Receptor for Activated C Kinase 1 counteracts ABSCISIC ACID INSENSITIVE5-mediated inhibition of seed germination and post-germinative growth in Arabidopsis.

ABSCISIC ACID INSENSITIVE5 (ABI5), a key regulator of the abscisic acid (ABA) signalling pathway, plays a fundamental role in seed germination and post-germinative development. However, the molecular mechanism underlying the repression function of ABI5 remains to be elucidated. In this study, we demonstrate that the conserved eukaryotic WD40 repeat protein Receptor for Activated C Kinase 1 (RACK1) is a novel negative regulator of ABI5 in Arabidopsis. The RACK1 loss-of-function mutant is hypersensitive to ABA, while this phenotype is rescued by a mutation in ABI5. Moreover, overexpression of RACK1 suppresses ABI5 transcriptional activation activity for ABI5-targeted genes. RACK1 may also physically interact with ABI5 and facilitate its degradation. Furthermore, we found that RACK1 and the two substrate receptors CUL4-based E3 ligases (DWA1 and DWA2) function together to mediate the turnover of ABI5, thereby efficiently reducing ABA signalling in seed germination and post-germinative growth. In addition, molecular analyses demonstrated that ABI5 may bind to the promoter of RACK1 to repress its expression. Collectively, our findings suggest that RACK1 and ABI5 might form a feedback loop to regulate the homeostasis of ABA signalling in acute seed germination and early plant development.

Nuclear Localization of G3BP6 Is Essential for the Flowering Transition in Arabidopsis.

The Ras GTPase-activating protein SH3 domain-binding protein (G3BP) belongs to the highly conserved family of RNA-binding proteins, which has been well-investigated in humans and animals. However, limited study of plant G3BP has been reported, and the precise biological function of the G3BP family has not been elucidated yet. In this study, the Arabidopsis G3BP family, comprising seven members, was comparatively analyzed. Transcriptome analysis showed that most G3BP genes are ubiquitously expressed in various tissues/organs. Transient expression analysis revealed that all G3BPs were presented in the cytoplasm, among which G3BP6 was additionally found in the nucleus. Further study revealed a conserved NLS motif required for the nuclear localization of G3BP6. Additionally, phenotypic analysis revealed that loss-of-function g3bp6 presented late-flowering phenotypes. RNA-sequencing analysis and qRT-PCR assays demonstrated that the expressions of abundant floral genes were significantly altered in g3bp6 plants. We also discovered that overexpression of G3BP6 in the nucleus, rather than in the cytoplasm, propelled bolting. Furthermore, we revealed that the scaffold protein Receptor for Activated C Kinase 1 (RACK1) interacted with and modulated the nuclear localization of G3BP6. Altogether, this study sheds new light on G3BP6 and its specific role in regulating the flowering transition in Arabidopsis.

Finite element analysis of Kirschner wire fixation for lateral condyle fracture in children in the sagittal plane.

Objective: This study aims to find the optimal arrangement of the Kirschner wire (K-wire) in the sagittal plane for fixation of a pediatric lateral condylar humeral fracture (Milch type II) by using finite element analysis (FEA). Methods: A model of lateral condyle fracture in a 6-year-old boy was developed, and an XYZ coordinate system was established based on this model. The YZ plane was defined as the sagittal plane to investigate the impact of the angle formed by the first and second K-wires on stability. Two configurations were studied for each angle: parallel and divergent. Evaluation indicators included the maximum displacement of the fracture fragment and the maximum von Mises stress in the pins and bone. Results: The model with a -60° angle showed the best performance in both evaluation indicators. The parallel and divergent pin configurations had different performances in each group. The displacement results for negative angles were similar, and this result was better than those for positive angles. Conclusion: We successfully created a model of pediatric lateral condyle humerus fracture (Milch type II) and performed K-wire fixation with varying sagittal plane configurations, combined with FEA. Our findings demonstrate that the angle of -60° between the two pins in the sagittal plane provided the highest level of stability, with divergent configurations proving superior to parallel pinning at this angle.

Histone Deacetylase GiSRT2 Negatively Regulates Flavonoid Biosynthesis in Glycyrrhiza inflata.

