Genetic and Epigenetic Fine-Tuning of TGFB1 Expression Within the Human Osteoarthritic Joint.

OBJECTIVE: Osteoarthritis (OA) is an age-related disease characterized by articular cartilage degeneration. It is largely heritable, and genetic screening has identified single-nucleotide polymorphisms (SNPs) marking genomic risk loci. One such locus is marked by the G>A SNP rs75621460, downstream of TGFB1. This gene encodes transforming growth factor ß1, the correct expression of which is essential for cartilage maintenance. This study investigated the regulatory activity of rs75621460 to characterize its impact on TGFB1 expression in disease-relevant patient samples (n = 319) and in Tc28a2 immortalized chondrocytes. METHODS: Articular cartilage samples from human patients were genotyped, and DNA methylation levels were quantified using pyrosequencing. Gene reporter and electrophoretic mobility shift assays were used to determine differential nuclear protein binding to the region. The functional impact of DNA methylation on TGFB1 expression was tested using targeted epigenome editing. RESULTS: The analyses showed that SNP rs75621460 was located within a TGFB1 enhancer region, and the OA risk allele A altered transcription factor binding, with decreased enhancer activity. Protein complexes binding to A (but not G) induced DNA methylation at flanking CG dinucleotides. Strong correlations between patient DNA methylation levels and TGFB1 expression were observed, with directly opposing effects in the cartilage and the synovium at this locus. This demonstrated biologic pleiotropy in the impact of the SNP within different tissues of the articulating joint. CONCLUSION: The OA risk SNP rs75621460 impacts TGFB1 expression by modulating the function of a gene enhancer. We propose a mechanism by which the SNP impacts enhancer function, providing novel biologic insight into one mechanism of OA genetic risk, which may facilitate the development of future pharmacologic therapies.

Prioritization of PLEC and GRINA as Osteoarthritis Risk Genes Through the Identification and Characterization of Novel Methylation Quantitative Trait Loci.

OBJECTIVE: To identify methylation quantitative trait loci (mQTLs) correlating with osteoarthritis (OA) risk alleles and to undertake mechanistic characterization as a means of target gene prioritization. METHODS: We used genome-wide genotyping and cartilage DNA methylation array data in a discovery screen of novel OA risk loci. This was followed by methylation, gene expression analysis, and genotyping studies in additional cartilage samples, accompanied by in silico analyses. RESULTS: We identified 4 novel OA mQTLs. The most significant mQTL contained 9 CpG sites where methylation correlated with OA risk genotype, with 5 of the CpG sites having P values <1 × 10-10 . The 9 CpG sites reside in an interval of only 7.7 kb within the PLEC gene and form 2 distinct clusters. We were able to prioritize PLEC and the adjacent gene GRINA as independent targets of the OA risk. We identified PLEC and GRINA expression QTLs operating in cartilage, as well as methylation-expression QTLs operating on the 2 genes. GRINA and PLEC also demonstrated differential expression between OA hip and non-OA hip cartilage. CONCLUSION: PLEC encodes plectin, a cytoskeletal protein that maintains tissue integrity by regulating intracellular signaling in response to mechanical stimuli. GRINA encodes the ionotropic glutamate receptor TMBIM3 (transmembrane BAX inhibitor 1 motif-containing protein family member 3), which regulates cell survival. Based on our results, we hypothesize that in a joint predisposed to OA, expression of these genes alters in order to combat aberrant biomechanics, and that this is epigenetically regulated. However, carriage of the OA risk-conferring allele at this locus hinders this response and contributes to disease development.

Genome-wide association study of developmental dysplasia of the hip identifies an association with GDF5.

Developmental dysplasia of the hip (DDH) is the most common skeletal developmental disease. However, its genetic architecture is poorly understood. We conduct the largest DDH genome-wide association study to date and replicate our findings in independent cohorts. We find the heritable component of DDH attributable to common genetic variants to be 55% and distributed equally across the autosomal and X-chromosomes. We identify replicating evidence for association between GDF5 promoter variation and DDH (rs143384, effect allele A, odds ratio 1.44, 95% confidence interval 1.34-1.56, P = 3.55 × 10-22). Gene-based analysis implicates GDF5 (P = 9.24 × 10-12), UQCC1 (P = 1.86 × 10- 10), MMP24 (P = 3.18 × 10-9), RETSAT (P = 3.70 × 10- 8) and PDRG1 (P = 1.06 × 10- 7) in DDH susceptibility. We find shared genetic architecture between DDH and hip osteoarthritis, but no predictive power of osteoarthritis polygenic risk score on DDH status, underscoring the complex nature of the two traits. We report a scalable, time-efficient recruitment strategy and establish for the first time to our knowledge a robust DDH genetic association locus at GDF5.

Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk.

