Impdh2 deficiency suppresses osteoclastogenesis through mitochondrial oxidative phosphorylation and alleviates ovariectomy-induced osteoporosis.

Abnormalities in osteoclastic generation or activity disrupt bone homeostasis and are highly involved in many pathologic bone-related diseases, including rheumatoid arthritis, osteopetrosis, and osteoporosis. Control of osteoclast-mediated bone resorption is crucial for treating these bone diseases. However, the mechanisms of control of osteoclastogenesis are incompletely understood. In this study, we identified that inosine 5'-monophosphate dehydrogenase type II (Impdh2) positively regulates bone resorption. By histomorphometric analysis, Impdh2 deletion in mouse myeloid lineage cells (Impdh2LysM-/- mice) showed a high bone mass due to the reduced osteoclast number. qPCR and western blotting results demonstrated that the expression of osteoclast marker genes, including Nfatc1, Ctsk, Calcr, Acp5, Dcstamp, and Atp6v0d2, was significantly decreased in the Impdh2LysM-/- mice. Furthermore, the Impdh inhibitor MPA treatment inhibited osteoclast differentiation and induced Impdh2-cytoophidia formation. The ability of osteoclast differentiation was recovered after MPA deprivation. Interestingly, genome-wide analysis revealed that the osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation, were impaired in the Impdh2LysM-/- mice. Moreover, the deletion of Impdh2 alleviated ovariectomy-induced bone loss. In conclusion, our findings revealed a previously unrecognized function of Impdh2, suggesting that Impdh2-mediated mechanisms represent therapeutic targets for osteolytic diseases.

A p38 MAP kinase inhibitor suppresses osteoclastogenesis and alleviates ovariectomy-induced bone loss through the inhibition of bone turnover.

Inhibition of excessive osteoclastic activity is an efficient therapeutic strategy for many bone diseases induced by increased bone resorption, such as osteoporosis. BMS-582949, a clinical p38α inhibitor, is a promising drug in Phase II studies for treating rheumatoid arthritis. However, its function on bone resorption is largely unknown. In this study, we find that BMS-582949 represses RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, BMS-582949 inhibits osteoclastic F-actin ring formation and osteoclast-specific gene expression. Mechanically, BMS-582949 treatment attenuates RANKL-mediated osteoclastogenesis through mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) signaling pathways without disturbing nuclear factor-κB (NF-κB) signaling. Interestingly, BMS-582949 impairs osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation (OXPHOS). Furthermore, BMS-582949 administration prevents bone loss in ovariectomized mouse mode by inhibiting both bone resorption and bone formation in vivo. Taken together, these findings indicate that BMS-582949 may be a potential and effective drug for the therapy of osteolytic diseases.

Preparation of dopamine-modified sea squirt cellulose hydrogel dust-fixing agent to prevent raising of dust.

Sea squirts, a tunicate, are found in all oceans and can foul marine ports and aquaculture, mainly affecting shipping and biodiversity. In this study, cellulose was extracted from sea squirts, and its hydrophilic properties were improved by substituting the hydrogen ions of the cellulose -OH with dopamine. The modified cellulose was used to prepare a hydrogel for use as a dust-fixing agent (CDP) to reduce air pollution caused by dust. After response surface method optimization, the proportions of binder, water-retaining agent, wetting agent, and antifreeze in CDP were 0.97, 1.44, 0.23, and 6.32%, respectively. This composition improved the wetting ability and permeability of CDP on particle surfaces. CDP exhibited good water retention at -11-50 °C. CDP reduced the wind erosion rate of dust at a wind speed of 12 m/s to 1.18%. The molecular dynamics method was used to analyze the wetting process and mechanism of CDP, revealing that hydrogen bonds were the dominant force at the solid-liquid interface. The adsorption of CDP onto the surface of coal increased the number of hydrophilic points. Water molecules were adsorbed on these hydrophilic points through hydrogen bonding, improving the binding energy between the solid and liquid interfaces. The application of ascidian cellulose in dust control makes full use of the biological value of ascidians, promoting sustainable development of the global biological economy.

Preparation and molecular simulation of an environmentally friendly dust-fixing agent based on chitosan-gelatin.

