Qingyu Wang

Two Wheat Glutathione Peroxidase Genes Whose Products Are Located in Chloroplasts Improve Salt and H2O2 Tolerances in Arabidopsis

Oxidative stress caused by accumulation of reactive oxygen species (ROS) is capable of damaging effects on numerous cellular components. Glutathione peroxidases (GPXs, EC 1.11.1.9) are key enzymes of the antioxidant network in plants. In this study, W69 and W106, two putative GPX genes, were obtained by de novo transcriptome sequencing of salt-treated wheat (Triticum aestivum) seedlings. The purified His-tag fusion proteins of W69 and W106 reduced H2O2 and t-butyl hydroperoxide (t-BHP) using glutathione (GSH) or thioredoxin (Trx) as an electron donor in vitro, showing their peroxidase activity toward H2O2 and toxic organic hydroperoxide. GFP fluorescence assays revealed that W69 and W106 are localized in chloroplasts. Quantitative real-time PCR (Q-RT-PCR) analysis showed that two GPXs were differentially responsive to salt, drought, H2O2, or ABA. Isolation of the W69 and W106 promoters revealed some cis-acting elements responding to abiotic stresses. Overexpression of W69 and W106 conferred strong tolerance to salt, H2O2, and ABA treatment in Arabidopsis. Moreover, the expression levels of key regulator genes (SOS1, RbohD and ABI1/ABI2) involved in salt, H2O2 and ABA signaling were altered in the transgenic plants. These findings suggest that W69 and W106 not only act as scavengers of H2O2 in controlling abiotic stress responses, but also play important roles in salt and ABA signaling.

Isolation and Characterization of the Brassinosteroid Receptor Gene (GmBRI1) from Glycine max

Brassinosteroids (BRs) constitute a group of steroidal phytohormones that contribute to a wide range of plant growth and development functions. The genetic modulation of BR receptor genes, which play major roles in the BR signaling pathway, can create semi-dwarf plants that have great advantages in crop production. In this study, a brassinosteroid insensitive gene homologous with AtBRI1 and other BRIs was isolated from Glycine max and designated as GmBRI1. A bioinformatic analysis revealed that GmBRI1 shares a conserved kinase domain and 25 tandem leucine-rich repeats (LRRs) that are characteristic of a BR receptor for BR reception and reaction and bear a striking similarity in protein tertiary structure to AtBRI1. GmBRI1 transcripts were more abundant in soybean hypocotyls and could be upregulated in response to exogenous BR treatment. The transformation of GmBRI1 into the Arabidopsis dwarf mutant bri1-5 restored the phenotype, especially regarding pod size and plant height. Additionally, this complementation is a consequence of a restored BR signaling pathway demonstrated in the light/dark analysis, root inhibition assay and BR-response gene expression. Therefore, GmBRI1 functions as a BR receptor to alter BR-mediated signaling and is valuable for improving plant architecture and enhancing the yield of soybean.

Acetate templating on calibrated standing digital radiograph improves accuracy of preoperative templating for total hip arthroplasty

BACKGROUND The accuracy of preoperative templating with respect to leg length, femoral offset, and the size of femoral and acetabular components is essential to the success of total hip arthroplasty (THA). Traditionally, templating has been performed using printed film with acetate templates. However, preoperative templating designed by different prosthetic manufacturers cannot be used directly on the film due to varying acetate template amplification ratios. Computer-based templating needs specialized digital templating software, which has cost implications. To address these shortcomings, we bring forward several questions: (1) the accuracy of traditional manual templating combined with the calibrated digital radiograph for preoperative templating, (2) the inter- and intraobserver reliability of this method. HYPOTHESIS Using calibrated digital radiograph with traditional manual templating improves the accuracy and reproducibility of preoperative templating for THA. PATIENTS AND METHODS We designed a stepwise method that combines the traditional manual templating with standing digital radiograph calibrated by a scaling ball. Two separate observers (XJL, QYG) analyzed data of 82 patients (109 THAs) who had undergone THA with preoperative templating using the calibrated digital templating. The intra- and interobserver reliability was assessed by intraclass correlation coefficient. RESULTS The size of the acetate template acetabular based on our method was identical to the actual implanted acetabular size in 55.0% (XJL 110/218 [50.5%]; QYG 130/218 [59.6%]) of the cases indicating moderate accuracy. The intraclass correlation coefficient (ICC) for acetabular templating indicated almost perfect interobserver (ICC=0.918 [95% CI, 0.893-0.937]) and intraobserver agreement (ICC=0.932 [95% CI, 0.912-0.947]). While the exact implanted femoral size was predicted in 55.3% (XJL 122/218 [56.0%]; QYG 119/218 [54.6%]) of the cases. The ICC for femoral component templating indicated almost perfect interobserver (ICC=0.944 [95% CI, 0.927-0.957]) and intraobserver agreement (ICC=0.909 [95% CI, 0.883-0.930]). DISCUSSION This new stepwise method may prove to be a more reliable preoperative design choice to accurately calibrate magnification with radiograph, and could solve the incompatibility of the preoperative template designed by different prosthetic companies for direct use with the x-ray film. The method described is practical, convenient, cost-effective and does not require specialized equipment or software, thus making it particularly suitable for use in underdeveloped settings. LEVEL OF EVIDENCE Level IV, case series without controls.