Glycyrrhiza inflata Batalin is a medicinal licorice species that has been widely used by humans for centuries. Licochalcone A (LCA) is a characteristic flavonoid that accumulates in G. inflata roots with high economical value. However, the biosynthetic pathway and regulatory network of its accumulation remain largely unknown. Here we found that a histone deacetylase (HDAC) inhibitor nicotinamide (NIC) could enhance the accumulation of LCA and total flavonoids in G. inflata seedlings. GiSRT2, a NIC-targeted HDAC was functionally analyzed and its RNAi transgenic hairy roots accumulated much more LCA and total flavonoids than its OE lines and the controls, indicating a negative regulatory role of GiSRT2 in the accumulation of LCA and total flavonoids. Co-analysis of transcriptome and metabolome of RNAi-GiSRT2 lines revealed potential mechanisms in this process. An O-methyltransferase gene, GiLMT1 was up-regulated in RNAi-GiSRT2 lines and the encoded enzyme catalyzed an intermediate step in LCA biosynthesis pathway. Transgenic hairy roots of GiLMT1 proved that GiLMT1 is required for LCA accumulation. Together, this work highlights the critical role of GiSRT2 in the regulation of flavonoid biosynthesis and identifies GiLMT1 as a candidate gene for the biosynthesis of LCA with synthetic biology approaches.

SIRT1 targeted by miR-211-5p regulated proliferation and apoptosis of Dex-treated growth plate chondrocytes via mediating SOX2.

Dexamethasone (Dex) is reported to cause bone growth retardation in children, which is associated with the increased apoptosis and decreased proliferation of growth plate chondrocytes. Sirtuin 1 (SIRT1) plays an important role in chondrocyte function and homeostasis. Thus, we further explored the regulatory mechanism of SIRT1 in Dex-induced growth plate chondrocyte dysfunction. SIRT1 expression was detected in Dex-treated growth plate chondrocytes using RT-qPCR and western blot assay. The modulation of SIRT1 on SOX2 expression was evaluated. Besides, we identified that SIRT1 was targeted by miR-211-5p using TargetScan and RNA pull-down assay. A loss-of-function assay was performed to evaluate the effects of miR-211-5p on Dex-induced growth plate chondrocyte dysfunction in vitro and in vivo. We found that SIRT1 was downregulated in Dex-treated growth plate chondrocytes. The expression of SOX2 was upregulated by overexpression SIRT1. Meanwhile, downregulation of SOX2 weakened the positive function of SIRT1 overexpression on Dex-induced growth plate chondrocytes dysfunction. Subsequently, we confirmed that SIRT1 was targeted by miR-211-5p. MiR-211-5p inhibitor increased the expression levels of SIRT1 and SOX2, and restored the Dex-treated growth plate chondrocyte function. Animal assays further demonstrated that the effects of miR-211-5p on the growth plate chondrogenesis. In conclusion, our data suggest that SIRT1 exerts a protective effect on growth plate chondrocyte under Dex stimulation. MiR-211-5p/SIRT1/SOX2 axis regulates the process of Dex-inhibited growth plate chondrogenesis.

Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza.

Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments.

Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), ß-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

[MiR-101-3p alleviates IL-1ß-induced chondrocyte injury by targeting stanniocalcin 1].

