Genetic control of root morphology in response to nitrogen across rapeseed diversity.

Rapeseed (Brassica napus L.) is an oil-containing crop of great economic value but with considerable nitrogen requirement. Breeding root systems that efficiently absorb nitrogen from the soil could be a driver to ensure genetic gains for more sustainable rapeseed production. The aim of this study is to identify genomic regions that regulate root morphology in response to nitrate availability. The natural variability offered by 300 inbred lines was screened at two experimental locations. Seedlings grew hydroponically with low or elevated nitrate levels. Fifteen traits related to biomass production and root morphology were measured. On average across the panel, a low nitrate level increased the root-to-shoot biomass ratio and the lateral root length. A large phenotypic variation was observed, along with important heritability values and genotypic effects, but low genotype-by-nitrogen interactions. Genome-wide association study and bulk segregant analysis were used to identify loci regulating phenotypic traits. The first approach nominated 319 SNPs that were combined into 80 QTLs. Three QTLs identified on the A07 and C07 chromosomes were stable across nitrate levels and/or experimental locations. The second approach involved genotyping two groups of individuals from an experimental F2 population created by crossing two accessions with contrasting lateral root lengths. These individuals were found in the tails of the phenotypic distribution. Co-localized QTLs found in both mapping approaches covered a chromosomal region on the A06 chromosome. The QTL regions contained some genes putatively involved in root organogenesis and represent selection targets for redesigning the root morphology of rapeseed.

IRAK4 inhibition dampens pathogenic processes driving inflammatory skin diseases.

Innate immunity not only shapes the way epithelial barriers interpret environmental cues but also drives adaptive responses. Therefore, modulators of innate immune responses are expected to have high therapeutic potential across immune-mediated inflammatory diseases. IRAK4 is a kinase that integrates signaling downstream of receptors acting at the interface between innate and adaptive immune responses, such as Toll-like receptors (TLRs), interleukin-1R (IL-1R), and IL-18R. Because effects of IRAK4 inhibition are stimulus, cell type, and species dependent, the evaluation of the therapeutic potential of IRAK4 inhibitors requires a highly translational approach. Here, we profiled a selective IRAK4 inhibitor, GLPG2534, in an extensive panel of models of inflammatory skin diseases, translationally expanding evidence from in vitro to in vivo and from mouse to human. In vitro, IRAK4 inhibition resulted in substantial inhibition of TLR and IL-1 responses in dendritic cells, keratinocytes, granulocytes, and T cells but only weakly affected dermal fibroblast responses. Furthermore, disease activity in murine models of skin inflammation (IL-23-, IL-33-, imiquimod-, and MC903-induced) was markedly dampened by IRAK4 inhibition. Last, inhibiting IRAK4 reversed pathogenic molecular signatures in human lesional psoriasis and atopic dermatitis biopsies. Over the variety of models used, IRAK4 inhibition consistently affected central mediators of psoriasis (IL-17A) and atopic dermatitis (IL-4 and IL-13). Overall, our data highlight IRAK4 as a central player in skin inflammatory processes and demonstrate the potential of IRAK4 inhibition as a therapeutic strategy in chronic inflammatory skin diseases.

A 24-Week, Phase IIa, Randomized, Double-Blind, Placebo-Controlled Study of Ziritaxestat in Early Diffuse Cutaneous Systemic Sclerosis.