Association of Genes Variants in RANKL/RANK/OPG Signaling Pathway with the Development of Osteonecrosis of the Femoral Head in Chinese Population: Erratum

The RANKL/RANK/OPG pathway plays an important role in regulating bone remodeling and bone turnover. However, the association of the genes variants with the risk of ONFH has rarely been reported. Here, we analyzed the correlation of the 10 SNPs polymorphisms of RANKL, RANK, OPG, TRAF6, and NFATC1 genes with the risk and development of ONFH in 200 ONFH patients and 177 health controls of Chinese population with using Mass ARRAY® platform. The results showed that the recessive model of NFATC1rs9518 was significantly associated with increased ONFH risk (OR:8.223, P=0.048); the proportion of stage Ⅳ patients in the rs9518TC genotype carriers was statistically higher than that of stage Ⅲ patients (P=0.03); in the T-C haplotype carriers of Naftac1, the proportion of bilateral hips lesions was also significantly enhanced than that of unilateral hip lesions(P=0.05). In addition, the proportion of idiopathic ONFH in the TT genotype carriers of OPGrs2073617 was significantly higher than that of steroid or alcohol-induced ONFH, respectively, while in the TC genotype carriers of the SNP, the proportion of idiopathic ONFH remarkably decreased compared with that of Alcohol-induced ONFH, P=0.021. Our results were first found that NFATC1rs9518 closely associated with the risk and the development of ONFH, while OPGrs2073617 statistically correlated with the etiological classification of ONFH.

Association of gene variants of transcription factors PPARγ, RUNX2, Osterix genes and COL2A1, IGFBP3 genes with the development of osteonecrosis of the femoral head in Chinese population

The molecular pathogenesis of osteonecrosis of the femoral head (ONFH) has been remained obscure so that its prevalence has been increasing in recent decades. Different transcription factors play critical roles in maintaining the balance between osteogenesis and adipogenesis. However, it has been unclear that the genes variants of the transcription factors exert the effects on the imbalance between steogenesis and adipogenesis during the development of ONFH. Here, we selected the 11SNPs from steogenesis, adipogenesis-specific transcription factors RUNX2, Osterix, and PPARγ genes, chondrogenesis or adipogenesis key factors COL2A1, IGFBP3 genes and analysed the genotypes, alleles, haplotypes and their association with the risk and clinical phenotypes of ONFH through Mass ARRAY® platformin in 200 ONFH patients and 177controls. The patients with ONFH (132 males, 68 females; age: 53.46±11.48yr) were consecutively enrolled at the Department of Orthopedics, the Second Clinical College of Jilin University, from March 2014 to June 2015 and were diagnosed and classified into 10 cases of stage II (5.6%), 54 cases of stage III (30.2%) and 115 cases (64.2%) of stage IV and alcohol-induced (71 cases (39.7%)), idiopathic (64 cases (34.0%)), and steroid-induced osteonecrosis (47 cases (26.3%)) subgroup, respectively. Our results showed that all models of logistical regression analysis, the co-dominants, dominants, and recessives of PPARγrs2920502, significantly associated with the increased risk of ONFH (p=0.004, p=0.013, p=0.016), respectively. Both the minor homozygous CC genotype and the allele C of rs2920502 were evidently correlated with the enhanced risk of ONFH (p=0.005, p=0.0005),respectively. The recessives models of IGFBP3rs2132572 (G/A) as well as RUNX2 rs3763190(G/A) were statistically associated with the higher ONFH risk, p=0.030, p=0.029, respectively; the minor homozygous(AA) of IGFBP3rs2132572 (G/A) was also related to the increased risk of bilateral hips lesions, p=0.039. Moreover, the ages on set of major homozygous(GG) and heterozygous(GT) of COL2A1rs2070739(G/A) were significantly younger than that of the minor homozygous(AA) of the SNP(p=0.008) while the A-T-G-A haplotype of COL2A1 gene revealed significant association with the decreased the risk of bilateral hip lesions, p=0.01, OR:0.258. More important, the serum HDL-c level and the ratio of LDL-c/HDL-c in the ONFH group were significantly decreased and increased compared with those of the control group (p=0.02, p=0.0001), respectively. Particularly, the CC genotype of PPARγ rs2920502 was statistically correlated with the enhanced serum TG level, p=0.011.These results suggest that the variants of PPARγ, RUNX2, COL2A1, and IGFBP3 genes closely associated with the development of ONFH.

