Synthesis and Structural Characterization of Copper Complexes Containing "R-Substituted" Bis-7-Azaindolyl Borate Ligands.

The coordination chemistry of scorpionate ligands based on borates containing the 7-azaindole heterocycle is relatively unexplored. Thus, there is a requirement to further understand their coordination chemistry. This article outlines the synthesis and characterization of a family of complexes containing anionic flexible scorpionate ligands of the type [(R)(bis-7-azaindolyl)borohydride]- ([RBai]-), where R = Me, Ph or naphthyl. The three ligands were coordinated to a series of copper(I) complexes containing a phosphine co-ligand to form the complexes, [Cu(MeBai)(PPh3)] (1), [Cu(PhBai)(PPh3)] (2), [Cu(NaphthBai)(PPh3)] (3), [Cu(MeBai)(PCy3)] (4), [Cu(PhBai)(PCy3)] (5) and [Cu(NaphthBai)(PCy3)] (6). Additional copper(II) complexes, namely, [Cu(MeBai)2] (7) and [Cu(PhBai)2] (8), were obtained during attempts to obtain single crystals from complexes 4 and 2, respectively. Complexes 7 and 8 were also prepared independently from CuCl2 and two equivalents of the corresponding Li[RBai] salt alongside an additional complex, namely, [Cu(NaphthBai)2] (9). The copper(I) and copper(II) complexes were characterized using spectroscopic and analytical methods. Furthermore, a crystal structure was obtained for eight of the nine complexes. In all cases, the boron-based ligand was found to bind to the metal centers via a κ3-N,N,H coordination mode.

Gut microbiota impact on the peripheral immune response in non-alcoholic fatty liver disease related hepatocellular carcinoma.

The gut microbiota is reported to modulate the immune response in hepatocellular carcinoma (HCC). Here, we employ metagenomic and metabolomic studies to characterise gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD) related cirrhosis, with or without HCC, and evaluate its effect on the peripheral immune response in an ex vivo model. We find that dysbiosis characterises the microbiota of patients with NAFLD-cirrhosis, with compositional and functional shifts occurring with HCC development. Gene function of the microbiota in NAFLD-HCC supports short chain fatty acid production, and this is confirmed by metabolomic studies. Ex vivo studies show that bacterial extracts from the NAFLD-HCC microbiota, but not from the control groups, elicit a T cell immunosuppressive phenotype, characterised by expansion of regulatory T cells and attenuation of CD8 + T cells. Our study suggest that the gut microbiota in NAFLD-HCC is characterised by a distinctive microbiome/metabolomic profile, and can modulate the peripheral immune response.

Reprogramming of human fibroblasts into osteoblasts by insulin-like growth factor-binding protein 7.

The induced pluripotent stem cell (iPSC) is a promising cell source for tissue regeneration. However, the therapeutic value of iPSC technology is limited due to the complexity of induction protocols and potential risks of teratoma formation. A trans-differentiation approach employing natural factors may allow better control over reprogramming and improved safety. We report here a novel approach to drive trans-differentiation of human fibroblasts into functional osteoblasts using insulin-like growth factor binding protein 7 (IGFBP7). We initially determined that media conditioned by human osteoblasts can induce reprogramming of human fibroblasts to functional osteoblasts. Proteomic analysis identified IGFBP7 as being significantly elevated in media conditioned with osteoblasts compared with those with fibroblasts. Recombinant IGFBP7 induced a phenotypic switch from fibroblasts to osteoblasts. The switch was associated with senescence and dependent on autocrine IL-6 signaling. Our study supports a novel strategy for regenerating bone by using IGFBP7 to trans-differentiate fibroblasts to osteoblasts.

Ablation of Perlecan Domain 1 Heparan Sulfate Reduces Progressive Cartilage Degradation, Synovitis, and Osteophyte Size in a Preclinical Model of Posttraumatic Osteoarthritis.

