[Mechanism of Qiwei Guibao Granules in treatment of premature ovarian failure based on proteomics].

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.

The roles of autophagy in osteogenic differentiation in rat ligamentum fibroblasts: Evidence and possible implications.

Autophagy, a macromolecular degradation process, plays a pivotal role in cell differentiation and survival. This study was designed to investigate the role of autophagy in the osteogenic differentiation in ligamentum fibroblasts. Rat ligamentum fibroblasts were isolated from the posterior longitudinal ligament and cultured in osteogenic induction medium. Ultrastructural analysis, immunofluorescence assay, western blot, flow cytometry, and lysosomal activity assessment were performed to determine the presence and activity of autophagy in the cells. The mineralization deposit and osteogenic gene expressions were evaluated to classify the association between autophagy activity and the bone formation ability of the spinal ligament cells. The influence of leptin and endothelin-1 on the autophagy activity was also evaluated. Our study demonstrated that autophagy was present and increased in the ligament cells under osteogenic induction. Inhibition of autophagy with either pharmacologic inhibitors (Bafilomycin A and 3-methyladenine) or Belcin1 (BECN1) knocking down weakened the mineralization capacity, decreased the gene expressions of COL1A1, osteocalcin (Ocn), and runt-related transcription factor 2 (Runx2) in the ligamentum fibroblasts and increased cell apoptosis. The Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-BECN1 autophagic pathway was activated in the osteogenic differentiating ligamentum fibroblasts. Leptin significantly increased the autophagy activity in the ligament cells under osteogenic induction. These discoveries might improve our understanding for the mechanism of ossification of the posterior longitudinal ligament (OPLL) and provide new approaches on the prevention and treatment of this not uncommon disease.

Intra-brachial ergonovine, not acetylcholine, is associated with radial artery vasospasm in patients with coronary vasospasm.

BACKGROUND: The intracoronary provocation test is expensive and may cause complications. Therefore, we investigated the sensitivity, specificity and safety of different drug- and dose-peripheral artery provocation tests in the diagnosis of coronary artery spasm (CAS). METHODS: The patients who had repeated chest pain as well as both coronary and radial stenoses <50% were selected. These patients were divided into CAS group (n = 24) and control group (n = 33) after the intracoronary ergonovine provocation test. All patients underwent radial artery provocation tests at different dose-acetylcholine (200 µg, 400 µg and 800 µg) and ergonovine (60 µg, 100 µg and 160 µg). The predictive values of radial provocation tests for CAS diagnosis were analysed using receiver operator characteristic (ROC) curves. RESULTS: In radial acetylcholine provocation tests, 200 µg of acetylcholine failed to induce radial artery spasm, and the radial artery stenosis degree was not significantly different between the CAS group and control group at 400 µg and 800 µg of acetylcholine (all p > 0.05). In the radial artery ergonovine provocation tests, the radial artery stenosis degree was all significantly higher in the CAS group than in the control group at the three different doses (all p < 0.05). The specificity and sensitivity of radial ergonovine provocation tests were 90.91% and 50.00% at 60 µg, 96.97% and 66.67% at 100 µg, and 90.91% and 95.83% at 160 µg. Only the radial 160 µg-ergonovine provocation test caused CAS in one case. CONCLUSION: The radial acetylcholine provocation test has no diagnostic value for CAS. The radial 160 µg-ergonovine provocation test has higher sensitivity and specificity for CAS diagnosis, but its safety should be paid attention to.

Sodium hydrosulfide alleviates dexamethasone-induced cell senescence and dysfunction through targeting the miR-22/sirt1 pathway in osteoblastic MC3T3-E1 cells.

