomal proteins such as collagen proteins and proteoglycans being found in this group. In conclusion, our results indicate that short-term regular bouts of high-intensity exercise have an impact on the intra-articular ACL but not extra-articular MCL ECM protein expression.

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PURPOSE/AIM: Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease characterized by extensive muscle weakness. Patients with DMD lack a functional dystrophin protein, which transmits force and organizes the cytoskeleton of skeletal muscle. Multiomic studies have been proposed as a way to obtain novel insight about disease processes from preclinical models, and we used this approach to study pathological changes in dystrophic muscles. MATERIALS AND METHODS: We evaluated hindlimb muscles of male mdx/mTR mice, which lack a functional dystrophin protein and have deficits in satellite cell abundance and proliferative capacity. Wild type (WT) C57BL/6 J mice served as controls. Muscle fiber contractility was measured, along with changes in the transcriptome using RNA sequencing, and in the proteome, metabolome, and lipidome using mass spectrometry. RESULTS: While mdx/mTR mice displayed gross pathological changes and continued cycles of degeneration and regeneration, we found no differences in permeabilized fiber contractility between strains. However, there were numerous changes in the transcriptome and proteome related to protein balance, contractile elements, extracellular matrix, and metabolism. There was only a 53% agreement in fold-change data between the proteome and transcriptome. Numerous changes in markers of skeletal muscle metabolism were observed, with dystrophic muscles exhibiting elevated glycolytic metabolites such as 6-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate, fructose bisphosphate, phosphorylated hexoses, and phosphoenolpyruvate. CONCLUSIONS: These findings highlight the utility of multiomics in studying muscle disease, and provide additional insight into the pathological changes in dystrophic muscles that might help to indirectly guide evidence-based nutritional or exercise prescription in DMD patients.

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Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.

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Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ) and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared with the WT Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.

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Tendons and ligaments play key roles in the musculoskeletal system in both man and animals. Both tissues can undergo traumatic injury, age-related degeneration and chronic disease, causing discomfort, pain and increased susceptibility to wider degenerative joint disease. To date, tendon and ligament ultrastructural biology is relatively under-studied in healthy, non-diseased tissues. This information is essential to understand the pathology of these tissues with regard to function-related injury and to assist with the future development of tissue-engineered tendon and ligament structures. This study investigated the morphological, compositional and extracellular matrix protein distribution differences between tendons and ligaments around the non-diseased canine stifle joint. The morphological, structural characteristics of different regions of the periarticular tendons and ligaments (the intra-articular anterior cruciate ligament, the extra-articular medial collateral ligament, the positional long digital extensor tendon and energy-storing superficial digital flexor tendons) were identified using a novel semi-objective histological scoring analysis and by determining their biochemical composition. Protein distribution of extracellular matrix collagens, proteoglycans and elastic fibre proteins in anterior cruciate ligament and long digital extensor tendon were also determined using immunostaining techniques. The anterior cruciate ligament was found to have significant morphological differences in comparison with the other three tissues, including less compact collagen architecture, differences in cell nuclei phenotype and increased glycosaminoglycan and elastin content. Intra- and interobserver differences of histology scoring resulted in an average score 0.7, indicative of good agreement between observers. Statistically significant differences were also found in the extracellular matrix composition in terms of glycosaminoglycan and elastin content, being more prominent in the anterior cruciate ligament than in the other three tissues. A different distribution of several extracellular matrix proteins was also found between long digital extensor tendon and anterior cruciate ligament, with a significantly increased immunostaining of aggrecan and versican in the anterior cruciate ligament. These findings directly relate to the different functions of tendon and ligament and indicate that the intra-articular anterior cruciate ligament is subjected to more compressive forces, reflecting an adaptive response to normal or increased loads and resulting in different extracellular matrix composition and arrangement to protect the tissue from damage.

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Tendons and ligaments (T/Ls) play key roles in the musculoskeletal system, but they are susceptible to traumatic or age-related rupture, leading to severe morbidity as well as increased susceptibility to degenerative joint diseases such as osteoarthritis. Tissue engineering represents an attractive therapeutic approach to treating T/L injury but it is hampered by our poor understanding of the defining characteristics of the two tissues. The present study aimed to determine differences in the proteomic profile between native T/Ls and tissue engineered (TE) T/L constructs. The canine long digital extensor tendon and anterior cruciate ligament were analyzed along with 3D TE fibrin-based constructs created from their cells. Native tendon and ligament differed in their content of key structural proteins, with the ligament being more abundant in fibrocartilaginous proteins. 3D T/L TE constructs contained less extracellular matrix (ECM) proteins and had a greater proportion of cellular-associated proteins than native tissue, corresponding to their low collagen and high DNA content. Constructs were able to recapitulate native T/L tissue characteristics particularly with regard to ECM proteins. However, 3D T/L TE constructs had similar ECM and cellular protein compositions indicating that cell source may not be an important factor for T/L tissue engineering.