OBJECTIVE: We undertook this study to explore the efficacy, safety, and tolerability of ziritaxestat, a selective autotaxin inhibitor, in patients with early diffuse cutaneous systemic sclerosis (dcSSc). METHODS: NOVESA was a 24-week, multicenter, phase IIa, double-blind, placebo-controlled study. Adults with dcSSc were randomized to oral ziritaxestat 600 mg once daily or matching placebo. The primary efficacy end point was change from baseline in modified Rodnan skin score (MRSS) at week 24. Secondary end points assessed safety and tolerability; other end points included assessment of skin and blood biomarkers. Patients in NOVESA could enter a 104-week open-label extension (OLE). RESULTS: Patients were randomized to ziritaxestat (n = 21) or placebo (n = 12). Reduction in MRSS was significantly greater in the ziritaxestat group versus the placebo group (-8.9 versus -6.0 units, respectively; P = 0.0411). Placebo patients switching to ziritaxestat in the OLE showed similar reductions in MRSS to those observed for ziritaxestat patients in the parent study. Ziritaxestat was well tolerated; the most frequent treatment-related treatment-emergent adverse events were headache and diarrhea. Circulating lysophosphatidic acid (LPA) C18:2 was significantly reduced, demonstrating ziritaxestat target engagement, and levels of fibrosis biomarkers were reduced in the blood. No differentially expressed genes were identified in skin biopsies. Significant changes in 109 genes were identified in blood samples. CONCLUSION: Ziritaxestat resulted in significantly greater reduction in MRSS at week 24 than placebo; no new safety signals emerged. Biomarker analysis suggests ziritaxestat may reduce fibrosis. Modulation of the autotaxin/LPA pathway could improve skin involvement in patients with dcSSc. A plain language summary is provided in the Supplementary Material, available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10.1002/art.42477.

Estimation of cell number based on metabolic activity: the MTT reduction assay.

The MTT reduction assay is used to determine the level of metabolic activity in eukaryotic cells, -including animal, plant, and fungal cells. If the metabolic rate is constant, the technique can be employed to count living cells in a sample. Once it is set up, the method is very robust, and can be automatized to be applied on a large number of samples.

Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering.

Articular cartilage injuries and degeneration affect a large proportion of the population in developed countries world wide. Stem cells can be differentiated into chondrocytes by adding transforming growth factor-beta1 and dexamethasone to a pellet culture, which are unfeasible for tissue engineering purposes. We attempted to achieve stable chondrogenesis without any requirement for exogenous growth factors. Human mesenchymal stem cells were transduced with an adenoviral vector containing the SRY-related HMG-box gene 9 (SOX9), and were cultured in a three-dimensional (3D) hydrogel scaffold composite. As an additional treatment, mechanical stimulation was applied in a custom-made bioreactor. SOX9 increased the expression level of its known target genes, as well as its cofactors: the long form of SOX5 and SOX6. However, it was unable to increase the synthesis of sulfated glycosaminoglycans (GAGs). Mechanical stimulation slightly enhanced collagen type X and increased lubricin expression. The combination of SOX9 and mechanical load boosted GAG synthesis as shown by (35)S incorporation. GAG production rate corresponded well with the amount of (endogenous) transforming growth factor-beta1. Finally, cartilage oligomeric matrix protein expression was increased by both treatments. These findings provide insight into the mechanotransduction of mesenchymal stem cells and demonstrate the potential of a transcription factor in stem cell therapy.

Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway.

This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-beta1 (TGF-beta(1)) concentrations (0, 1 or 10 ng/ml).The role of the TGF-beta signalling pathway in this process was also studied. Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 x 10(6) cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression, DNA content and glycosaminoglycan amount. Addition of TGF-beta(1) in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-beta(1) concentration of the culture medium, mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds, with a much stronger effect on gene expression at lower TGF-beta(1) concentrations. With TGF-beta(1) absent in the culture medium, mechanical load stimulated gene transcripts and protein synthesis of TGF-beta(1) and TGF-beta(3). TGF-beta type I receptor inhibitor LY364947 blocked the up-regulation on TGF-beta(1) and TGF-beta(3) production stimulated by mechanical load, and also blocked the chondrogenesis of hMSCs. Taken together, these findings suggest that mechanical load promotes chondrogenesis of hMSCs through TGF-beta pathway by up-regulating TGF-beta gene expression and protein synthesis.

Statin-induced calcification in human mesenchymal stem cells is cell death related.