Circular RNAs hsa_circ_0032462, hsa_circ_0028173, hsa_circ_0005909 are predicted to promote CADM1 expression by functioning as miRNAs sponge in human osteosarcoma

Background Osteosarcoma (OS) is a primary malignant bone tumor with a high fatality rate. Many circRNAs have been proved to play important roles in the pathogenesis of some diseases. However, the occurrence of circRNAs in OS remains little known. Methods The circular RNA (circRNA) expression file GSE96964 dataset, which included seven osteosarcoma cell lines and one control sample (osteoblast cell line), was downloaded from the Gene Expression Omnibus (GEO) database to explore the potential function of circRNAs in osteosarcoma by competing endogenous RNA (ceRNA) analysis. Three gene expression profiles of OS were downloaded from GEO database and then used for the pathway enrichment analysis, Venn analysis and protein-protein interaction (PPI) network analysis. Real-time qPCR validation and RNA interference were conducted to verify our prediction. Results Differentially expressed circRNAs between OS and control, including 8 up-regulated and 102 down-regulated circRNAs, were generated and ceRNA analysis for 5 most up-regulated or 5 most down-regulated circRNAs in OS were then performed. The pathway enrichment analysis of gene expression profiles indicated differentially expressed genes (DEGs) of three gene profiles significantly enriched in cell cycle pathway, cell adhesion molecules (CAMs) pathway, oxidative phosphorylation pathway, cytokine-cytokine receptor interaction pathway, p53 signaling pathway and proteoglycans in cancer pathway, which were critical important pathways in the pathogenesis of OS. The Venn analysis showed that 2 (one is a pseudogene) up-regulated and 39 down-regulated DEGs were co-expressed in all three gene profiles. Then PPI networks of 41 co-expressed DEGs (up- and down-regulated DEGs) were constructed to predict their functions using the GeneMANIA. The expression levels of these related RNAs also matched our predictions really well. Conclusion Ultimately, we found cell adhesion molecule 1 (CADM1) gene was not only a co-expression mRNA of the three mRNA expression profiles of OS, but also are predicted to be regulated by hsa_circ_0032462, hsa_circ_0028173, hsa_circ_0005909 by functioning as miRNAs ‘Sponge’ in human osteosarcoma. These over-expressed circRNAs may result in the over expression of CADM1 which promote the development of OS. We envision this discovery of these important moleculars, incuding hsa_circ_0032462, hsa_circ_0028173, hsa_circ_0005909 and CADM1 may lead to further development of new concepts, thus allowing for more opportunities in diagnosis and therapy of OS.

Isolation and characterization of GmMYBJ3, an R2R3-MYB transcription factor that affects isoflavonoids biosynthesis in soybean

Isoflavonoids are secondary metabolites that play a variety of roles in plant-microbe interactions and plant defenses against abiotic stresses. Here we report a new MYB transcription factor (TF) gene, GmMYBJ3, that is involved in the isoflavonoids biosynthesis. The GmMYBJ3 gene is 1,002 bp long and encodes a protein of 333 amino acids. Amino acid sequence analysis showed that GmMYBJ3 is a typical R2R3 MYB TF. Yeast expression experiment demonstrated that GmMYBJ3 has its transcription activity in the nucleus and is transiently expressed in onion epidermal cells. The GmMYBJ3 gene was transformed into soybean and the expression activity of the GmMYBJ3 gene was significantly positively correlated with total isoflavonoid accumulation in soybean. Transient expression assays indicated that GmMYBJ3 can activate CHS8 expression. Furthermore, we analyzed the expressions of several genes known involved in the isoflavonoid biosynthesis, including CHS8, CHI1A, PAL1, IFS2 and F3H, in the GmMYBJ3 transgenic plants. The results showed that the expression levels of CHS8 and CHI1A were significantly increased in the transgenic plants compared to wild-type plants, but those of PAL1, IFS2 and F3H remained similar between the transgenic and wild-type plants. These results suggest that GmMYBJ3 participates in the isoflavonoid biosynthesis through regulation of CHS8 and CHI1A in soybean.