Osteoarthritis (OA) is a common, multifactorial and polygenic skeletal disease that, in its severest form, requires joint replacement surgery to restore mobility and to relieve chronic pain. Using tissues from the articulating joints of 260 patients with OA and a range of in vitro experiments, including CRISPR-Cas9, we have characterized an intergenic regulatory element. Here, genotype at an OA risk locus correlates with differential DNA methylation, with altered gene expression of both a transcriptional regulator (RUNX2), and a chromatin remodelling protein (SUPT3H). RUNX2 is a strong candidate for OA susceptibility, with its encoded protein being essential for skeletogenesis and healthy joint function. The OA risk locus includes single nucleotide polymorphisms (SNPs) located within and flanking the differentially methylated region (DMR). The OA association SNP, rs10948172, demonstrates particularly strong correlation with methylation, and two intergenic SNPs falling within the DMR (rs62435998 and rs62435999) demonstrate genetic and epigenetic effects on the regulatory activity of this region. We therefore posit that the OA signal mediates its effect by modulating the methylation of the regulatory element, which then impacts on gene expression, with RUNX2 being the principal target. Our study highlights the interplay between DNA methylation, OA genetic risk and the downstream regulation of genes critical to normal joint function.

A consistent and potentially exploitable response during chondrogenesis of mesenchymal stem cells from osteoarthritis patients to the protein encoded by the susceptibility gene GDF5.

Osteoarthritis (OA) is a common joint disease characterised by the focal loss of the protective cartilage layer at the ends of the bones. It is painful, disabling, multifactorial and polygenic. The growth differentiation factor 5 gene GDF5 was one of the first reported OA susceptibility signals that showed consistent association to OA, with the transcript single nucleotide polymorphism (SNP) rs143383 demonstrating association in Asians and Europeans. The functional effect of the signal is reduced expression of the gene. The GDF5 protein is an extracellular matrix signalling molecule that is active during chondrogenesis and in mature chondrocytes. Due to the functional impact of the susceptibility, we previously assessed the effect of supplementing chondrocytes from OA patients with exogenous GDF5. Their response was highly discordant, precluding the application of GDF5 as a simple means of attenuating the genetic deficit. Since GDF5 is also active during development, we have now assessed the effect of exogenous GDF5 on bone marrow derived mesenchymal stem cells (MSCs) that are undergoing chondrogenesis during cartilage disc formation. MSCs from healthy donors and OA patients were studied and the effect of GDF5 was assessed by measuring the wet mass of the discs, by histological staining, and by monitoring the change in expression of anabolic, catabolic and hypertrophic protein-coding genes. The MSCs expressed the three principal GDF5 receptor genes and responded in a significantly anabolic manner (increase in wet mass, p = 0.0022; Bonferroni corrected p = 0.018) to a variant form of GDF5 that targets the most abundantly expressed receptor, BMPR-IA. GDF5 elicited significant (p < 0.05) changes in the expression of anabolic, catabolic and hypertrophic genes with several consistent effects in healthy donors and in OA patients. Our data implies that, unlike OA chondrocytes, OA MSCs do respond in a predictable, anabolic manner to GDF5, which could therefore provide a route to modulate the genetic deficit mediated by the rs143383 association signal.

Cotransport of human adenoviruses with clay colloids and TiO2 nanoparticles in saturated porous media: Effect of flow velocity.

This study focuses on the effects of two clay colloids (kaolinite, KGa-1b and montmorillonite, STx-1b) and titanium dioxide (TiO2) nanoparticles (NPs) on human adenovirus transport and retention in water saturated porous media at three different pore water velocities (0.38, 0.74, and 1.21cm/min). Transport and cotransport experiments were performed in 30-cm long laboratory columns packed with clean glass beads with 2mm diameter. The experimental results suggested that the presence of KGa-1b, STx-1b and TiO2 NPs increased human adenovirus inactivation and attachment onto the solid matrix, due to the additional attachment sites available. Retention by the packed column was found to be highest (up to 99%) in the presence of TiO2 NPs at the highest pore water velocity, and lowest in the presence of KGa-1b. The experimental results suggested that adenoviruses would undergo substantial aggregation or heteroaggregation during cotransport. However, no distinct relationships between mass recoveries and water velocity could be established from the experimental cotransport data. Note that for the cotransport experiments, collision efficiency values were shown to be higher for the higher flow rate examined in this study.

Evaluation of Antimicrobial Efficiency of New Polymers Comprised by Covalently Attached and/or Electrostatically Bound Bacteriostatic Species, Based on Quaternary Ammonium Compounds.

In the present work a detailed study of new bacteriostatic copolymers with quaternized ammonium groups introduced in the polymer chain through covalent attachment or electrostatic interaction, was performed. Different copolymers have been considered since beside the active species, the hydrophobic/hydrophilic nature of the co-monomer was also evaluated in the case of covalently attached bacteriostatic groups, aiming at achieving permanent antibacterial activity. Homopolymers with quaternized ammonium/phosphonium groups were also tested for comparison reasons. The antimicrobial activity of the synthesized polymers after 3 and 24 h of exposure at 4 and 22 °C was investigated on cultures of Gram-negative (P. aeruginosa, E. coli) and Gram-positive (S. aureus, E. faecalis) bacteria. It was found that the combination of the hydrophilic monomer acrylic acid (AA), at low contents, with the covalently attached bacteriostatic group vinyl benzyl dimethylhexadecylammonium chloride (VBCHAM) in the copolymer P(AA-co-VBCHAM88), resulted in a high bacteriostatic activity against P. aeruginosa and E. faecalis (6 log reduction in certain cases). Moreover, the combination of covalently attached VBCHAM units with electrostatically bound cetyltrimethylammonium 4-styrene sulfonate (SSAmC16) units in the P(SSAmC16-co-VBCHAMx) copolymers led to efficient antimicrobial materials, especially against Gram-positive bacteria, where a log reduction between 4.9 and 6.2 was verified. These materials remain remarkably efficient even when they are incorporated in polysulfone membranes.