OBJECTIVE: To explore the effects of miR-101-3p on IL-1ß-induced chondrocyte injury and its underlying mechanisms.
 Methods: Chondrocytes were divided into 4 groups: a control group (NC group), a IL-1ß group, a negative control group (IL-1ß+miR-NC group), and a miR-101-3p group (IL-1ß+miR-101-3p group), which were treated with IL-1ß after transfecting with miR-101-3p mimic or negative mimic. The expressions of miR-101-3p-5p and stanniocalcin 1 (STC1) at different concentrations of IL-1ß (1, 5, 10 ng/mL)-induced chondrocytes were detected by Western blotting and real-time PCR. MTT assay was used to detect cell proliferation rate, while caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein, such as matrix metalloproteinase 9 (MMP9) and collagen Type II. In addition, 3'-untranslated regions (UTR) of wild-type STC1 (STC1-3'-UTR-WT) or 3'-UTR of mutant STC1 (STC1-3'-UTR-MUT) were co-transfected with miR-101-3p mimic or miR-NC, respectively, while luciferase reporter assay was used to examine the regulative role of miR-101-3p in STC1. In order to detect whether STC1 was involved in the effect of miR-101-3p on chondrocytes, miR-NC (miR-NC group), miR-101-3p (miR-101-3p group), anti-NC (anti-NC group) and anti-miR-101-3p (anti-miR-101-3p group) were respectively transfected into the cells, and the expression of STC1 protein was detected by Western blotting. Subsequently, the cells were randomly divided into a miR-101-3P group (IL-1ß+miR-101-3p group), an over-expression control group (IL-1ß+miR-101-3p+ad-GFP group), and an over-expression STC1 group (IL-1ß+miR-101-3p+ad-STC1 group) to investigate whether STC1 was involved in the role of miR-101-3p in chondrocyte. Similarly, MTT assay was used to detect cell proliferation rate, caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein MMP9 and collagen Type II.
 Results: Compared with the 0 ng/mL IL-1ß, the expression of miR-101-3p was decreased in chondrocyte at different concentration of IL-1ß (1, 5, 10 ng/mL) (all P<0.05), while the level of STC1 was increased (P<0.05). Compared with the NC group, the chondrocyte proliferation rate was down-regulated (P<0.05), while the apoptosis rate, the levels of caspases, IL-6 and TNF-α were increased in the IL-1ß group (P<0.05). Moreover, the MMP9 levels were increased obviously, and the protein levels of collagen Type II were decreased in the IL-1ß group compared with the NC group (both P<0.05). Compared with the IL-1ß+miR-NC group, the proliferation rate was increased (P<0.05), whereas the apoptosis rates, the caspase-3/9 levels, the IL-6 and TNF-α levels were increased in the IL-1ß+miR-101-3p group (all P<0.05). Then MMP9 levels were decreased obviously (P<0.05), and the protein levels of collagen Type II were increased in IL-1ß+miR-101-3p group compared with the IL-1ß+miR-NC group (both P<0.05). In addition, the double luciferase assay showed that the STC1 levels could be inhibited in the miR-101-3p group compared with the miR-NC group (P<0.05). STC1 levels were decreased in the miR-101-3p group compared with the miR-NC group (P<0.05), and the STC1 levels were increased in the anti-miR-101-3p group compared with those in the anti-NC group (P<0.05). The results of miR-101-3p+ad-STC1 group showed that compared with the miR-101-3p+ad-GFP group, the STC1 could reverse the effects of miR-101-3p on IL-1ß-induced proliferation, apoptosis, inflammatory responses and ECM protein of chondrocytes.
 Conclusion: The regulation of miR-101-3p/STC1 signal pathway may have a role in reducing the IL-1ß-induced chondrocyte injury.

Double ulnar osteomy for the treatment of congenital radial head dislocation.

OBJECTIVE: The aim of this study was to retrospectively evaluate the effects of our double osteotomy technique in the treatment of congenital radial head dislocation (CRHD). METHODS: A total 14 children (14 elbows; 71.42% male; mean age: 9.31 ± 3.06 years) with CRHD who underwent double osteotomy of the proximal ulna between April 2010 and June 2015 were included in the study. The patients with CRHD were identified according to medical history, plain radiographs or magnetic resonance imagings. The outcomes were evaluated through comparison of the preoperative and postoperative motion range of elbow and Mayo Elbow Performance Score (MEPS). RESULTS: After a follow-up of 13-35 months (22.29 ± 5.80), compared with pre-operation, the flexion (132.14 ± 3.23° vs 123.21 ± 7.75°, P = 0.003), extension (8.21 ± 4.21° vs 1.07 ± 3.50°, P = 0.003), and pronation of elbow (83.21 ± 4.21° vs 80.36 ± 4.14°, P = 0.011) improved significantly in all patients. Furthermore, the carrying angle was recovered to the normal level (5-15°) in all of these patients (18.57 ± 5.69° vs 8.21 ± 2.49°, P = 0.001). MEPS score was significantly increased postoperatively (96.79 ± 2.49 vs. 90.71 ± 1.82, P = 0.000), with the good outcome in CRHD patients. CONCLUSION: The results of our study suggested that this double osteotomy on the proximal ulna might be an effective method for the treatment of CRHD. LEVEL OF EVIDENCE: Level IV, Therapeutic Study.

miR-421 promotes apoptosis and suppresses metastasis of osteosarcoma cells via targeting LTBP2.