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OBJECTIVES: To investigate HLA-DRB1, DQA1 and DQB1 allelic polymorphism in Iranian patients with pulmonary tuberculosis (PTB). METHODS: Forty patients with smear-positive PTB and 100 healthy individuals as a control group were studied for MHC class II allelic polymorphism by polymerase chain reaction with sequence-specific primers (PCR-SSP). The primer was supplied by biotest in the standard kit. DRB low resolution SSP and DQA, DQB intermediate resolution SSP was applied. RESULTS: The comparison of the patients and the control group showed a significant increase in the frequency of the HLA-DRB1*07 and DQA1*0101 alleles (OR 2.7, 95%CI 1.19-6.13, P = 0.025 and OR 2.66, 95%CI 1.15-6.44, P = 0.04, respectively) in the patient group. The frequency of DQA1*0301 and DQA1*0501 was also significantly decreased (OR 0.254, 95%CI 0.075-0.865, P = 0.033 and OR 0.53, 95%CI 0.3-0.95, P = 0.045, respectively) in the PTB patients. Concerning haplotype frequency, DRB1*11501, QDQA1*0103 and DQB1*0601 were increased, but this difference was not statistically significant. In the DQB1 locus, DQB1*0501 was non-significantly over-represented. CONCLUSIONS: HLA-DRB1*07 and HLA-DQA1*0101 appeared to be the predisposing alleles and HLA-DQA1*0301 and 0501 the protective alleles in our patients with TB.

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1. Four experiments were carried out to investigate the effects on growth, food conversion efficiency (FCE) and apparent diet digestibility, of wetting food before offering it to individually caged growing chickens. 2. Female broiler chicks (8/treatment) were given grower food ad libitum from 28-49 d of age either in the dry form or wetted with 2.0 kg water/kg air dry food, or wet food restricted to the same daily amount of dry matter as eaten by the dry-fed birds. Ad libitum feeding of the wet food significantly increased food intake and body weight gain, compared to dry feeding, while weight gains of birds with restricted feeding of wet food were intermediate. 3. Experiments 2 and 3 studied the effects of the time interval between mixing the food with water and offering it to the birds. When pre-soaking times of 0, 12 and 24 h were compared with dry food for male broilers (8/treatment) from 25 to 40 d all wet treatments increased body weight gains significantly, the best results coming from the zero soaking time, when DM digestibility was increased significantly from 677 to 714 g/kg. When restricted amounts of food were offered hourly for 8 h on each of 4 d, DM digestibility was significantly increased from 634 for dry food to 659 for that freshly mixed with water and 664 g/kg for that soaked for 1 h between mixing and offering. 4. In a factorial experiment with wet and dry food, either in the standard form or with added enzyme or 400 g/kg cornflour, there were significant positive effects on growth and FCE of broilers (7/treatment) attributable to enzyme and wetting, while cornflour significantly reduced growth. However, wet cornflour-diluted food gave better growth than dry standard food. Wetting significantly increased the apparent digestibility of dry matter and protein while dilution with cornflour significantly reduced protein digestibility. 5. These results confirm those previously presented in terms of improved growth and FCE with wet feeding and demonstrate a large improvement in the proportion of the food absorbed from the digestive tract, of similar magnitude to the improvement in FCE. They also show that it is not necessary to pre-soak food in order to attain the maximum effect.

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1. Individually caged growing chickens were offered a commercial grower food mixed with 1.5 to 2.25 times the weight of water and the effects, compared to giving the same food in air-dry form, on food intake, body weight gain and carcase composition were investigated. 2. Male broilers (24) were given either a grower food in the air-dry form with access to drinking water or the same food mixed with 2.0 parts of water (700 g water/kg of mixed food) with no drinking water from 28 to 49 d of age. From 49 to 63 d all birds were given dry food and drinking water and were then killed for carcase analysis. Food intake and weight gain were significantly increased during the wet-feeding period, as was carcase protein but not abdominal or carcase fat at the end of the experiment. 3. Five male broilers were given each of 5 dietary treatments containing 0 (control), 1.5, 1.75, 2.0 and 2.25 times added water (640, 673, 700 and 723 g water/kg) from 28 to 49 d. Food intakes, body weight gains and carcase weights were significantly increased for all water additions compared with dry food, but there were no significant differences between different water additions. 4. Female broiler chicks responded to wet feeding (700 g water/kg) in a similar manner to males and the dry matter approximate digestibility was increased from 0.65 for the dry food to 0.73 for the wet. 5. Cockerels of an egg-laying strain did not increase their intake of dietary dry matter when it was fed in the wet form (700 g water/kg), but there was a significant increase in body weight gain. 6. Male broilers were offered wet food (700 g water/kg) with or without access to drinking water. There was equal stimulation of food intake, growth and carcase weight with both wet-feeding treatments. 7. Providing food mixed with sufficient water to give a porridge-like consistency significantly increased weight gains in each of the five experiments and significantly improved food conversion efficiency in three of the five. It is not necessary to withhold drinking water in order to obtain this effect.

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