Glucocorticoid-induced osteoporosis is characterized by osteoblastic cell and microarchitecture dysfunction, as well as a loss of bone mass. Cell senescence contributes to the pathological process of osteoporosis and sodium hydrosulfide (NaHS) regulates the potent protective effects through delaying cell senescence. The aim of the present study was to investigate whether senescence could contribute to dexamethasone (Dex)-induced osteoblast impairment and to examine the effect of NaHS on Dex-induced cell senescence and damage. It was found that the levels of the senescence-associated markers, p53 and p21, were markedly increased in osteoblasts exposed to Dex. A p53 inhibitor reversed Dex-induced osteoblast injury, a process that was mitigated by NaHS administration through alleviating osteoblastic cell senescence. MicroRNA (miR)-22 blocked the impact of NaHS on Dex-induced osteoblast damage and senescence through targeting the regulation of Sirtuin 1 (sirt1) expression, as shown by the decreased cell viability and alkaline phosphatase activity, as well as an increased expression of p53 and p21. It was revealed that the sirt1 gene was the target of miR-22 in osteoblastic MC3T3-E1 cells through combining the results of dual luciferase reporter assays and reverse transcription-quantitative PCR, as well as western blot analyses. Silencing of sirt1 abolished the protective effect of NaHS against Dex-associated osteoblast senescence and injury. Taken together, the present study showed that NaHS prevents Dex-induced cell senescence and damage through targeting the miR-22/sirt1 pathway in osteoblastic MC3T3-E1 cells.

Oxidative stress induces downregulation of TP53INP2 and suppresses osteogenic differentiation of BMSCs during osteoporosis through the autophagy degradation pathway.

Oxidative stress plays an important role in the pathogenesis of osteoporosis and impaired bone formation. However, the mechanisms behind which oxidative stress represses bone formation remains unclear. TP53INP2, a target of the tumor suppressor p53, is ubiquitously expressed in various cell types including BMSCs and contributes to autophagosome formation by recruiting ubiquitinated substrates to autophagosomes for degradation. However, little is known about its function in BMSCs and its relation to osteoporosis. In this study, first, we verified that the expression of TP53INP2 was persistently decreased in BMSCs derived from osteoporosis patients and OVX mice, and that the antioxidant N-acetylcysteine could ameliorate this decreased TP53INP2 level in vitro. Second, we identified that the mRNA and protein levels of TP53INP2 decreased in BMSCs under H2O2 induced oxidative stress in a dose-dependent manner, with resultant co-location of LC3 and TP53INP2. Additionally, the autophagy-lysosome system was involved in the degradation process of TP53INP2 and applying autophagy inhibitors (Baf-A1) could significantly increase the TP53INP2 levels in murine and human BMSCs under oxidative stress. Third, gain- and loss-of-function assays revealed that knockdown of TP53INP2 inhibited osteogenic differentiation of BMSCs, while overexpression of TP53INP2 promoted osteogenic differentiation of BMSCs in vitro. Further analysis demonstrated that TP53INP2 promoted osteogenic differentiation of BMSCs by activating Wnt/ß-cantenin signaling. DKK1, an inhibitor of Wnt signaling, resulted in osteogenic defects of BMSCs that had over-expressed TP53INP2. Lithium, a Wnt/ß-catenin activator, improved the mineralization ability in TP53INP2-knockdown BMSCs. Moreover, restoring TP53INP2 levels recovered OVX-derived BMSCs osteogenic differentiation and attenuated bone loss in OVX mice. Taken together, our study indicated that oxidative stress-induced downregulation of TP53INP2 suppressed osteogenic differentiation of BMSCs during osteoporosis and was mediated by the autophagy degradation pathway. These findings may introduce a novel therapeutic target for osteoporosis.

Preparation of RGD-modified long circulating liposome loading matrine, and its in vitro anti-cancer effects.

