Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast‐derived ADAMTS‐7 and ‐12

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS‐7 and ‐12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK‐Runx2 axis and Wnt/β‐catenin signalling in ADAMTS‐12 and ADAMTS‐7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL‐1β or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS‐12 expression in OA‐SF, also reducing Fn‐fs‐induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS‐7 and COMP degradation in OA‐SF as well. In addition, Wnt7B expression was induced by IL‐1β and by itself, also increasing ADAMTS‐7. Our results could contribute to the development of disease‐modifying OA drugs targeting ADAMTS‐7 and ‐12 for the prevention of extracellular matrix components degradation like COMP.     

Disulfide and amide-bridged cyclic peptide analogues of the VEGF₈₁₋₉₁ fragment: synthesis, conformational analysis and biological evaluation

The design, synthesis, conformational studies and binding affinity for VEGFR-1 receptors of a collection of linear and cyclic peptide analogues of the β-hairpin fragment VEGF(81-91) are described. Cyclic 11-mer peptide derivatives were prepared from linear precursors with conveniently located Cys, Asp or Dap residues, by the formation of disulfide and amide bridges, using solid-phase synthesis. Molecular modelling studies indicated a tendency to be structured around the central β-turn of the VEGF(81-91) β-hairpin in most synthesized cyclic compounds. This structural behavior was confirmed by NMR conformational analysis. The NHCO cyclic derivative 7 showed significant affinity for VEGFR-1, slightly higher than the native linear fragment, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF/VEGFR-1 interaction.     

Vicia faba L. in the Bejaia region of Algeria is nodulated by Rhizobium leguminosarum sv. viciae, Rhizobium laguerreae and two new genospecies

Fifty-eight rhizobial strains were isolated from root nodules of Vicia faba cv. Equina and Vicia faba cv. Minor by the host-trapping method in soils collected from eleven sites in Bejaia, Eastern Algeria. Eleven genotypic groups were distinguished based on the combined PCR/RFLP of 16S rRNA, 16S–23S rRNA intergenic spacer and symbiotic (nodC and nodD-F) genes and further confirmed by multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and rpoB), the 16S rRNA gene and the nodulation genes nodC and nodD. Of the 11 genotypes, 5 were dominant and 2 were the most represented. Most of the strains shared high nodD gene sequence similarity with Rhizobium leguminosarum sv. viciae; their nodC sequences were similar to both Rhizobium leguminosarum and Rhizobium laguerreae. Sequence analyses of the 16S–23S rRNA intergenic spacer showed that all the new strains were phylogenetically related to those described from Vicia sativa and V. faba in several African, European, American and Asian countries, with which they form a group related to Rhizobium leguminosarum. Phylogenetic analysis based on MLSA of 16S rRNA, recA, atpD and rpoB genes allowed the affiliations of strain AM11R to Rhizobium leguminosarum sv. viciae and of strains EB1 and ES8 to Rhizobium laguerreae. In addition, two separate clades with <97% similarity may represent two novel genospecies within the genus Rhizobium.     

Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.     

Clinical Relevance of VPAC1 Receptor Expression in Early Arthritis: Association with IL-6 and Disease Activity

Background

The vasoactive intestinal peptide (VIP) receptors VPAC1 and VPAC2 mediate anti-inflammatory and immunoregulatory responses in rheumatoid arthritis (RA). Data on the expression of these receptors could complement clinical assessment in the management of RA. Our goal was to investigate the correlation between expression of both receptors and the 28-Joint Disease Activity Score (DAS28) in peripheral blood mononuclear cells (PBMCs) from patients with early arthritis (EA). We also measured expression of IL-6 to evaluate the association between VIP receptors and systemic inflammation.

Methods

We analyzed 250 blood samples collected at any of the 5 scheduled follow-up visits from 125 patients enrolled in the Princesa Early Arthritis Register Longitudinal study. Samples from 22 healthy donors were also analyzed. Sociodemographic, clinical, and therapeutic data were systematically recorded. mRNA expression levels were determined using real-time PCR. Then, longitudinal multivariate analyses were performed.

Results

PBMCs from EA patients showed significantly higher expression of VPAC2 receptors at baseline compared to healthy donors (p<0.001). With time, however, VPAC2 expression tended to be significantly lower while VPAC1 receptor expression increased in correlation with a reduction in DAS28 index. Our results reveal that more severe inflammation, based on high levels of IL-6, is associated with lower expression of VPAC1 (p<0.001) and conversely with increased expression of VPAC2 (p<0.001). A major finding of this study is that expression of VPAC1 is lower in patients with increased disease activity (p = 0.001), thus making it possible to differentiate between patients with various degrees of clinical disease activity.

Conclusion

Patients with more severe inflammation and higher disease activity show lower levels of VPAC1 expression, which is associated with patient-reported impairment. Therefore, VPAC1 is a biological marker in EA.     