OBJECTIVE: To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG-2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene-knockout model. METHODS: Maturational changes were evaluated histologically in the knees of 3-, 6-, and 12-week-old wild-type (WT) mice and Hspg2(Δ3-/Δ3-) mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2(Δ3-/Δ3-) mice. Changes in cartilage expression of FGF-2, FGF-18, HSPG-2, FGF receptor 1 (FGFR-1), and FGFR-3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin-1α (IL-1α), FGF-2, and FGF-18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified. RESULTS: No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2(Δ3-/Δ3-) mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA-induced loss of chondrocyte expression of FGF-2, FGF-18, and HSPG-2 occurred in both genotypes. Expression of FGFR-1 after OA induction was maintained in WT mice, while FGFR-3 loss after OA induction was significantly reduced in Hspg2(Δ3-/Δ3-) mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL-1α-stimulated cartilage. However, IL-1α-induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2(Δ3-/Δ3-) mice. Cartilage GAG release in either the presence or absence of IL-1α was unaltered by FGF-2 in both genotypes. In cartilage cultures with FGF-18, IL-1α-stimulated GAG loss was significantly reduced only in Hspg2(Δ3-/Δ3-) mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA. CONCLUSION: Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR-3 and increased FGF signaling.

Activation of matrix metalloproteinases 2, 9, and 13 by activated protein C in human osteoarthritic cartilage chondrocytes.

OBJECTIVE: Levels of activated protein C (APC) are elevated in the synovial fluid of patients with osteoarthritis (OA), and increased APC levels are correlated with the levels of active matrix metalloproteinase 2 (MMP-2). This study sought to investigate whether APC is a relevant protein for activation of MMPs in the degradation of human OA cartilage, and to elucidate its mechanisms of action. METHODS: Human articular cartilage was cultured with or without interleukin-1α (IL-1α), in the presence or absence of APC or protein C, and an MMP or serine proteinase inhibitor. Aggrecan and collagen release and chondrocyte gene expression levels were quantified. Aggrecanase and MMP cleavage of aggrecan was examined with neoepitope-specific antibodies, and MMP activity was measured using gelatin zymography and fluorogenic peptide assay. RESULTS: In human OA cartilage, APC induced aggrecan and collagen release, whereas in non-OA cartilage, costimulation with IL-1α was required. Inhibition of MMP activity reduced APC-induced cartilage proteolysis, and MMP-induced aggrecanolysis was confirmed by Western blotting. In cultures with APC alone, the activity of MMPs 2, 9, and 13 was significantly increased in OA cartilage, although APC could not directly activate MMPs 2 or 9. Expression of MMP1, MMP2, MMP9, MMP13, TIMP1, and TIMP3 was not altered by APC in OA cartilage. Human OA chondrocytes expressed messenger RNA for protein C, endothelial protein C receptor, thrombomodulin, and protease-activated receptor 1, but these were unaltered or down-regulated by APC. The induction of MMP activation and cartilage degradation by APC was dependent on its serine protease activity. CONCLUSION: APC is a physiologically relevant activator of MMPs and cartilage breakdown in human OA. The effects of APC are dependent on its proteolytic activity and as-yet-undefined cell and/or cartilage matrix factors, and inhibition of this pathway may provide a novel therapeutic target to halt the progression of cartilage damage in OA.

Depletion of protease-activated receptor 2 but not protease-activated receptor 1 may confer protection against osteoarthritis in mice through extracartilaginous mechanisms.

OBJECTIVE: To explore the involvement of protease-activated receptor 1 (PAR-1) and PAR-2 in the pathologic processes of osteoarthritis (OA) and to identify the cells/tissues primarily affected by ablation of PAR-1 or PAR-2 in mice. METHODS: OA was induced in the joints of wild-type (WT), PAR-1(+/+) , PAR-1(-/-) , and PAR-2(-/-) mice by destabilization of the medial meniscus (DMM), and scores of histologic features (cartilage aggrecan loss and erosion, subchondral bone sclerosis, osteophytes, and synovitis) were compared at 1, 4, and 8 weeks post-DMM. The effects of PAR ablation on cartilage degradation and chondrocyte metalloproteinase expression/activity were studied in cultures of mouse femoral head tissue with or without interleukin-1α (IL-1α). At 1 week post-DMM, synovial expression of cytokines and metalloproteinase genes was measured by reverse transcription-polymerase chain reaction, and populations of inflammatory cells were quantified by flow cytometry. RESULTS: Deletion of PAR-2, but not that of PAR-1, in mice significantly delayed the progression of cartilage damage and inhibited subchondral bone sclerosis following DMM. There was no inhibitory effect of PAR-1 or PAR-2 ablation on IL-1α-induced cartilage degradation or chondrocyte metalloproteinase expression/activation. A low but significant level of synovitis persisted in mice subjected to DMM compared to that in control mice subjected to sham surgery, but no differences between the genotypes were seen 4 or 8 weeks post-DMM. One week after DMM, increased synovial expression of proinflammatory cytokines and metalloproteinase genes, along with increased levels of CD4+ T cells, inflammatory monocytes, and activated macrophages, were seen in all genotypes. However, there was a significant reduction in the percentage of activated macrophages in PAR-2(-/-) mice compared to PAR-1(-/-) and WT mice. CONCLUSION: Deletion of PAR-2, but not that of PAR-1, results in a significant decrease in DMM-induced cartilage damage. The chondroprotection in PAR-2(-/-) mice appears to occur indirectly through modulation of extracartilaginous events such as subchondral bone remodeling and synovial macrophage activation, rather than through alteration of chondrocyte catabolic responses.

