CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65.

The E3 ubiquitin ligase complex CDC20-activated anaphase-promoting complex/Cyclosome (APC/CCDC20 ) plays a critical role in governing mitotic progression by targeting key cell cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co-activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well-known cell cycle-related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA-binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11-dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell cycle-independent function of CDC20, establishes APC11CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone-related diseases.

Stressful Life Events and Problem Gambling Among Chinese Lottery Gamblers: The Mediating Effects of Coping Strategies and Magical Thinking.

Problem gambling poses serious harm to individuals and societies worldwide. This study aims to investigate the relationship between stressful life events and problem gambling, and further explore the mediating role of coping strategies and magical thinking. Currently, the research on problem gambling is widely conducted worldwide. However, due to the unique characteristics of China's gambling industry, research on problem gambling conducted in the Chinese mainland has always been an underrepresented area in international gambling research. This study recruited participants from a province in central China, and data from 483 of them were ultimately analyzed. The data analysis results indicate that task-oriented coping, emotion-oriented coping, avoidance-oriented coping, and magical thinking all serve as mediators in the relationship between stressful life events and problem gambling. Emotion-oriented coping and magical thinking, avoidance-oriented coping and magical thinking, all serve as serial mediators in the relationship between stressful life events and problem gambling. Task-oriented coping and magical thinking did not act as serial mediators in this relationship. This study demonstrates that helping problem gamblers develop effective coping strategies and reduce their level of magical thinking is crucial for treating their problem gambling.

ZIM1 Combined with Hydrogel Inhibits Senescence of Primary PαS Cells during In Vitro Expansion.

Bone marrow stem cells (BMSCs) are a promising source of seed cells in bone tissue engineering, which needs a great quantity of cells. Cell senescence occurs as they are passaged, which could affect the therapeutic effects of cells. Therefore, this study aims to explore the transcriptomic differences among the uncultured and passaged cells, finding a practical target gene for anti-aging. We sorted PαS (PDGFR-α+SCA-1+CD45-TER119-) cells as BMSCs by flow cytometry analysis. The changes in cellular senescence phenotype (Counting Kit-8 (CCK-8) assay, reactive oxygen species (ROS) test, senescence-associated ß-galactosidase (SA-ß-Gal) activity staining, expression of aging-related genes, telomere-related changes and in vivo differentiation potential) and associated transcriptional alterations during three important cell culture processes (in vivo, first adherence in vitro, first passage, and serial passage in vitro) were studied. Overexpression plasmids of potential target genes were made and examed. Gelatin methacryloyl (GelMA) was applied to explore the anti-aging effects combined with the target gene. Aging-related genes and ROS levels increased, telomerase activity and average telomere length decreased, and SA-ß-Gal activities increased as cells were passaged. RNA-seq offered that imprinted zinc-finger gene 1 (Zim1) played a critical role in anti-aging during cell culture. Further, Zim1 combined with GelMA reduced the expression of P16/P53 and ROS levels with doubled telomerase activities. Few SA-ß-Gal positive cells were found in the above state. These effects are achieved at least by the activation of Wnt/ß-catenin signaling through the regulation of Wnt2. The combined application of Zim1 and hydrogel could inhibit the senescence of BMSCs during in vitro expansion, which may benefit clinical application.

DUSP5 promotes osteogenic differentiation through SCP1/2-dependent phosphorylation of SMAD1.

Dual-specificity phosphatases (DUSPs) are defined by their capability to dephosphorylate both phosphoserine/phosphothreonine (pSer/pThr) and phosphotyrosine (pTyr). DUSP5, a member of DUSPs superfamily, is located in the nucleus and plays crucially regulatory roles in the signaling pathway transduction. In our present study, we discover that DUSP5 significantly promotes osteogenic differentiation of mesenchymal stromal cells (MSCs) by activating SMAD1 signaling pathway. Mechanistically, DUSP5 physically interacts with the phosphatase domain of small C-terminal phosphatase 1/2 (SCP1/2, SMAD1 phosphatases) by the linker region. In addition, we further confirm that DUSP5 activates SMAD1 signaling through a SCP1/2-dependent manner. Specifically, DUSP5 attenuates the SCP1/2-SMAD1 interaction by competitively binding to SCP1/2, which is responsible for the SMAD1 dephosphorylation, and thus results in the activation of SMAD1 signaling. Importantly, DUSP5 expression in mouse bone marrow MSCs is significantly reduced in ovariectomized (OVX) mice in which osteogenesis is highly passive, and overexpression of Dusp5 via tail vein injection reverses the bone loss of OVX mice efficiently. Collectively, this work demonstrates that the linker region of DUSP5 maybe a novel chemically modifiable target for controlling MSCs fate choices and for osteoporosis treatment.

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer.

A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.

Deficiency of the ribosomal protein uS10 (RPS20) reorganizes human cells translatome according to the abundance, CDS length and GC content of mRNAs.