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (K_D) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG''s variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG''s serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.     

Concordance among stream assemblages and spatial autocorrelation along a fragmented gradient

Patterns of spatial autocorrelation of biota and distributional similarity (concordance) between assemblages of different organism groups have important implications in both theoretical ecology and biodiversity conservation. Here we report environmental gradients and spatial distribution patterns of taxonomic composition among stream fish, benthic macroinvertebrate, and diatom assemblages along a fragmented stream in south‐western France. We quantified spatial patterns of lotic assemblage structure along this stream, and we tested for concordance in distribution patterns among the three taxonomic groups. Our results showed that both environmental characteristics and stream assemblages were spatially autocorrelated. For stream fish and diatom assemblages, these patterns reflected assemblage changes along the longitudinal stream gradient, whereas environmental variables and benthic macroinvertebrates exhibited a more patchy spatial pattern. Cross‐taxa concordance was significant between stream fish and diatoms, and between stream fish and benthic macroinvertebrates. The assemblage concordance between stream fish and diatoms could be attributed to similar responses along the longitudinal gradient, whereas those between stream fish and benthic macroinvertebrates may result from biotic interactions. Based on potential dispersal capacities of taxa, our results validated the hypotheses that weakly dispersing taxa exhibit greater concordance than highly dispersing ones and that dispersal capacities affect how taxonomic groups respond to their local environment. Both diatoms and highly dispersing stream fish were affected by stream fragmentation (i.e. the number of dams between sites), while the effect of fragmentation was not significant for invertebrates that fly well in their adult stage, thus emphasizing the importance of the way of dispersal. These results suggest that addressing the effects of dispersal capacity on stream assemblage patterns is crucial to identifying mechanisms behind patterns and to better understanding the determinants of stream biodiversity.     

VIP balances innate and adaptive immune responses induced by specific stimulation of TLR2 and TLR4

Crohn's disease (CD) is a chronic intestinal inflammatory pathology, which develops as a result of innate immune signals, such as the activation of Toll-like receptors (TLRs), and adaptive immune signals, including Th1 cytokine release. We have recently demonstrated in TNBS-induced colitis, a murine model of CD, that VIP plays a homeostatic role, by reducing TNBS-induced TLR2 and TLR4 expression to control levels. The purpose of this paper is to elucidate for the first time, the physiological relevance of VIP specific control of innate and adaptive immune responses through TLR2 and TLR4 ligands. In addition, we investigated the effect of VIP on regulatory activity of T regulatory (Treg) cells in the TNBS-colitis model. First, we found that VIP downregulated the inflammatory response elicited in mesenteric lymph node cell cultures by treatment with the TLR2 ligand Pam3Cys, or the TLR4 ligand lipopolysaccharide (LPS), reducing the production of the chemokine CXCL1. Also, treatment with VIP impaired the induction of Th1 responses by decreasing p70 interleukin (IL)-12 and interferon gamma (IFN-γ) levels after TLR2/TLR4 stimulation in culture. Besides, VIP treatment restored in vivo the numbers of TLR2 and TLR4 positive CD4⁺CD25⁺ T lymphocytes, augmented by TNBS administration, and increased the expression of molecules involved in regulatory T cell function, such as Foxp3 and TGF-β. In conclusion, the ability of VIP to down-regulate uncontrolled inflammation by targeting TLR-mediated responses and regulatory T cell activity could be used as a new alternative therapy for intestinal inflammatory/autoimmune disorders.     

Perovskite Tandem Solar Cells

The meteoric rise of perovskite single‐junction solar cells has been accompanied by similar stunning developments in perovskite tandem solar cells. Debuting with efficiencies less than 14% in 2014, silicon–perovskite solar cells are now above 25% and will soon surpass record silicon single‐junction efficiencies. Unconstrained by the Shockley–Quiesser single‐junction limit, perovskite tandems suggest a real possibility of true third‐generation thin‐film photovoltaics; monolithic all‐perovskite tandems have reached 18% efficiency and will likely pass perovskite single‐junction efficiencies within the next 5 years. Inorganic–organic metal–halide perovskites are ideal candidates for inclusion in tandem solar cells due to their high radiative recombination efficiencies, excellent absorption, long‐range charge‐transport, and broad ability to tune the bandgap. In this progress report, the development of perovskite tandem cells is reviewed, with presentation of their key motivations and challenges. In detail, it presents an overview of recombination layer materials, bandgap‐tuneability, transparent contact architectures, and perovskite compounds for use in tandems. Theoretical estimates of efficiency for future tandem and triple‐junction perovskite cells are presented, outlining roadmaps for future focused research.     

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2alpha or GFP-CK2beta revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2beta, CK2alpha can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2alpha is dramatically changed by its association with CK2beta, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.