Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics

Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2 μl of sample. We further compare the performance of labeled linear probes, quantum dot labeled probes, molecular beacons (MBs) and tentacle probes (TPs). Each probe technology was compared and contrasted with traditional hybridization methods using labeled sample. All probes demonstrated similar sensitivity and greater specificity than traditional hybridization techniques. MBs and TPs were able to bypass a wash step due to their ‘on–off’ signaling mechanism. TPs demonstrated reaction kinetics 37.6 times faster than MBs, resulting in the fastest assay time of 5 min. Our data further indicate TPs had the most sensitive detection limit (<1 nM) as well as the highest specificity (>1 × 104 improvement) among all tested probes in these experiments. By matching the enhanced extraction efficiencies of PPM with the selectivity of TPs, we have created a format for improved sandwich assays

Age-dependent alteration of TGF-β signalling in osteoarthritis

Osteoarthritis (OA) is a disease of articular cartilage, with aging as the main risk factor. In OA, changes in chondrocytes lead to the autolytic destruction of cartilage. Transforming growth factor-β has recently been demonstrated to signal not only via activin receptor-like kinase 5 (ALK5)-induced Smad2/3 phosphorylation, but also via ALK1-induced Smad1/5/8 phosphorylation in articular cartilage. In aging cartilage and experimental OA, the ratio ALK1/ALK5 has been found to be increased, and the expression of ALK1 is correlated with matrix metalloproteinase-13 expression. The age-dependent shift towards Smad1/5/8 signalling might trigger the differentiation of articular chondrocytes with an autolytic phenotype

Protein-Tyrosine Kinase Activity Profiling in Knock Down Zebrafish Embryos

BACKGROUND: Protein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other. METHODOLOGY: We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles. RESULTS AND CONCLUSION: Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo

A systematic review evaluating the psychometric properties of measures of social inclusion

Introduction: Improving social inclusion opportunities for population health has been identified as a priority area for international policy. There is a need to comprehensively examine and evaluate the quality of psychometric properties of measures of social inclusion that are used to guide social policy and outcomes. Objective: To conduct a systematic review of the literature on all current measures of social inclusion for any population group, to evaluate the quality of the psychometric properties of identified measures, and to evaluate if they capture the construct of social inclusion. Methods: A systematic search was performed using five electronic databases: CINAHL, PsycINFO, Embase, ERIC and Pubmed and grey literature were sourced to identify measures of social inclusion. The psychometric properties of the social inclusion measures were evaluated against the COSMIN taxonomy of measurement properties using pre-set psychometric criteria. Results: Of the 109 measures identified, twenty-five measures, involving twenty-five studies and one manual met the inclusion criteria. The overall quality of the reviewed measures was variable, with the Social and Community Opportunities Profile-Short, Social Connectedness Scale and the Social Inclusion Scale demonstrating the strongest evidence for sound psychometric quality. The most common domain included in the measures was connectedness (21), followed by participation (19); the domain of citizenship was covered by the least number of measures (10). No single instrument measured all aspects within the three domains of social inclusion. Of the measures with sound psychometric evidence, the Social and Community Opportunities Profile-Short captured the construct of social inclusion best. Conclusions: The overall quality of the psychometric properties demonstrate that the current suite of available instruments for the measurement of social inclusion are promising but need further refinement. There is a need for a universal working definition of social inclusion as an overarching construct for ongoing research in the area of the psychometric properties of social inclusion instruments

Catabolic and proinflammatory effects of leptin in chondrocytes are regulated by suppressor of cytokine signaling-3

BACKGROUND: Previous studies provide evidence that adipokine leptin increases production of catabolic and proinflammatory factors in chondrocytes and serves as a link between obesity and osteoarthritis (OA). However, the magnitude of the response to leptin treatment varies greatly between chondrocytes from different donor patients. In the present study, we investigated the regulatory role of suppressor of cytokine signaling-3 (SOCS-3) in the leptin-induced responses in OA cartilage. METHODS: Cartilage and synovial fluid samples from 97 patients with OA undergoing knee replacement surgery were collected. Cartilage samples were cultured with leptin (10 μg/ml), and the levels of proinflammatory and catabolic factors in synovial fluid and in the cartilage culture media, and SOCS-3 expression in the cartilage were measured. The role of SOCS-3 in leptin signaling was further studied in H4 murine chondrocytes by downregulating SOCS-3 with siRNA. RESULTS: Leptin-induced expression of matrix metalloproteinases MMP-1, MMP-3, MMP-13, interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were higher in the cartilage samples with low SOCS-3 expression. Accordingly, downregulation of SOCS-3 by siRNA in H4 chondrocytes led to enhanced leptin-induced expression of MMP-3, MMP-13, IL-6 and iNOS. Synovial fluid leptin was associated positively, and cartilage SOCS-3 negatively with synovial fluid levels of MMPs in a multivariate model in obese (body mass index (BMI) >30 kg/m2) but not in non-obese (BMI <30 kg/m2) patients. CONCLUSIONS: Our results show, for the first time, that SOCS-3 regulates leptin-induced responses in cartilage, and could thus be a future drug target in the treatment or prevention of OA, especially in obese patientsBioMed Central open acces