The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

Concentrations of C4d, C3bBbP and sTCC in synovial fluid. (DOCX 37 kb

Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity

Background A hallmark of osteoarthritis is increased proteolytic cleavage of aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC. Methods Bovine cartilage explants were untreated; coincubated with SJC; or subjected to mechanical injury and coincubated with SJC, mechanical injury alone, or mechanical injury and incubated with tumor necrosis factor-α (TNF-α). To compare the patterns of aggrecan proteolysis between 6 h and 16 days, release of sulfated glycosaminoglycans and specific proteolytic aggrecan fragments into medium or remaining in cartilage explants was measured by dimethylmethylene blue and Western blot analysis. Results Aggrecanase activity toward aggrecan was observed in all conditions, but it was directed toward the TEGE↓ARGS interglobular domain (IGD) site only when cartilage was coincubated with SJC or TNF-α. Matrix metalloproteinase (MMP) activity at the aggrecan IGD site (IPES↓FFGV) was not detected when cartilage was exposed to TNF-α (up to 6 days), but it was in all other conditions. Compared with when bovine cartilage was left untreated or subjected to mechanical injury alone, additional aggrecan fragment types were released into medium and proteolysis of aggrecan started at an earlier time when SJC was present. Conclusions Indicative of different proteolytic pathways for aggrecan degradation, the SJC increases both aggrecanase and MMP activity toward aggrecan, whereas TNF-α inhibits MMP activity against the IGD of aggrecan.National Institutes of Health (U.S.) (AR060331

Integrated acoustic immunoaffinity-capture (IAI) platform for detection of PSA from whole blood samples.

On-chip detection of low abundant protein biomarkers is of interest to enable point-of-care diagnostics. Using a simple form of integration, we have realized an integrated microfluidic platform for the detection of prostate specific antigen (PSA), directly in anti-coagulated whole blood. We combine acoustophoresis-based separation of plasma from undiluted whole blood with a miniaturized immunoassay system in a polymer manifold, demonstrating improved assay speed on our Integrated Acoustic Immunoaffinity-capture (IAI) platform. The IAI platform separates plasma from undiluted whole blood by means of acoustophoresis and provides cell free plasma of clinical quality at a rate of 10 uL/min for an online immunoaffinity-capture of PSA on a porous silicon antibody microarray. The whole blood input (hematocrit 38-40%) rate was 50 μl min(-1) giving a plasma volume fraction yield of ≈33%. PSA was immunoaffinity-captured directly from spiked female whole blood samples at clinically significant levels of 1.7-100 ng ml(-1) within 15 min and was subsequently detected via fluorescence readout, showing a linear response over the entire range with a coefficient of variation of 13%

The RHO-1 RhoGTPase Modulates Fertility and Multiple Behaviors in Adult C. elegans

The Rho family of small GTPases are essential during early embryonic development making it difficult to study their functions in adult animals. Using inducible transgenes expressing either a constitutively active version of the single C. elegans Rho ortholog, RHO-1, or an inhibitor of endogenous Rho (C3 transferase), we demonstrate multiple defects caused by altering Rho signaling in adult C. elegans. Changes in RHO-1 signaling in cholinergic neurons affected locomotion, pharyngeal pumping and fecundity. Changes in RHO-1 signaling outside the cholinergic neurons resulted in defective defecation, ovulation, and changes in C. elegans body morphology. Finally both increased and decreased RHO-1 signaling in adults resulted in death within hours. The multiple post-developmental roles for Rho in C. elegans demonstrate that RhoA signaling pathways continue to be used post-developmentally and the resulting phenotypes provide an opportunity to further study post-developmental Rho signaling pathways using genetic screens

On the Role of Faith in Sustainability Management: A Conceptual Model and Research Agenda

International audienceThe objective of this article is to develop a faith development perspective on corporate sustainability. A firm’s management of sustainability is arguably determined by the way decision-makers relate to the other and the natural environment, and this relationship is fundamentally shaped by faith. This study advances theoretical understanding of the approach managers take on sustainability issues by explaining how four distinct phases of faith development—improvidence, obedience, irreverence and providence—determine a manager’s disposition towards sustainability. Combining insights from intentional and relational faith development theories, the analysis reveals that a manager’s faith disposition can be measured according to four interrelated process criteria: (1) connectivity as a measure of a manager’s actual engagement and activities aimed at relating to sustainability; (2) inclusivity as a measure of who and what is included or excluded in a manager’s moral consideration; (3) emotional affinity as a measure of a manager’s sensitivity and affection towards the well-being of others and ecological welfare; and (4) reciprocity as a measure of the degree to which a manager is rewarded for responding to the needs and concerns of ‘Others’, mainly in the form of a positive emotional (and relational) stimulus. The conceptual model consolidates earlier scholarly works on the psychological drivers of sustainability management by illuminating our search for a process of faith development that connects with an increasingly complex understanding of the role of business in society