In-Field Implementation of a Recombinant Factor C Assay for the Detection of Lipopolysaccharide as a Biomarker of Extant Life within Glacial Environments.

The discovery over the past two decades of viable microbial communities within glaciers has promoted interest in the role of glaciers and ice sheets (the cryosphere) as contributors to subglacial erosion, global biodiversity, and in regulating global biogeochemical cycles. In situ or in-field detection and characterisation of microbial communities is becoming recognised as an important approach to improve our understanding of such communities. Within this context we demonstrate, for the first time, the ability to detect Gram-negative bacteria in glacial field-environments (including subglacial environments) via the detection of lipopolysaccharide (LPS); an important component of Gram-negative bacterial cell walls. In-field measurements were performed using the recently commercialised PyroGene® recombinant Factor C (rFC) endotoxin detection system and used in conjunction with a handheld fluorometer to measure the fluorescent endpoint of the assay. Twenty-seven glacial samples were collected from the surface, bed and terminus of a low-biomass Arctic valley glacier (Engabreen, Northern Norway), and were analysed in a field laboratory using the rFC assay. Sixteen of these samples returned positive LPS detection. This work demonstrates that LPS detection via rFC assay is a viable in-field method and is expected to be a useful proxy for microbial cell concentrations in low biomass environments.

Activation of cartilage matrix metalloproteinases by activated protein C.

OBJECTIVE: To investigate the role of activated protein C (APC) in cartilage degradation. METHODS: Chondrocyte expression of protein C, endothelial protein C receptor (EPCR), and thrombomodulin (TM) were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). APC was immunolocalized in developing joints and in osteoarthritic (OA) cartilage from humans. The effect of APC on aggrecan and collagen degradation was examined in explant cultures of ovine cartilage in control cultures and in cultures stimulated with interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNFalpha), or retinoic acid (RetA), using colorimetric assays and Western blotting. Chondrocyte expression of matrix metalloproteinases (MMPs), ADAMTS, and tissue inhibitor of metalloproteinases (TIMPs) was measured by RT-PCR. MMP-2 and MMP-9 activity was evaluated by gelatin zymography and MMP-13 by fluorogenic assay. RESULTS: Positive cellular immunostaining for APC was found at sites of MMP activity in developing joints and in OA, but not normal, cartilage. Chondrocytes expressed messenger RNA for protein C, EPCR, and TM, with the latter 2 levels increased by IL-1alpha and TNFalpha stimulation. APC augmented aggrecan release and initiated collagen breakdown in IL-1alpha-treated and TNFalpha-treated cartilage, but not in normal or in RetA-treated cartilage. APC-stimulated aggrecan and collagen breakdown were due to MMP activity but were not associated with modulation of MMP, ADAMTS, or TIMP expression. APC resulted in MMP-13 activation in cartilage cultures. APC could not directly activate proMMP-13, but it was associated with increased MMP-2 and MMP-9 activity. CONCLUSION: APC may be a relevant activator of MMPs in cartilage and may play a role in progressive cartilage degradation in arthritis.

Effects of basal debris on glacier flow.

Glacier movement is resisted partially by debris, either within glaciers or under glaciers in water-saturated layers. In experiments beneath a thick, sliding glacier, ice containing 2 to 11% debris exerted shear traction of 60 to 200 kilopascals on a smooth rock bed, comparable to the total shear traction beneath glaciers and contrary to the usual assumption that debris-bed friction is negligible. Imposed pore-water pressure that was 60 to 100% of the normal stress in a subglacial debris layer reduced shear traction on the debris sufficiently to halt its deformation and cause slip of ice over the debris. Slip resistance was thus less than debris shearing resistance.