In order to solve the hazard of coal mine dust, a dust-fixing agent (GC-TG-JFC) was prepared with gelatin, chitosan, octadecanol polyoxyethylene ether, and glutamine transaminase. The experimental conditions and the formulation were optimized by response surface method. The ratio of gelatin, chitosan, octadecanol polyoxyethylene ether, and glutamine transaminase was 0.405:0.211:0.095:0.286 and the dilution ratio was 1:30. The results of product performance test showed that the dust fixation rate could reach 99.95% when the wind speed was 9 m/s. The viscosity of the diluted solution was 42.5 mPa·s. The Forcite module in Materials studio software was used to analyze and calculate the radial distribution concentration, diffusion coefficient, and binding energy of the solution. The results showed that GC-TG-JFC migrated more water molecules to the surface of coal through the action of van der Waals force and hydrogen bond. In addition, the binding energy of water molecules increased and the diffusion coefficient decreased, which improved the binding ability of water molecules with coal. It could be found that GC-TG-JFC had good dust fixation performance by combining experiment and molecular dynamics method.

Prognostic Factors for Primary Localized Gastrointestinal Stromal Tumors After Radical Resection: Shandong Gastrointestinal Surgery Study Group, Study 1201.

BACKGROUND: Most previous risk-prediction models for gastrointestinal stromal tumors (GISTs) were based on Western populations. In the current study, we collected data from 23 hospitals in Shandong Province, China, and used the data to examine prognostic factors in Chinese patients and establish a new recurrence-free survival (RFS) prediction model. METHODS: Records were analyzed for 5285 GIST patients. Independent prognostic factors were identified using Cox models. Receiver operating characteristic curve analysis was used to compare a novel RFS prediction model with current risk-prediction models. RESULTS: Overall, 4216 patients met the inclusion criteria and 3363 completed follow-up. One-, 3-, and 5-year RFS was 94.6% (95% confidence interval [CI] 93.8-95.4), 85.9% (95% CI 84.7-87.1), and 78.8% (95% CI 77.0-80.6), respectively. Sex, tumor location, size, mitotic count, and rupture were independent prognostic factors. A new prognostic index (PI) was developed: PI = 0.000 (if female) + 0.270 (if male) + 0.000 (if gastric GIST) + 0.350 (if non-gastric GIST) + 0.000 (if no tumor rupture) + 1.259 (if tumor rupture) + 0.000 (tumor mitotic count < 6 per 50 high-power fields [HPFs]) + 1.442 (tumor mitotic count between 6 and 10 per 50 HPFs) + 2.026 (tumor mitotic count > 10 per 50 HPFs) + 0.096 × tumor size (cm). Model-predicted 1-, 3-, and 5-year RFS was S(12, X) = 0.9926exp(PI), S(36, X) = 0.9739exp(PI) and S(60, X) = 0.9471exp(PI), respectively. CONCLUSIONS: Sex, tumor location, size, mitotic count, and rupture were independently prognostic for GIST recurrence. Our RFS prediction model is effective for Chinese GIST patients.

Influence of temperature on the pathogenicity of Ostreid herpesvirus-1 in ark clam, Scapharca broughtonii.

OsHV-1 is an epidemic pathogen of molluscs, and temperature has been recognized as a decisive environmental factor in its pathogenicity. In recent years, ark clam, Scapharca broughtonii, emerged as a host for OsHV-1. In the north of China, massive summer mortalities of ark clams infected with OsHV-1 have been continuously reported since 2012. However, the interaction between temperature and the pathogenicity of OsHV-1 was unknown in ark clams. In this study, the effect of temperature (10 °C to 18 °C stepped by 2 °C) on the occurrence of OsHV-1 disease in ark clams was analyzed. OsHV-1 infection led to gill erosion but not below the critical low temperature (between 12 °C and 14 °C). However, OsHV-1 persisted for more than 2 weeks at 12 °C post inoculation and replication was reactivated when the temperature was elevated to 18 °C. No significant reduction of OsHV-1 DNA load was found when the temperature descended to 12 °C from 18 °C, while the gill erosion remained unchanged. Ark clams failed to show the capability of effective clearance of OsHV-1 below the critical low temperature. Our results demonstrated that the pathogenicity of OsHV-1 was influenced significantly by temperature. Moreover, high temperature favored infection, which could provide more information to understand summer mortality of ark clams.

A feedback loop involving FREP and NF-κB regulates the immune response of sea cucumber Apostichopus japonicus.