Significant Associations of SOX9 Gene Polymorphism and Gene Expression with the Risk of Osteonecrosis of the Femoral Head in a Han Population in Northern China

Sex determining region Y-box 9 (SOX9) is a key transcription factor involved in cartilage formation during the embryonic development stage and cartilage growth and repair after birth. To explore the roles of polymorphism and expression of the SOX9 gene in the development of osteonecrosis of the femoral head (ONFH), we analyzed the polymorphism of rs12601701 [A/G] and rs1042667 [A/C] and the serum protein expression of the SOX9 gene in 182 patients with ONFH and 179 healthy control subjects. Results revealed that the A-A haplotype of SOX9 gene as well as the GG and AA genotypes of rs12601701 was significantly associated with increased ONFH risk (P = 0.038) and the risk of bilateral hip lesions of ONFH (P = 0.009), respectively. The C-A, A-A, and A-G haplotypes were also statistically associated with the decreased and increased risk of bilateral hip lesions of ONFH (P = 0.03, P = 0.048, and P = 0.013), respectively, while the A-A haplotype closely related to the clinical stages of ONFH (P = 0.041). More importantly, the serum SOX9 protein expression of the ONFH group was greatly decreased compared to control group (P = 0.0001). Our results first showed that the gene polymorphism and gene expression of SOX9 were significantly associated with the risk and clinical phenotypes of ONFH and also indicate that the SOX9 gene may play a key role in the development of ONFH.

Functional Analysis of GmCPDs and Investigation of Their Roles in Flowering

The onset of floral development is a pivotal switch in the life of soybean. Brassinosteroids (BRs), a group of steroidal phytohormones with essential roles in plant growth and development, are associated with flowering induction. Genes involved in BR biosynthesis have been studied to a great extent in Arabidopsis, but the study of these genes has been limited in soybean. In this study, four CPD homologs (GmCPDs) catalyzing BR synthesis were isolated from soybean. Transcripts were mainly confined to cotyledons and leaves and were down-regulated in response to exogenous BR. Bioinformatic analysis showed strong sequence and structure similarity between GmCPDs and AtCPD as well as CPDs of other species. Overexpression of GmCPDs in an Arabidopsis BR-deficient mutant rescued the phenotype by restoring the biosynthesis pathway, revealing the functional roles of each GmCPDs in. Except for the rescue of root development, leaf expansion and plant type architecture, GmCPDs in expression also complemented the late flowering phenotype of Arabidopsis mutants deficient in CPD. Further evidence in soybean plants is that the expression levels of GmCPDs in are under photoperiod control in Zigongdongdou, a photoperiod-sensitive variety, and show a sudden peak upon floral meristem initiation. Together with increased GmCPDs in expression in the leaves and cotyledons of photoperiod-insensitive early-maturity soybean, it is clear that GmCPDs in contribute to flowering development and are essential in the early stages of flowering regulation.

LncRNA expression profiling of BMSCs in osteonecrosis of the femoral head associated with increased adipogenic and decreased osteogenic differentiation

Long noncoding RNAs (lncRNAs) are critical gene expression regulators and are involved in several bone diseases. To explore the potential roles of lncRNAs in osteonecrosis of the femoral head (ONFH), we investigated for the first time the lncRNA expression profile of bone marrow mesenchymal stem cells (BMSCs) from patients with steroid-induced ONFH (SONFH) with microarray and bioinformatics analysis. A total of 1878 lncRNAs and 838 mRNAs were significantly up-regulated while 1842 lncRNAs and 1937 mRNAs were statistically down-regulated in the SONFH group compared with control group. The results validated by qRT-PCR were consistent with the microarray profiling data, especially involved in upregulation and downregulation of critical lncRNAs as well as mRNAs expression related to adipogenic and osteogenic differentiation. Pathway analyses revealed 40 signaling pathways with significant differences, especially the signaling pathways to regulate stem cell pluripotency. The CNC and ceRNA network indicated that lncRNA RP1-193H18.2, MALAT1 and HOTAIR were associated with abnormal osteogenic and adipogenic differentiation of BMSCs in the patients with SONFH. Our results suggest the lncRNA expression profiles were closely associated with the abnormal adipogenic and osteogenic transdifferentiation of BMSCs during the development of SONFH and explore a new insight into the molecular mechanisms of SONFH.