Angular Power Spectrum of Gravitational-Wave Transient Sources as a Probe of the Large-Scale Structure.

We present a new, simulation-based inference method to compute the angular power spectrum of the distribution of foreground gravitational-wave transient events. As a first application of this method, we use the binary black hole mergers observed during the LIGO, Virgo, and KAGRA third observation run to test the spatial distribution of these sources. We find no evidence for anisotropy in their angular distribution. We discuss further applications of this method to investigate other gravitational-wave source populations and their correlations to the cosmological large-scale structure.

The recombinant Link module of human TSG-6 suppresses cartilage damage in models of osteoarthritis: A potential disease-modifying OA drug.

OBJECTIVE: To investigate the role of endogenous TSG-6 in human osteoarthritis (OA) and assess the disease-modifying potential of a TSG-6-based biological treatment in cell, explant and animal models of OA. DESIGN: Knee articular cartilages from OA patients were analyzed for TSG-6 protein and mRNA expression using immunohistochemistry and RNAscope, respectively. The inhibitory activities of TSG-6 and its isolated Link module (Link_TSG6) on cytokine-induced degradation of OA cartilage explants were compared. Human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures were used to determine the effects of Link_TSG6 and full-length TSG-6 on IL-1α-, IL-1ß-, or TNF-stimulated ADAMTS4, ADAMTS5, and MMP13 mRNA expression. Link_TSG6 was administered i.a. to the rat ACLTpMMx model; cartilage damage and tactile allodynia were assessed. RESULTS: TSG-6 is predominantly associated with chondrocytes in regions of cartilage damage where high TSG-6 expression aligns with low MMP13, the major collagenase implicated in OA progression. Link_TSG6 is more potent than full-length TSG-6 at inhibiting cytokine-mediated matrix breakdown in human OA cartilage explants;>50% of donor cartilages, from 59 tested, were responsive to Link_TSG6 treatment. Link_TSG6 also displayed more potent effects in 3D pellet cultures, suppressing ADAMTS4, ADAMTS5, and MMP13 gene expression, which was consistent with reduced aggrecanase and collagenase activities in explant cultures. Link_TSG6 treatment reduced touch-evoked pain behavior and dose-dependently inhibited cartilage damage in a rodent model of surgically-induced OA. CONCLUSIONS: Link_TSG6 has enhanced chondroprotective activity compared to the full-length TSG-6 protein and shows potential as a disease modifying OA drug via its inhibition of aggrecanase and collagenase activity.

Disease modification and symptom relief in osteoarthritis using a mutated GCP-2/CXCL6 chemokine.

We showed that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP-2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP-2 expression was retained in adult articular cartilage. GCP-2 loss-of-function inhibited extracellular matrix production. GCP-2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo. To exploit the chondrogenic activity of GCP-2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP-2 haptotactic gradient on endothelia. This mutated version (GCP-2-T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra-articular adenoviral overexpression of GCP-2-T, but not wild-type GCP-2, reduced pain and cartilage loss in instability-induced osteoarthritis in mice. We suggest that GCP-2-T may be used for disease modification in osteoarthritis.

The Link module of human TSG-6 (Link_TSG6) promotes wound healing, suppresses inflammation and improves glandular function in mouse models of Dry Eye Disease.

PURPOSE: To investigate the potential of the Link_TSG6 polypeptide comprising the Link module of human TSG-6 (TNF-stimulated gene/protein-6) as a novel treatment for dry eye disease (DED). METHODS: We analyzed the therapeutic effects of topical application of Link_TSG6 in two murine models of DED, the NOD.B10.H2b mouse model and the desiccating stress model. The effects of Link_TSG6 on the ocular surface and DED were compared with those of full-length TSG-6 (FL_TSG6) and of 0.05% cyclosporine (Restasis®). Additionally, the direct effect of Link_TSG6 on wound healing of the corneal epithelium was evaluated in a mouse model of corneal epithelial debridement. RESULTS: Topical Link_TSG6 administration dose-dependently reduced corneal epithelial defects in DED mice while increasing tear production and conjunctival goblet cell density. At the highest dose, no corneal lesions remained in ∼50% of eyes treated. Also, Link_TSG6 significantly suppressed the levels of inflammatory cytokines at the ocular surface and inhibited the infiltration of T cells in the lacrimal glands and draining lymph nodes. Link_TSG6 was more effective in decreasing corneal epithelial defects than an equimolar concentration of FL_TSG6. Link_TSG6 was significantly more potent than Restasis® at ameliorating clinical signs and reducing inflammation. Link_TSG6 markedly and rapidly facilitated epithelial healing in mice with corneal epithelial debridement wounds. CONCLUSION: Link_TSG6 holds promise as a novel therapeutic agent for DED through its effects on the promotion of corneal epithelial healing and tear secretion, the preservation of conjunctival goblet cells and the suppression of inflammation.

