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Nitisinone (NIT) produces inevitable but varying degree of tyrosinaemia. However, the understanding of the dynamic adaptive relationships within the tyrosine catabolic pathway has not been investigated fully. The objective of the study was to assess the contribution of protein intake, serum NIT (sNIT) and tyrosine pathway metabolites to nitisinone-induced tyrosinaemia in alkaptonuria (AKU). Samples of serum and 24-h urine collected during SONIA 2 (Suitability Of Nitisinone In Alkaptonuria 2) at months 3 (V2), 12 (V3), 24 (V4), 36 (V5) and 48 (V6) were included in these analyses. Homogentisic acid (HGA), tyrosine (TYR), phenylalanine (PHE), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and sNIT were analysed at all time-points in serum and urine. Total body water (TBW) metabolites were derived using 60% body weight. 24-h urine and TBW metabolites were summed to obtain combined values. All statistical analyses were post-hoc. 307 serum and 24-h urine sampling points were analysed. Serum TYR from V2 to V6, ranging from 478 to 1983 µmol/L were stratified (number of sampling points in brackets) into groups < 701 (47), 701-900 (105), 901-1100 (96) and > 1100 (59) µmol/L. The majority of sampling points had values greater than 900 µmol/L. sPHE increased with increasing sTYR (p < 0.001). Tyrosine, HPPA and HPLA in serum and TBW all increased with rising sTYR (p < 0.001), while HPLA/TYR ratio decreased (p < 0.0001). During NIT therapy, adaptive response to minimise TYR formation was demonstrated. Decreased conversion of HPPA to HPLA, relative to TYR, seems to be most influential in determining the degree of tyrosinaemia., (© 2022. The Author(s).)

A key feature of osteoarthritis is the gradual loss of articular cartilage and bone deformation, resulting in the impairment of joint function. The primary cause of cartilage destruction is considered to be the presence of elevated proteases, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). However, clinically tested global MMP inhibitors have low efficacy that may be due to their lack of selectivity. We previously demonstrated in vitro that a variant of tissue inhibitor of metalloproteinase-3 ([-1A]TIMP3) inhibits ADAMTSs but not MMPs. In this study, we tested whether the selectivity of [-1A]TIMP3 is beneficial compared with that of the wild-type TIMP3 in preventing or delaying the onset of the degenerative effects in a mouse model of osteoarthritis. We generated transgenic mice that overexpressed TIMP3 or [-1A]TIMP3 driven by a chondrocyte-specific type II collagen promoter. TIMP3 transgenic mice showed compromised bone integrity as opposed to [-1A]TIMP3 mice. After surgically induced joint instability, TIMP3 overexpression proved to be less protective in cartilage destruction than [-1A]TIMP3 at late stages of OA. The selective inhibition of ADAMTSs provides the possibility of modifying TIMP3 to specifically target a class of cartilage-degrading proteinases and to minimize adverse effects on bone and possibly other tissues.

Osteoarthritis (OA) is the most common form of arthritis worldwide with no effective treatment. Ageing is the primary risk factor for OA. We sought to investigate if there is a distinct and functional convergence of ageing-related mechanisms SIRT1 and autophagy in chondrocytes. Our results show that, levels of SIRT1 are decreased in human normal aged and OA cartilage compared with young cartilage. Moreover, silencing SIRT1 in chondrocytes lead to decreased expression of chondrogenic markers but did not alter the expression of catabolic proteases. In contrast, activation of SIRT1 increased autophagy in chondrocytes by the deacetylation of lysine residues on crucial autophagy proteins (Beclin1, ATG5, ATG7, LC3). This activation was shown to be mTOR/ULK1 independent. Our results indicate that maintenance of autophagy in chondrocytes by SIRT1 is essential for preserving cartilage integrity throughout life and therefore is a target for drug intervention to protect against OA., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

The transcriptional mechanism through which chondrocytes control the spatial and temporal composition of the cartilage tissue has remained largely elusive. The central aim of this study was to identify whether transcriptional enhancers played a role in the organisation of the chondrocytes in cartilaginous tissue. We focused on the Aggrecan gene (Acan) as it is essential for the normal structure and function of cartilage and it is expressed developmentally in different stages of chondrocyte maturation. Using transgenic reporter studies in mice we identified four elements, two of which showed individual chondrocyte developmental stage specificity. In particular, one enhancer (-80) distinguishes itself from the others by being predominantly active in adult cartilage. Furthermore, the -62 element uniquely drove reporter activity in early chondrocytes. The remaining chondrocyte specific enhancers, +28 and -30, showed no preference to chondrocyte type. The transcription factor SOX9 interacted with all the enhancers in vitro and mutation of SOX9 binding sites in one of the enhancers (-30) resulted in a loss of its chondrocyte specificity and ectopic enhancer reporter activity. Thus, the Acan enhancers orchestrate the precise spatiotemporal expression of this gene in cartilage types at different stages of development and adulthood.

Ever since the publication of the first reports in 1990 using skeletal muscle as a direct target for expressing foreign transgenes, an avalanche of papers has identified a variety of proteins that can be synthesized and correctly processed by skeletal muscle. The impetus to the development of such applications is not only amelioration of muscle diseases, but also a range of therapeutic applications, from immunization to delivery of therapeutic proteins, such as clotting factors and hormones. Although the most efficient way of introducing transgenes into muscle fibres has been by a variety of recombinant viral vectors, there are potential benefits in the use of non-viral vectors. In this review we assess the recent advances in construction and delivery of naked plasmid DNA to skeletal muscle and highlight the options available for further improvements to raise efficiency to therapeutic levels.

Muscle can be used for systemic delivery of non-muscle proteins. In order to investigate the relative effectiveness of direct DNA plasmid injection versus implantation of genetically modified myogenic cell lines, we have used the human alpha 1 anti-trypsin (alpha1AT) cDNA driven by either cytomegalovirus (CMV) or the muscle creatine kinase 3.3 kb (MCK) promoter in immunodeficient mice. We demonstrate that the implantation of transfected myoblasts stably expressing the human alpha1AT cDNA generates a more persistent production of alpha1AT than does direct intramuscular injection of the same construct as plasmid DNA. Moreover, immunohistological labelling of muscle sections implanted with myoblasts show that the newly formed muscle fibres are those containing the human protein.