TENDINopathy Severity Assessment - Achilles (TENDINS-A):Development and Content Validity Assessment of a New Patient-Reported Outcome Measure for Achilles Tendinopathy

OBJECTIVE: To develop a new patient-reported outcome measure (PROM) assessing TENDINopathy Severity of the Achilles (TENDINS-Achilles) and evaluate its content validity. DESIGN: Mixed-methods, modified Delphi. METHODS: We performed 1 round of semistructured one-on-one interview responses with professionals and patients, for initial item generation. This was followed by 1 round of survey responses for professionals and a final round of semistructured one-on-one interviews with patients. The work culminated in a PROM to quantify Achilles tendinopathy severity under the core health domain of disability. Participants identified 3 subdomains contributing to the severity of disability of Achilles tendinopathy: pain, symptoms, and functional capacity. RESULTS: All 8 patient participants invited to participate were enrolled. Forty professional participants (50% women, six different continents) were invited to participate and 30 were enrolled (75% response rate). Therefore, a total of 30 professionals and 8 patients were included within this study. Following 3 rounds of qualitative or quantitative feedback, this study has established the content validity of TENDINS-A (good relevance, comprehensibility, and comprehensiveness) as a new PROM to assess the severity of Achilles tendinopathy, which assesses aspects of pain, symptoms, and functional capacity. CONCLUSION: TENDINS-A has established content validity and is appropriate for use with clinical and research populations. We recommend users interpret TENDINS-A results cautiously, until further testing evaluates the most appropriate scoring scale, reliability, construct validity, criterion validity, and responsiveness of TENDINS-A. Until these psychometric properties are established, we suggest using TENDINS-A alongside existing tools. J Orthop Sports Phys Ther 2023;53(11):1-16. Epub: 24 August 2023. doi:10.2519/jospt.2023.11964.</p

Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes

OBJECTIVE—O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals

A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay

In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5'-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (Em = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT

Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis

O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP–OGT. Elevated GFP–OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration

Memory and synaptic plasticity are impaired by dysregulated hippocampal O-GlcNAcylation

O-GlcNAcylated proteins are abundant in the brain and are associated with neuronal functions and neurodegenerative diseases. Although several studies have reported the effects of aberrant regulation of O-GlcNAcylation on brain function, the roles of O-GlcNAcylation in synaptic function remain unclear. To understand the effect of aberrant O-GlcNAcylation on the brain, we used Oga+/- mice which have an increased level of O-GlcNAcylation, and found that Oga+/- mice exhibited impaired spatial learning and memory. Consistent with this result, Oga+/- mice showed a defect in hippocampal synaptic plasticity. Oga heterozygosity causes impairment of both long-term potentiation and long-term depression due to dysregulation of AMPA receptor phosphorylation. These results demonstrate a role for hyper-O-GlcNAcylation in learning and memory.ope

Adipocyte extracellular matrix composition, dynamics and role in obesity

The central role of the adipose tissue in lipid metabolism places specific demands on the cell structure of adipocytes. The protein composition and dynamics of the extracellular matrix (ECM) is of crucial importance for the functioning of those cells. Adipogenesis is a bi-phasic process in which the ECM develops from a fibrillar to a laminar structure as cells move from the commitment phase to the growth phase characterized by storage of vast amounts of triglycerides. Mature adipocytes appear to spend a lot of energy on the maintenance of the ECM. ECM remodeling is mediated by a balanced complement of constructive and destructive enzymes together with their enhancers and inhibitors. ECM remodeling is an energy costing process regulated by insulin, by the energy metabolism, and by mechanical forces. In the obese, overgrowth of adipocytes may lead to instability of the ECM, possibly mediated by hypoxia

Extensive Crosstalk between O-GlcNAcylation and Phosphorylation Regulates Akt Signaling

O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes