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Objective: Lubrication and free radical scavenging are key features of biomaterials used for viscosupplementation (VS) of joints affected by osteoarthritis (OA). The objective of this study was to describe the non-clinical performance characterization of KiOmedine® CM-Chitosan, a non-animal carboxymethyl chitosan, in order to assess its intended action in VS and to compare it to existing viscosupplements based on crosslinked hyaluronan (HA) formulations., Method: The lubrication capacity of the tested viscosupplements (VS) was evaluated in-vitro and ex-vivo. In-vitro, the coefficient of friction (COF) was measured using a novel tribological system. Meanwhile, an ex-vivo biomechanical model in ovine hindlimbs was developed to assess the recovery of join mobility after an intra-articular (IA) injection. Free radical scavenging capacity of HA and KiOmedine® CM-Chitosan formulations was evaluated using the Trolox Equivalent Antioxidant Capacity (TEAC) assay., Results: In the in-vitro tribological model, KiOmedine® CM-Chitosan showed high lubrication capacity with a significant COF reduction than crosslinked HA formulations. In the ex-vivo model, the lubrication effect of KiOmedine® CM-Chitosan following an IA injection in the injured knee was proven again by a COF reduction. The recovery of joint motion was optimal with an IA injection of 3 ml of KiOmedine® CM-Chitosan, which was significantly better than the crosslinked HA formulation at the same volume. In the in-vitro TEAC assay, KiOmedine® CM-Chitosan showed a significantly higher free radical scavenging capacity than HA formulations., Conclusion: Overall, the results provide a first insight into the mechanism of action in terms of lubrication and free radical scavenging for the use of KiOmedine® CM-Chitosan as a VS treatment of OA. KiOmedine® CM-Chitosan demonstrated a higher capacity to scavenge free radicals, and it showed a higher recovery of mobility after a knee lesion than crosslinked HA formulations. This difference could be explained by the difference in chemical structure between KiOmedine® CM-Chitosan and HA and their formulations., Competing Interests: The sheep study and biomechanical characterization (at Namur university and Université Libre de Bruxelles) were sponsored by KiOmed Pharma, Herstal, Belgium. Jean-Michel Vandeweerd and Fanny Hontoir are paid consultants for KiOmed Pharma. This did not affect the way in which the results of this paper were analysed and reported. Bernardo Innocenti reports no conflicts of interest that could impact the research. Guillem Rocasalbas, Sandrine Gautier and Mickaël Chausson are full-time employees of KiOmed Pharma. Laurence Hermitte is a full-time consultant for KiOmed Pharma. Pierre Douette was, at the time of the study, a full-time consultant for KiOmed Pharma. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling.

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.

Mitochondria are the major sites of ATP synthesis through oxidative phosphorylation, a process that is weakened by proton leak. Uncoupling proteins are mitochondrial membrane proteins specialized in inducible proton conductance. They dissipate the proton electrochemical gradient established by the respiratory chain at the expense of reducing substrates. Several physiological roles have been suggested for uncoupling proteins, including roles in the control of the cellular energy balance and in preventive action against oxidative stress. This review focuses on new leads emerging from comparative proteomics about the involvement of uncoupling protein in the mitochondrial physiology. A brief overview on uncoupling proteins and on proteomics applied to mitochondria is also presented herein.

Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein.