TLR4 stimulation by LPS enhances angiogenesis in a co-culture system consisting of primary human osteoblasts and outgrowth endothelial cells

The development of new approaches leading to fast and successful vascularization of tissue-engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS-EB) significantly enhances microvessel formation in a co-culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS-EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT-PCR have shown that the level of two adhesion molecules (ICAM-1 and E-selectin), two cytokines (IL-6 and IL-8) and two growth factors (VEGF and PDGF-BB) related to angiogenesis increase significantly after LPS-EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering

Cell adhesion and proliferation onto chitosan-based membranes treated by plasma surface modification

Surface properties play a vita role in the functioning of a biomaterial. Cellular adherence and growth onto biomaterials can be enhanced in biomaterial modifications of their surface. In this work, the cell behaviour on chitosan membranes modified by argon and nitron-plasma treatments was investigated. Characterization of the membranes was performed using atomic force microscopy, contact angle measurements, and X-ray photoelectron spectroscopy. Cytotoxicity assessment and direct contact assay were carried out for untreated and treated chitosan membranes using L929 fibroblast-like cells. Cell morphology and cell viability were assessed to evaluate the cell attachment and proliferation. Changes in terms of roughness, surface chemistry, and hydrophilicity/hydrophobic balance of chitosan-modified membranes were observed. Regarding cell studies, the findings revealed that the extracts of all membranes do not induce cytotoxicity effects. Moreover, the in vitro assays evidenced an improvement of the L929 adhesion, and attachment when compared to untreated chitosan membranes. overall, the data obtained clearly demonstrated that plasma treatments constitute an effective way of improving the biocompatibility of chitosan membranes towards to their use in biomedical applications.S. M. Luna acknowledges the support of the Programme Alssan - The European Union Programme of High Level Scholarships for Latin America (scholarship No E04M041362CO) and Silva SS acknowledge the support of the Portuguese Foundation for Science and Technology, (SFRH/BPD/45307/2008). This work was also partially supported by the European Union-funded - STREP project HIPPOCRATES (NMP3-CT-2003-505758) and was carried out under the scope of European NoE EXPERTISSUES (NMP3-CT-2004-500283)

In vivo biocompatibility and biodegradability of dextrin-based hydrogels

The in vivo biocompatibility of dextrin hydrogels obtained by polymerization of dextrin-hydroxyethylmethacrylate (dextrin-HEMA) and dextrin-vinyl acrylate (dextrin-VA) are reported in this work. The histological analysis of subcutaneous implants of these hydrogels, featuring inflammatory and reabsorption events, were carried out over a 16-week period in mice. The dextrin-HEMA hydrogel was quickly and completely degraded and reabsorbed, whereas the dextrin-VA degradation occurred slowly and a thin fibrous capsule surrounded the nondegradable hydrogel. The dextrin-HEMA was degraded after 16 weeks with only mild inflammation and a few detectable foamy macrophages around the implant. These events were followed by complete resorption and no sign of capsule formation or fibrosis associated to the implants. The results indicate that the dextrin hydrogels are biocompatible because no toxicity on the tissues surrounding the implants was found. It may be speculated that a controlled degradation rate of the hydrogels may be obtained by grafting dextrin to HEMA and VA in different proportions.Funding from FCT through POCTI program is acknowledged. The authors Susana Moreira and Rui M. Gil da Costa are recipients of a PhD fellowship from Fundacao para a Ciencia e a Tecnologia (FCT, Portugal)

Spectral density analysis of time correlation functions in lattice QCD using the maximum entropy method

We study various aspects of extracting spectral information from time correlation functions of lattice QCD by means of Bayesian inference with an entropic prior, the maximum entropy method (MEM). Correlator functions of a heavy-light meson-meson system serve as a repository for lattice data with diverse statistical quality. Attention is given to spectral mass density functions, inferred from the data, and their dependence on the parameters of the MEM. We propose to employ simulated annealing, or cooling, to solve the Bayesian inference problem, and discuss practical issues of the approach.Comment: 11 pages, 30 figure files (eps), some references added and updated, requires REVTeX 4.0 and prerequisites (AMS-LaTeX 2.0, graphicx, dcolumn, bm) see http://publish.aps.org/revtex4

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6

The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia

Genome-wide association study in frontal fibrosing alopecia identifies four susceptibility loci including HLA-B*07:02

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07:02