Ribosomal protein uS10, a product of the RPS20 gene, is an essential constituent of the small (40S) subunit of the human ribosome. Disruptive mutations in its gene are associated with a predisposition to hereditary colorectal carcinoma. Here, using HEK293T cells, we show that a deficiency of this protein leads to a decrease in the level of ribosomes (ribosomal shortage). RNA sequencing of the total and polysome-associated mRNA samples reveals hundreds of genes differentially expressed in the transcriptome (t)DEGs and translatome (p)DEGs under conditions of uS10 deficiency. We demonstrate that the (t)DEG and (p)DEG sets partially overlap, determine genes with altered translational efficiency (TE) and identify cellular processes affected by uS10 deficiency-induced ribosomal shortage. We reveal that translated mRNAs of upregulated (p)DEGs and genes with altered TE in uS10-deficient cells are generally more abundant and that their GC contents are significantly lower than those of the respective downregulated sets. We also observed that upregulated (p)DEGs have longer coding sequences. Based on our findings, we propose a combinatorial model describing the process of reorganization of mRNA translation under conditions of ribosomal shortage. Our results reveal rules according to which ribosomal shortage reorganizes the transcriptome and translatome repertoires of actively proliferating cells.

BHLHE40 Maintains the Stemness of PαS Cells In Vitro by Targeting Zbp1 through the Wnt/ß-Catenin Signaling Pathway.

Primary bone mesenchymal stem cells (BMSCs) gradually lose stemness during in vitro expansion, which significantly affects the cell therapeutic effects. Here, we chose murine PαS (SCA-1+PDGFRα+CD45-TER119-) cells as representative of BMSCs and aimed to explore the premium culture conditions for PαS cells. Freshly isolated (fresh) PαS cells were obtained from the limbs of C57/6N mice by fluorescence-activated cell sorting (FACS). We investigated the differences in the stemness of PαS cells by proliferation, differentiation, and stemness markers in vitro and by ectopic osteogenesis and chondrogenesis ability in vivo, as well as the changes in the stemness of PαS cells during expansion in vitro. Gain- and loss-of-function experiments were applied to investigate the critical role and underlying mechanism of the basic helix-loop-helix family member E40 (BHLHE40) in maintaining the stemness of PαS cells. The stemness of fresh PαS cells representative in vivo was superior to that of passage 0 (P0) PαS cells in vitro. The stemness of PαS cells in vitro decreased gradually from P0 to passage 4 (P4). Moreover, BHLHE40 plays a critical role in regulating the stemness of PαS cells during in vitro expansion. Mechanically, BHLHE40 regulates the stemness of PαS cells by targeting Zbp1 through the Wnt/ß-catenin signaling pathway. This work confirms that BHLHE40 is a critical factor for regulating the stemness of PαS cells during expansion in vitro and may provide significant indications in the exploration of premium culture conditions for PαS cells.

In Situ Synthesized Particulates on a Zirconia Ceramic Surface and Their Effect on Bonding to Porcelain.

PURPOSE: To explore the effect of in situ synthesized particulates on a zirconia surface on the bonding properties between zirconia and porcelain. MATERIALS AND METHODS: Presintered yttrium-stabilized tetragonal zirconia (Y-TZP) was cut into slices and bars and polished with 1,200-grit silicon carbide abrasive paper. Samples were randomly divided into six groups (C, I1, I3, I5, I7, and I9) according to immersion time in hydrofluoric acid solution (0, 10, 30, 50, 70, and 90 seconds, respectively). Then, the samples were placed in calcium chloride solution for 90 seconds and dipped in sodium hydroxide solution at 80°C for 2 hours. After sintering, the surface topography and roughness were examined. After the porcelain was fired, the bonding interface was observed, and cross-sectional microhardness was measured. The shear bond strength of the zirconia to porcelain was evaluated, and failure modes were classified. A 3-point bending test was applied to confirm the effects of the treatment on the mechanical properties. The above data were statistically analyzed. RESULTS: Polycrystalline particulates were synthesized on the zirconia surface. The surface roughness values increased as the immersion time of the samples in hydrofluoric acid increased. The cross-sectional microhardness decreased gradually in the experimental groups. Group I7 showed an elevated bond strength (27.02 ± 2.44 MPa). Mainly mixed failure mode was obtained in the experimental groups. The Weibull characteristic strength for the experimental groups was higher than that of group C. The flexural strengths were not significantly different among the groups. CONCLUSIONS: In situ synthesized polycrystalline particulates on zirconia could effectively improve the bonding between zirconia ceramics and porcelain without significantly decreasing the mechanical properties.

Improvement of Pit-and-Fissure Sealant Bonding to Enamel with Subpressure Treatment.