Our previous work indicated that fibrinogen-related protein (AjFREP) from sea cucumber (Apostichopus japonicus) plays important roles in innate immunity. To understand AjFREP expression regulation in A. japonicus, we cloned the AjFREP promoter and characterized the putative transcription-factor binding motifs. The AjFREP promoter region spans 1365bp, containing several transcription-factor binding sites. The full-length sequence and all truncated fragments exhibited high promoter activity in HEK-293 cells. Luciferase activity significantly increased for P1(-1365/+16) after LPS exposure, suggesting that the promoter responded to LPS. We also found that two potential NF-κB binding sites were involved in the promoter region, and co-transfection assay demonstrated that the first binding site was necessary for AjFREP transcription. The expression of AjNF-κB/Rel after AjFREP knock-down followed by LPS injection in vivo was further investigated. We found that AjNF-κB/Rel transcript significantly decreased after silencing AjFREP with LPS challenge compared with the control at 4 and 8h, suggesting that activated AjFREP in turn affected the NF-κB pathway under immune responses. These results provided novel insights into the activation mechanism of AjFREP, i.e., that selectively changing AjFREP expression may prevent pathogen infection in A. japonicus.

Molecular characteristics, expression, and antimicrobial activities of i-type lysozyme from the razor clam Sinonovacula constricta.

Lysozyme is a key component of the innate immune system, which plays a pivotal role in early defense against pathogen infection. In this study, an i-type lysozyme homology was identified from the razor clam Sinonovacula constricta (designated as ScLYZ) through RACE approaches. The full-length cDNA of ScLYZ was 768 bp and encoded a polypeptide of 140 amino acid residues. SMART analysis revealed that ScLYZ processed a signal peptide (1-18 aa) and a destabilase domain from 25 to 133 aa. Two catalytic residues (Glu36 and Asp47) and two specific motifs ["CL(E/L/R/H)C(I/M)C" and "MDVGSLSCG(P/Y) (F/Y)QIK"] of the i-type lysozyme were highly conserved in the ScLYZ sequence. Multiple sequence alignments and phylogenetic analysis indicated that ScLYZ could be a new member of the i-type lysozyme subfamily. Tissue distribution analysis revealed that ScLYZ was constitutively expressed in all examined tissues, and the highest expression was found in the hepatopancreas. After the razor clams were challenged by Vibrio parahaemolyticus, the mRNA levels of ScLYZ increased in the gill and hepatopancreas. Moreover, the recombinant protein was expressed in Escherichia coli, and the refolded ScLYZ showed highly antimicrobial activities against V. parahaemolyticus and Vibrio splendidus. The minimal inhibitory concentration toward V. parahaemolyticus was 8.2 µmol/mL. All our results supported that ScLYZ was involved in the innate immune defense of razor clam by inhibiting the growth of invasive pathogens.

A novel C1q-domain-containing protein from razor clam Sinonovacula constricta mediates G-bacterial agglutination as a pattern recognition receptor.

Complement component 1q (C1q) with a characteristic C1q globular domain is an important pattern recognition molecule in the classical complement systems and plays a major role in the crosslinking between innate immunity and specific immunity in vertebrates. In this study, a homologous gene encoding typically C1q domains was obtained from the razor clam Sinonovacula constricta (designated ScC1qDC) by rapid amplification of the cDNA end. The full-length cDNA of ScC1qDC was 1225 bp in length with a 5'UTR of 258 bp, a 3'UTR of 223 bp, and an open reading frame of 744 bp encoding a polypeptide of 247 amino acids containing a typical C1q globular domain. The mRNA transcripts of ScC1qDC were constitutively transcribed in all examined tissues with higher expression in the hepatopancreas. Time-course expression analysis indicated that ScC1qDC was significantly up-regulated both in hepatopancreas and gills after Vibrio parahaemolyticus challenge. The recombinant ScC1qDC (rScC1qDC) displayed high binding activities to various pathogen-associated molecular patterns, including LPS, PGN, and MAN. Recombinant ScC1qDC showed no agglutinating activity to Gram-positive bacterium of Micrococcus luteus but showed obvious activities towards all the three examined Gram-negative bacteria. All our results indicated that ScC1qDC might be served as a pattern recognition receptor and promoted Gram-negative bacteria agglutination during the pathogen challenge.

Microsomal glutathione transferase 2 modulates LTC4 synthesis and ROS production in Apostichopus japonicus.