Increasing evidence has confirmed that microRNAs (miRs) are involved in tumor development and progression. A previous study reported that miR-421 could serve as a diagnostic marker in patients with osteosarcoma (OS). The present study explored the potential roles of miR-421 in the regulation of cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition of OS cells. Our results showed that miR-421 was upregulated in OS tissues and cell lines (MG63, U2OS, HOS, and Saos-2) compared with the corresponding adjacent tissues or human osteoblast cells hFOB1.19, while the latent transforming growth factor ß-binding protein 2 (LTBP2) expression was reduced. In MG63 and U2OS cells, CCK8 assay displayed that cell proliferation was repressed by the miR-421 inhibitor, conversely increased by miR-421 mimics. Inhibition of miR-421 promoted cell apoptosis rate, caspase 3 activity, cleaved-caspase 3 (c-caspase 3) expression, and Bax/Bcl-2 ratio, restoration of miR-421 showed the opposite functions. Suppression of miR-421 blocked migration and invasion, whereas miR-421 overexpression promoted the migration and invasion of MG63 and U2OS cells. In addition, real-time polymerase chain reaction and Western blot analysis revealed that miR-421 negatively regulated E-cadherin expression, and positively regulated the expression of N-cadherin and vimentin. The luciferase reporter assay determined that miR-421 could target LTBP2-3'-UTR, and LTBP2 expression was regulated negatively by miR-421 both in mRNA and protein levels. Depletion of LTBP2 partly abolished the biological functions of miR-421 inhibitor in OS. In conclusion, miR-421 plays an oncogenic role in OS via targeting LTBP2, suggesting that miR-421 may be a potential therapeutic target against OS.

[Reconstruction finger web with dorsal two wing-shaped advancement flap for the treatment of congenital syndactyly].

Objective: To discuss the effectiveness of using dorsal two wing-shaped advancement flap to reconstruct finger web for treatment of congenital syndactyly. Methods: Between August 2014 and August 2017, 30 cases of congenital syndactyly were treated, including 18 males and 12 females with an average age of 2.5 years (range, 1.5-5 years). Eight cases were of bilateral hands syndactyly and 22 cases of single hand syndactyly. There were 39 webs of syndactyly (including 1 case of syndactyly of middle finger, ring finger, and little finger). Among them, 11 webs were complete and 28 webs were incomplete. At the dorsum, a flap with V-shaped tip and two wing-shaped pedicle was designed and was just sewed up with an anchor-shaped incision at the palm. Distal end of fingers were separated by serrated flap and were sutured after removal of fatty tissue. In 11 cases with tight skin connection, the defect area at lateral and distal end of fingers was repaired by small pieces of full-thickness skin graft. Results: All the flaps survived completely after operation, and no flap necrosis occurred. The skin grafts on the distal side of the finger survived and the wound healed by first intension. All 30 cases were followed up 6-12 months, with an average of 9 months. Postoperative flexion and extension function of fingers were good, and the web depth and width were normal. At last follow-up, according to the Swanson et al. standard, 20 fingers were graded as excellent, 8 as good, and 2 as fair, with an excellent and good rate of 93.3%. Conclusion: The effectiveness of using dorsal two wing-shaped advancement flap to reconstruction finger web for treatment of congenital syndactyly is satisfactory.

SENP1/HIF-1α feedback loop modulates hypoxia-induced cell proliferation, invasion, and EMT in human osteosarcoma cells.

Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of osteosarcoma (OS), and is associated with resistance to therapy, poor survival, and a malignant phenotype. The purpose of the present study was to investigate the role and underlying mechanism of SUMO-specific protease 1 (SENP1)/hypoxia-inducible factor-1α (HIF-1α) feedback loop in hypoxic microenvironment of OS. We observed that the expression of SENP1 was remarkably upregulated in OS cells. Additionally, there was a concomitant high expression of HIF-1α and SENP1 in MG-63 cells under a hypoxic microenvironment. Interestingly, blockage of HIF-1α repressed the enhancement of SENP1. Moreover, knockdown of SENP1 with siRNA notably inhibited cell viability and accelerated cell apoptosis accompanied by a decrease in the expression of Bcl-2 and an increase in the expression of Bax in MG-63 cells following exposure to hypoxia. Furthermore, repression of SENP1 dramatically suppressed cell invasive ability through modulating epithelial-mesenchymal transition (EMT) marked genes as reflected by the upregulation of E-cadherin, as well as the downregulation of vimentin and N-cadherin under hypoxic conditions. Most importantly, SENP1 positively regulated HIF-1α expression level in the setting of hypoxic; subsequently, depletion of SENP1 expression markedly ameliorated vascular endothelial growth factor (VEGF) production triggered by hypoxia. Taken together, positive feedback loop between HIF-1α and SENP1 in the regulating of the process of cell proliferation, invasion, and EMT in OS cells under hypoxic conditions, suggesting that the SENP1/HIF-1α axis may serve as a new potential therapeutic agent for OS treatments.