This research evaluated the effects of subpressure on the shear bond strength (SBS) of 80 specimens with flat enamel surfaces and on AgNO3 microleakage of 40 specimens with flat enamel surfaces and 40 specimens with 1 mm deep cavities before and after thermocycling. The enamel of 168 specimens was grounded to a flat surface. Two types of sealants (E and H) were selected. Sealants were applied to enamel surface (88 specimens, group F) either subjected or not to subpressure. The bonding interfaces were observed using scanning electron microscopy (SEM) and the SBS was examined using a universal testing machine before and after thermocycling. The failure mode was also analyzed. For the microleakage test, 80 specimens were grouped as group A (original enamel flat surface) and group B (a round cavity of 1 mm in depth) (40 per group). Sealants were applied to the teeth either subjected or not to subpressure. The specimens were submitted to a microleakage protocol with AgNO3 and analyzed before and after thermocycling. Statistical analysis was performed for the data. The results showed that subpressure eliminated voids on the interface between the enamel and sealants and significantly enhanced specimens' SBS. Although thermocycling reduced SBS significantly, specimens under subpressure after thermocycling still showed higher SBS than specimens under nonsubpressure before thermocycling. The subpressure groups showed a lower microleakage level compared to nonsubpressure groups, though thermocycling caused deeper silver infiltration. In addition, different sealants showed no significant effect on the SBS and microleakage performance. Overall, subpressure application improves sealant bonding and retention rate and has potential to prevent secondary caries.

Ca-P spots modified zirconia by liquid precursor infiltration and the effect on osteoblast-like cell responses.

Ca-P spots modified zirconia by liquid precursor infiltration and the cell responses were investigated. Pre-sintered zirconia specimens were immersed in Ca-P precursor solution. After dense sintering, scanning electron microscopy showed Ca-P spots were formed on the zirconia and anchored with zirconia substrates. The distribution density was increased with the extension of immersion time. Energy dispersive spectrometer confirmed the stoichiometric Ca/P ratio was about 1.67. After hydrothermal treatment, Ca-P spots turned into rod crystals where diffraction peaks of tricalcium phosphate and hydroxyapatite were detected by X-ray diffraction, and Ca2+ and PO43- release decreased slightly (p>0.05). There was no significant decrease on three-point bending strength (p>0.05). Osteoblast-like MC3T3-E1 cells attached and spread well and showed higher proliferation on Ca-P spots modified zirconia (p<0.05), though its initial alkaline phosphatase activity was not significant high (p>0.05). In conclusion, Ca-P liquid precursor infiltration is a potential method to modify the zirconia ceramics for improving bioactivity.

Improvement of Total Etching Dentin Bonding with Subpressure.

This study aimed to investigate the effects of subpressure on the bond properties of total-etching adhesive to dentin. Thirty-six caries-free premolars were sectioned parallel to the occlusal plane and randomly divided into four groups (n = 9): a control group (C, no treatment) and three subpressure groups, which were treated under 0.8, 0.6 or 0.4 bar after applying adhesives, named S8, S6 and S4, respectively. Afterward, resin was bonded to the dentin surface, and 27 beams (1.0 mm × 1.0 mm) of each group were sectioned. One was selected to observe the bonding interface from each group by SEM. Each group was divided into two subgroups (n = 13): 24 hours of water storage (I) and 10,000 thermocycling (A). The microtensile bond strength (µTBS), failure modes and nanoleakage expression were evaluated. SEM results showed that the subpressure groups had longer and denser resin tags. The µTBS of the subpressure groups was higher than that of the control group (p < 0.05). The subpressure groups were dominated by mixed failure, whereas main interfacial failure appeared in group C. The subpressure groups showed less silver deposition than the control group (p < 0.05). The subpressure technique may remarkably improve bonding strength and decrease nanoleakage on total-etching bonding.

The effect of subpressure on the bond strength of resin to zirconia ceramic.

OBJECTIVE: This study was conducted to investigate the effect of subpressure on the bond strength of resin to zirconia ceramic. The subpressure would create a pressure gradient which could clean out the bubbles in the adhesives or bonding interface. METHODS: Twenty-eight pre-sintered zirconia discs were fabricated. Half of them were polished (group P, n = 14), and the rest were sandblasted (group S, n = 14). After sintered,the surface roughness of the zirconia discs was measured. Then, they were randomly divided into two subgroups (n = 7). The groups were named as follows: PC: P + no additional treatments; PP: P + 0.04 MPa after application of adhesives; SC: S + no additional treatments; and SP: S + 0.04 MPa after application of adhesives. Resin columns were bonded to the zirconia specimens to determine shear bond strength (SBS). The bonding interfaces were observed and the fracture modes were evaluated. Statistical analysis was performed on all data. RESULTS: The surface roughness of group S was significantly higher than that of group P (P<0.05). The SBS values were PC = 13.48 ± 0.7 MPa, PP = 15.22 ± 0.8 MPa, SC = 17.23 ± 0.7 MPa and SP = 21.68 ± 1.4 MPa. There were significant differences among the groups (P<0.05). Scanning electron microscopy (SEM) results showed that the adhesives of group SP and PP were closer and denser to the zirconia ceramic than that of group PC and SC. The proportion of the mixed fracture mode significantly increased after adding subpressure (P< 0.05). CONCLUSION: Subpressure can improve the shear bond strength of resin to zirconia ceramics and increase micro-infiltration between the adhesives and the zirconia ceramics, especially on the rough surfaces.