Microsomal glutathione transferase 2 (mGST2) is an integral membrane protein involved in detoxication of xenobiotics, and has also been suggested to catalyze the biosynthesis of pro-inflammatory mediator leukotriene C4 (LTC4) as homologous to LTC4 synthase (LTC4S) in mammals. In the present study, a novel mGST2 homology was identified from Apostichopus japonicus (designated as AjmGST2) by RACE approaches. The full-length cDNA of AjmGST2 was of 1917bp encoding a polypeptide of 161 amino acids residues. Multiple sequences alignment and phylogenetic analysis together supported that AjmGST2 belonged to a new member in invertebrate mGSTs family and close to mammalian LTC4S. Spatial expression analysis revealed that AjmGST2 was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. AjmGST2 transcripts in coelomocytes were slightly induced post 6h challenge of pathogenic Vibrio splendidus and reached the peak expression at 48h. The increased expression profiles of AjmGST2 were also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. Consistently, LTC4 contents were also induced by a 1.56-fold increase in the same condition. Functional assay further revealed that AjmGST2 might be functioned as LTC4S to promote LTC4 synthesis. AjmGST2 knock-down by specific siRNA significantly depressed LTC4 contents with 27.0% decrease at 24h. Meantime, ROS levels were elevated by 40.1% in vitro. All of these results indicated that AjmGST2 performed dual functions roles as LTC4S and ROS eliminator in sea cucumber immune response.

[Analysis of gastric gastrointestinal stromal tumors in Shandong Province: a midterm report of multicenter GISSG1201 study].

OBJECTIVE: To summarize the treatment status of gastric gastrointestinal stromal tumor (GIST) in Shandong province,by analyzing the clinicopathological features and prognostic factors. METHODS: Clinicopathological and follow-up data of 1 165 patients with gastric GIST between January 2000 and December 2013 from 23 tertiary referral hospitals in Shandong Province were collected to establish a database. The risk stratification of all cases was performed according to the National Institutes of Health(NIH) criteria proposed in 2008. Kaplan-Meier method was used to calculate the survival rate. Log-rank test and Cox regression model were used for univariate and multivariate prognostic analyses. RESULTS: Among 1 165 cases of gastric GIST, 557 were male and 608 were female. The median age of onset was 60 (range 15-89) years. Primary tumors were located in the gastric fundus and cardia in 623 cases(53.5%), gastric body in 346 cases(29.7%), gastric antrum in 196 cases(16.8%). All the cases underwent resection of tumors, including endoscopic resection (n=106), local resection (n=589), subtotal gastrectomy(n=399), and total gastrectomy(n=72). Based on the NIH risk stratification, there were 256 cases (22.0%) at very low risk, 435 (37.3%) at low risk, 251 cases (21.5%) at intermediate risk, and 223 cases (19.1%) at high risk. A total of 1 116 cases(95.8%) were followed up and the median follow-up period was 40 (range, 1-60) months. During the period, 337 patients relapsed and the median time to recurrence was 34 (range 1-60) months. The 1-, 3-, and 5-year survival rates were 98.6%, 86.1% and 73.4%, respectively. The 5-year survival rates of patients at very low, low, intermediate, and high risk were 93.1%, 85.8%, 63.0% and 42.3% respectively, with a statistically significant difference (P=0.000). Multivariate analysis showed that primary tumor site (RR=0.580, 95%CI:0.402-0.835), tumor size (RR=0.450, 95%CI:0.266-0.760), intraoperative tumor rupture(RR=0.557, 95%CI:0.336-0.924), risk classification (RR=0.309, 95%CI:0.164-0.580) and the use of imatinib after surgery (RR=1.993, 95%CI:1.350-2.922) were independent prognostic factors. CONCLUSIONS: The choice of surgical procedure for gastric GIST patients should be based on tumor size. All the routine procedures including endoscopic resection, local excision, subtotal gastrectomy and total gastrectomy can obtain satisfactory curative outcomes. NIH classification has a high value for the prediction of prognosis. Primary tumor site, tumor size, intraoperative tumor rupture, risk stratification and postoperative use of imatinib are independent prognostic factors in gastric GIST patients.

HMGB3 modulates ROS production via activating TLR cascade in Apostichopus japonicus.