Association of plasma trace element levels with neovascular age-related macular degeneration.

Although the triggers causing angiogenesis in the context of neovascular age-related macular degeneration (nAMD) are not fully understood, oxidative stress is likely involved. Oxidative stress in the eye can occur through exposure of macular tissues to sunlight and local or systemic exposure to oxidative stressors associated with environmental or lifestyle factors. Because trace elements have been implicated as regulators of oxidative stress and cellular antioxidant defense mechanisms, we hypothesized that they may play a role as a risk factor, modifying the progression toward nAMD. Herein, we determined whether levels of human plasma trace elements are different in 236 individuals with nAMD compared to 236 age-matched controls without AMD. Plasma levels of 16 trace elements including arsenic, barium, calcium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, molybdenum, lead, antimony, selenium, vanadium and zinc were measured using inductively coupled plasma mass spectrometry. Associations of trace elements with demographic, environmental and lifestyle factors and AMD-associated genetic variants were assessed. Elevated levels of barium and cadmium and reduced levels of chromium were observed in nAMD patients compared to controls. Mean plasma concentrations of barium were 1.35 µg/L (standard deviation [SD] 0.71) in nAMD and 1.15 µg/L (SD 0.63) in controls (P = 0.001). Mean levels of chromium were 0.37 µg/L (SD 0.22) in nAMD and 0.46 µg/L (SD 0.34) in controls (P = 0.001). Median levels for cadmium, which were not normally distributed, were 0.016 µg/L (interquartile range [IQR] 0.001-0.026) in nAMD and 0.012 µg/L (IQR 0.001-0.022) in controls (P = 0.002). Comparison of the Spearman's correlation coefficients between nAMD patients and controls identified a difference in correlations for 8 trace elements. Cadmium levels were associated with the smoking status (P < 0.001), while barium levels showed a trend of association with the usage of antihypertensive drugs. None of the AMD-associated genetic variants were associated with any trace element levels. In conclusion, in this case-control study we detected elevated plasma levels of barium and cadmium and reduced plasma levels of chromium in nAMD patients. An imbalance in plasma trace elements, which is most likely driven by environmental and lifestyle factors, might have a role in the pathogenesis of AMD. These trace elements may be incorporated as biomarkers into models for prediction of disease risk and progression. Additionally, population-based preventive strategies to decrease Cd exposure, especially by the cessation of smoking, could potentially reduce the burden of nAMD. Future studies are warranted to investigate whether supplementation of Cr would have a beneficial effect on nAMD.

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer.

In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(-) binding site on the heteromeric low sensitivity α2ß2 nAChR and validated the functional importance of specific residues in α2 and ß2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.

Purification and functional characterization of a truncated human α4ß2 nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChR) are abundant in the brain and are essential in cognitive function, learning and memory. Previous efforts on α4ß2 nAChR had been focused on functional and pharmacological characterization, where high expression yield is not essential. For structural studies though, large amounts of pure protein is important but heterologous overexpression of membrane proteins can be a burdensome task, especially if high amounts are required. In the current study, a truncated mutant of the human α4ß2 nAChR was designed in order to improve expression and solubility and to obtain material suitable for high resolution structural studies. We showed that the wild type α4ß2 nAChR presented low expression and solubilization yield both of which were improved with the truncated construct. The truncated nAChR showed similar binding profile to the wild type, was purified by a two-step chromatography and isolated in high purity and adequate quantity.

Bile acid interactions with rabbit ileal lipid binding protein and an engineered helixless variant reveal novel ligand binding properties of a versatile beta-clam shell protein scaffold.

The intracellular ileal lipid binding proteins (ILBPs) are involved in the transport and enterohepatic circulation of bile acids. ILBPs from different species show high sequence and structural homology and have been shown to bind multiple bile acid ligands with differing degrees of selectivity and positive co-operativity. Human ILBP binds bile acid derivatives in a well-characterised 2:1 ligand:protein complex, however, we show that the highly homologous rabbit ILBP (82% sequence identity) with seven conservative substitutions preferentially binds multiple conjugated deoxycholate ligands in a novel 3:1 binding mode essentially within the same beta-clam shell structure. We have extended these studies to investigate the role of the alpha-helical capping motif (residues 9-35) in controlling the dimensions of the binding cavity and ligand uptake. Substituting the alpha-helical motif (residues 9-35) with a short Gly-Gly-Ser-Gly linker dramatically affects the protein stability such that under physiological conditions the mutant (Deltaalpha-ILBP) is highly disordered. However, we show that the inability of the mutant to adopt a stable three-dimensional structure under these conditions is no barrier to binding ligands with near-native affinity. These structural modifications not only demonstrate the possibility of strong coupling between ligand binding and protein folding, but result in changes in bile acid selectivity and binding stoichiometry, which we characterise in detail using isothermal calorimetry and mass spectrometry.

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.