Genome-Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR-140-5p.

microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF-ß, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR-140 and miR-455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR-140, especially the -5p strand. We provide a detailed identification and validation of direct targets of miR-140-5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3'UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR-140-5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR-140-5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR-140-5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering.

Interaction of human adenoviruses and coliphages with kaolinite and bentonite.

Human adenoviruses (hAdVs) are pathogenic viruses responsible for public health problems worldwide. They have also been used as viral indicators in environmental systems. Coliphages (e.g., MS2, ΦX174) have also been studied as indicators of viral pollution in fecally contaminated water. Our objective was to evaluate the distribution of three viral fecal indicators (hAdVs, MS2, and ΦΧ174), between two different phyllosilicate clays (kaolinite and bentonite) and the aqueous phase. A series of static and dynamic experiments were conducted under two different temperatures (4, 25°C) for a time period of seven days. HAdV adsorption was examined in DNase I reaction buffer (pH=7.6, and ionic strength (IS)=1.4mM), whereas coliphage adsorption in phosphate buffered saline solution (pH=7, IS=2mM). Moreover, the effect of IS on hAdV adsorption under static conditions was evaluated. The adsorption of hAdV was assessed by real-time PCR and its infectivity was tested by cultivation methods. The coliphages MS2 and ΦΧ174 were assayed by the double-layer overlay method. The experimental results have shown that coliphage adsorption onto both kaolinite and bentonite was higher for the dynamic than the static experiments; whereas hAdV adsorption was lower under dynamic conditions. The adsorption of hAdV increased with decreasing temperature, contrary to the results obtained for the coliphages. This study examines the combined effect of temperature, agitation, clay type, and IS on hAdV adsorption onto clays. The results provide useful new information on the effective removal of viral fecal indicators (MS2, ΦX174 and hAdV) from dilute aqueous solutions by adsorption onto kaolinite and bentonite. Factors enabling enteric viruses to penetrate soils, groundwater and travel long distances within aquifers are important public health issues. Because the observed adsorption behavior of surrogate coliphages MS2 and ΦΧ174 is substantially different to that of hAdV, neither MS2 nor ΦΧ174 is recommended as a suitable model for adenovirus.

Catalytic inhibition of eukaryotic topoisomerases I and II by flavonol glycosides extracted from Vicia faba and Lotus edulis.

Topoisomerases are essential enzymes involved in all processes of DNA metabolism, and their inhibitors have been identified as potential anticancer agents. The present study examined the effect of nine polyphenolic compounds derived from parts of two unique varieties of the Leguminosae, Vicia faba and Lotus edulis, on the activity of eukaryotic topoisomerases. We identified polyphenolic compounds that act as catalytic inhibitors of wheat germ topoisomerase I (IC50: 120-350 µM), human topoisomerase I (IC50: 110-260 µM), and human topoisomerase II (IC50: 240-600 µM) activities. Some compounds inhibited all enzymatic activities to a similar extent, while others exhibited specificity toward individual enzymes. The strongest catalytic inhibitor of all the examined enzymes was a kaempherol glycoside with an acetyl group linked to a sugar moiety. In addition, this compound inhibited the growth of human cancer cell lines MCF7, HeLa, and HepG2. The inhibition of topoisomerase I and II activities observed by the specific compounds possibly implies a role as potential agents in the prevention and therapy of cancer.

Fatty acid composition in lipid fractions lengthwise the mycelium of Mortierella isabellina and lipid production by solid state fermentation.

This paper investigates the correlation between mycelial age and fatty acid biosynthesis. The correlation was investigated by analyzing the lipid composition lengthwise the mycelium of the oleaginous fungus Mortierella isabellina, a potential producer of gamma-linolenic acid (GLA). Young mycelia were rich in polar lipids (glycolipids plus sphingolipids and phospholipids), while neutral lipid content increased in aged mycelia. In young mycelia, each polar lipid fraction contained almost 40% (w/w) polyunsaturated fatty acids (PUFAs), but this content decreased to less than 30% (w/w) in aged mycelia. On the other hand, PUFA content in neutral lipids fluctuated slightly with age. These results indicate that PUFA biosynthesis is favored in young, fast growing mycelia, while it decreases significantly in aged mycelia. This trend was also observed when we grew M. isabellina on pear pomace, an agro-industrial waste. Pear pomace cultures yielded significant amounts of lipid, which reached 12% (w/w) in dry fermented mass. The produced lipid was rich in GLA and the maximum GLA content in dry fermented mass was 2.9 mg/g.