High mobility group box protein 3 (HMGB3) regulates proliferation and inflammatory response in vertebrates. However, its functional roles in invertebrates are largely unknown. In this study, a HMGB3 homologue molecule was identified from Apostichopus japonicus (designated as AjHMGB3) by RACE approach. The full-length cDNA of AjHMGB3 was of 2298 bp with an open reading frame of 1320 bp encoding a 439-amino-acid (aa) residue protein. Structural analysis then conducted and the results revealed that AjHMGB3 processed two conserved HMGBs (133-204 and 210-279 aa) and an acidic tail. The results of subsequent multiple sequence alignment and phylogenetic analysis both indicated that AjHMGB3 belongs to a new member of HMGB3 protein subfamily. Furthermore, AjHMGB3 was expressed in all examined tissues except in tentacles and particularly highly expressed in the intestine, as indicated by spatial expression analysis results. The Vibrio splendidus challenge in vivo and lipolysaccharide (LPS) stimulation in vitro can significantly upregulate the mRNA expression of AjHMGB3 in coelomocytes. This finding is consistent with the expression profiles of TLR cascade members. We further investigated the expression profiles of AjMyD88 and Ajp105 after the gain- or loss-of-function of AjHMGB3 in coelomocytes. The results showed that AjMyD88 and Ajp105 were upregulated 2.19- and 2.83-fold in AjHMGB3 overexpressed treatment and downregulated 0.38- and 0.43-fold in the AjHMGB3 silencing group. The p50 subunit displayed expression profiles that are identical to those of AjMyD88 and Ajp105 according to the Western blot results. In the same condition, the respiratory burst was increased by 37.5% in the AjHMGB3 overexpressed group and depressed by 28.2% in the AjHMGB3 knock-down group. Our present findings collectively suggested that AjHMGB3 acted as an NF-κB activator and produced ROS production in sea cucumbers.

Characterization of NLRP3-like gene from Apostichopus japonicus provides new evidence on inflammation response in invertebrates.

Inflammatory/defensive response after pathogen invasion is considered a local defense reaction in vertebrates. Inflammation response in Apostichopus japonicus was hardly determined due to scarce information available for nucleotide binding domain-like receptor family, pyrin domain-containing (NLRP) family. In the present study, invertebrate NLRP homologue was identified from A. japonicus (designated as AjNLRP3-like) by rapid amplification of cDNA ends. Full-length cDNA of AjNLRP3-like measured 2970 bp with 2265 bp open reading frame encoding a 754-amino acid (aa) residue protein. Structural analysis revealed that AjNLRP3-like processed characteristic domains of pyrin (32-102aa) and NACHT (183-339aa). Multiple sequence alignment and phylogenetic analysis supported that AjNLRP3-like belongs to a new member of NLRP3 protein subfamily. Spatial expression analysis revealed that AjNLRP3-like was ubiquitously expressed in all examined tissues with larger magnitude in coelomocytes. Both Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly upregulated mRNA expression of AjNLRP3-like when compared with the control group. NLRP3-mediated inflammation response depended on release of lysosomal cathepsin B (CTSB) and subsequent activation of high-mobility group box (HMGB) in vertebrates. We investigated expression profiles of AjNLRP3-like and AjHMGB after AjCTSB knock-down and discovered that AjNLRP3-like was depressed by 0.66-fold and 0.47-fold, whereas AjHMGB was depressed by 0.70-fold and 0.50-fold at 24 and 48 h in AjCTSB-silenced group, respectively. Similarly, down-regulation of AjHMGB was also observed after AjNLRP3-like knock-down. This study therefore suggests that A. japonicus feature similar inflammatory events as those in vertebrates, and activation of AjNLRP3-like depends on AjCTSB expression and release of AjHMGB.

Mice overexpressing hepatic Abcb11 rapidly develop cholesterol gallstones.

Cholelithiasis is a polygenic disease, although the genes responsible for gallstone formation have not yet been clearly identified. QTL analysis has identified the Lith 1 loci on mouse Chromosome 2, and the hepatic bile salt transporter Abcb11 maps to the Lith 1 locus. We have used recently developed TTR-Abcb11 transgenic mice that overexpress Abcb11 to determine the effects of Abcb11 overexpression on cholesterol gallstone formation. TTR-Abcb11 and FVB/NJ strain control mice were fed a lithogenic or chow diet and cholesterol crystal and gallstone formation were measured. Biliary lipids in gallbladder bile and gene expression of canalicular lipid transporters were also analyzed. TTR-Abcb11 mice fed a lithogenic diet had an increased rate of cholesterol crystal and gallstone formation. This was associated with an increase in both the hydrophobic bile salt and cholesterol content of gallbladder bile. Expression of Abcb4, Abcg5, and Abcg8 did not change before gallstone formation. These data indicate that hepatic overexpression of Abcb11 increases the rate of cholesterol gallstone formation. This is likely because of increases in bile salt hydrophobicity but not because of alterations of other biliary lipid transporters. These findings strongly support Abcb11 as a Lith 1 gene.