Statins are widely used in clinics to lower cholesterol levels. Recently, they have been shown to positively affect bone formation and bone mass in a rat model. The aim of this study was to investigate the effect of pravastatin, simvastatin and lovastatin on the osteoblastic differentiation of human mesenchymal stem cells (MSCs) in vitro. Cell number, alkaline phosphatase (ALP) activity, matrix mineralization and gene expression pattern were determined. Pravastatin did not affect cell differentiation. Simvastatin and lovastatin enhanced bone morphogenetic protein 2 (BMP-2) mRNA levels. In contrast, ALP activity and mRNA levels were suppressed by statins, as well as the DNA content and cell activity (MTT). An increase in apoptotic events was observed at high concentrations of statins, along with high Ca-45 incorporation. Lower concentrations of statins did not increase apoptotic staining, but also failed to induce calcification. When statin-induced calcification did occur, the morphology of the deposits was very different from the conventional nodule formation; the calcium was laid down along the membranes of the rounded cells suggesting it was as a result of cell death. Our results indicate that statins are not able to differentiate human MSCs into osteoblasts and that high concentrations of statins (>1 microM) have a cytotoxic effect.

Chondrogenesis of human bone marrow mesenchymal stem cells in fibrin-polyurethane composites.

This study investigated whether a three-dimensional (3D) fibrin gel-polyurethane scaffold composite can provide an environment for chondrogenesis of human bone marrow mesenchymal stem cells (hMSCs) that is as supportive as pellet culture, which is an established model for evaluating chondrogenesis. Pellet culture was carried out in serum-free medium in the absence or presence of transforming growth factor beta 1 (TGF-beta1) and dexamethasone. hMSCs were seeded into a fibrin gel-biodegradable polyurethane scaffold at cell densities of 2 x 10(6), 5 x 10(6), and 10 x 10(6) cells per scaffold and cultured in serum-free medium supplemented with TGF-beta1 and dexamethasone. With comparable proteoglycan synthesis and type I and type X collagen gene expression levels, scaffolds seeded with 5 x 10(6) cells expressed higher type II collagen and aggrecan gene transcripts than pellets on day 14. The deposition of proteoglycan and type II collagen was detected on the top layer of scaffolds seeded with 10 x 10(6) cells and was more evenly distributed in the scaffolds seeded with 5 x 10(6) cells. The scaffold composite culture system shows chondrogenesis of hMSCs comparable with that of pellet culture. Initial cell seeding density influences the ability and process of hMSC chondrogenesis. This study founded a basic system for cartilage neo-tissue formation in vitro.

Epsilon-aminocaproic acid is a useful fibrin degradation inhibitor for cartilage tissue engineering.

Fibrin is a hydrogel carrier widely used in cartilage tissue engineering. It is rapidly degraded by plasmin, which is produced by the cells. epsilon-Aminocaproic acid (EACA) can be used to inhibit this enzyme and thus save the fibrin carrier. In this study we investigated the effect of EACA on the transforming growth factor beta-1-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). To assess this, we used the standard pellet culture system, and EACA treatment was compared to an untreated chondrogenic control. To investigate differentiation, real-time RT-PCR was used on chondrocytic marker genes: aggrecan, collagen types II and X, and the SRY-related HMG-box gene 9 (SOX9). Also, specific glycosaminoglycan production was measured. Safranin-O/fast green staining was used to localize proteoglycans and collagens within the pellet. All results concur that EACA did not affect the chondrogenic differentiation process at 5 muM concentration, which is adequate to inhibit fibrin degradation. Therefore, it is a useful plasmin inhibitor for cartilage tissue engineering with hMSCs.

The use of biodegradable polyurethane scaffolds for cartilage tissue engineering: potential and limitations.

The aim of the present study was to evaluate the capability of novel biodegradable polyurethane scaffolds to support attachment, growth and maintenance of differentiated chondrocytes in vitro for up to 42 days. After an initial decrease, although not significant, the DNA content of the constructs remained constant over the culture time. A progressive increase in glycosaminoglycans and collagen was observed during the culture period. However, a significant release of matrix molecules into the culture medium was also noticeable. At the transcriptional level, a decrease in aggrecan and procollagen II mRNA expression was noticeable, whereas procollagen I expression was increased. To conclude, the present data demonstrate that biodegradable polyurethane porous scaffolds seeded with articular chondrocytes support cell attachment and the production of extracellular matrix proteins. The limitations of the system are the diffusion of large amounts of matrix molecules into the culture medium and the dedifferentiation of the chondrocytes with prolonged time in culture. However, due to the favourable mechanical properties of the polyurethane scaffold, stimulation of chondrocytes by mechanical loading can be considered in order to improve the formation of a functional cartilage-like extracellular matrix.