Effect of glibenclamide on membrane response to metabolic inhibition in smooth muscle of rat portal vein

Vascular smooth muscle tone is dependent on oxidative metabolism, a phenomenon of potential importance for the metabolic regulation of blood flow to tissues. The response of the rat portal vein to inhibition of cell respiration by cyanide (0.1-1 mM) is a reduction of its spontaneous myogenic activity. The trains of action potentials triggering phasic contractions are reduced in duration, while the frequency of trains is often somewhat increased as the resting membrane potential in the intervals between spike trains is less negative by 6.5 mV. Glibenclamide (10(-7) M) did not affect the resting membrane potential or spontaneous mechanical activity of oxygenated portal veins, but partly restored the depressed myogenic activity in the presence of cyanide (0.5 mM). The spike trains were longer, while the membrane was depolarized by 3 mV compared with the effects of cyanide alone. Inhibition of both oxidative and glycolytic metabolism by 2 mM NaCN in a medium where glucose was replaced by beta-hydroxybutyrate caused a hyperpolarization which was abolished by 10(-7) M glibenclamide. The relaxing effect of the K+ channel opener cromakalim (5 x 10(-9) to 6.25 x 10(-7) M) was partly antagonized by glibenclamide. Basal cytosolic [Ca2+] was increased by cyanide, while the Ca2+ transients associated with phasic contractions were reduced in duration. This latter effect was partially reversed by glibenclamide. The effect of cyanide on high-K+ contractures, which are associated with sustained membrane depolarization and not dependent on repetitive spike activity, was not influenced by 10(-7) M glibenclamide. The effects of inhibited cell respiration on spontaneous electrical activity seem to reflect a depolarizing drive caused by inhibited active ion exchange mechanisms, modified by a repolarizing drive, possibly from ATP-regulated K+ channels, causing reduced duration of the spike trains. While glibenclamide affects spontaneous activity at all levels of oxidative blockade, glibenclamide-sensitive hyperpolarization is seen only when both oxidative and glycolytic metabolism is inhibited

Technical performance of a proximity extension assay inflammation biomarker panel with synovial fluid

The plasma proteome can provide a deeper understanding of the pathophysiology of different diseases and is a key source for detection of new biomarkers. Proximity extension assay (PEA), developed by Olink Proteomics (Uppsala, Sweden), detects proteins by using pairs of antibodies that are linked to oligonucleotides; upon target binding, the probes anneal when in proximity and the oligonucleotides are extended by DNA polymerase and the newly formed antigen is quantified by real-time PCR [1]. PEA provides high specificity and sensitivity, and the possibility to measure the relative abundance of a large number of proteins (92 up to 384 biomarkers), only using a few μL of biofluid per sample. This technique makes it possible to assess low-abundant proteins which are not accessible using mass spectrometry techniques [2]. The multi-assay/plex capacity of PEA is comparable with other multiplex assays like Meso Scale Discovery (MSD); MSD uses electrochemiluminescence measuring the concentration of up to 10 biomarkers per well in 25 ​μl per sample. The PEA has an advantage over enzyme-linked immunosorbent assay (ELISA) and MSD assays in that PEA has a high number of biomarkers which are quantifiable in very low sample volume.In plasma and serum, the correlation between the PEA technique and other techniques, including ELISA and electrochemiluminescence has been shown to be strong [[3], [4], [5], [6], [7], [8]]. The technical performance of the PEA technique in synovial fluid is unknown. The characteristics of synovial fluid differs from plasma in that some proteins are more abundant, whereas other proteins are less abundant. Compared to plasma, synovial fluid is very viscous due to high levels of hyaluronic acid [9]. These differences could interfere with the PEA analysis technique. To interpret protein data generated by the PEA technique in synovial fluid, information on the technical performance of this assay on synovial fluid samples is needed [10].The purpose of the present study was to evaluate the technical performance of Olink's inflammation PEA with synovial fluid samples and compare it with the performance of the same PEA in the more commonly used biofluids, serum and plasma. The second aim was to compare some of the PEA data with data obtained by immunoassays using MSD