AIM: To prepare RGD-modified long circulating liposome (LCL) loading matrine (RGD-M-LCL) to improve the tumor-targeting and efficacy of matrine. METHODS: LCL which was prepared with HSPC, cholesterol, DSPE-PEG2000 and DSPE-PEG-MAL was modified with an RGD motif confirmed by high performance liquid chromatography (HPLC). The encapsulation efficiency of RGD-M-LCL was also detected by HPLC. MTT assay was used to examine the effects of RGD-M-LCL on the proliferation of Bcap-37, HT-29 and A375 cells. The percentage of apoptotic cells and morphological changes in Bcap-37 cells treated with RGD-M-LCL were detected by Annexin-V-FITC/PI affinity assay and observed under light microscope, respectively. RESULTS: Spherical or oval single-chamber particles of uniform sizes with little agglutination or adhesion were observed under transmission electronic microscope. The RGD motif was successfully coupled to the DSPE-PEG-MAL on liposomes, as confirmed by HPLC. An encapsulation efficiency of 83.13% was obtained when the drug-lipid molar ratio was 0.1, and the encapsulation efficiency was negatively related to the drug-lipid ratio in the range of 0.1-0.4, and to the duration of storage. We found that, compared with free matrine, RGD-M-LCL had much stronger in vitro activity, leading to anti-proliferative and pro-apoptotic effects against cancer cells (P<0.01). CONCLUSION: RGD-M-LCL, a novel delivery system for anti-cancer drugs, was successfully prepared, and we demonstrated that the use of this material could augment the effects of matrine on cancer cells in vitro.

Synthesis and evaluation of novel substituted 5-hydroxycoumarin and pyranocoumarin derivatives exhibiting significant antiproliferative activity against breast cancer cell lines.

A library of novel 5-hydroxycoumarin and pyranocoumarin derivatives was constructed via silica sulfuric acid-catalyzed pechmann reaction and Pd(0)-catalyzed suzuki coupling in tandem, and their antiproliferative activities against breast cancer cells MCF-7 and MDA-MB-231 were evaluated. The results showed that compounds such as 6b, 6d, 6h, and 6k possess significant antiproliferative activity against MCF-7 cell line with the IC(50) values of 7.2, 5.3, 3.3, and 6.5 microM, respectively.

[Echocardiographic evaluation of crisscross heart].

OBJECTIVE: To explore atrioventricular connection and atrioventricular segmental situs in patients with crisscross heart (CCH) and to evaluate the diagnostic value of echocardiography for this anomaly. METHODS: Ten consecutive patients with crisscross heart were enrolled into this retrospective study. Their echocardiographic data were analyzed and compared with the results of X-ray angiocardiography and 64-slice multi-detector row computed tomography (MDCT) or MRI. RESULTS: The crossing of atrioventricular valves could be seen in each case by scanning in a subxiphoid or apical 4-chamber view. Both the positive rate and the specificity were 100%. Horizontal ventricular septum was in 9 cases and vertical (sagittal) ventricular septum in 1 case. The segmental set of 8 patients with concordant atrioventricular connection was {S. D. L} in 5 cases, {S. D. D} 1 case, {S. D. S} 1 case and {S. L. D} 1 case. The segmental set of 1 case with discordant atrioventricular connection was {I. D. D} and another 1 case with ambiguous atrioventricular connection was {A. L. L}. In 1 case, the atrioventricular connection was inconsistent with the atrioventricular segmental situs. Ventriculoarterial connections were concordant in 1, DORV in 6, TGA in 2 and C-TGA in 1. CONCLUSION: Echocardiography is proven quite helpful in diagnosis of CCH, and continuous sweeps in subxiphoid long-axis plane or apical 4-chamber view play a key role. Both the atrioventricular connection and the atrioventricular segmental situs are complicated so that they are not always concordance with each other. It is necessary to account for separately.

Incidence of metastatic disease and survival among patients with newly diagnosed primary CNS tumors in the United States from 2004-2013.

Background: Population-based estimates of the incidence and prognosis of metastatic disease at the initial diagnosis of primary central nervous system (CNS) tumors are currently lacking. Methods: A total of 43,455 patients diagnosed with a primary CNS tumor were enrolled to evaluate metastatic rates utilizing the data from the Surveillance, Epidemiology, and End Results (SEER) program. We used multivariate logistic regression to analyze the risk factors associated with the presence of metastasis at the first visit of patients with metastatic medulloblastoma (MB), atypical teratoid/rhabdoid tumor (ATRT), glioblastoma multiforme (GBM), or pilocytic astrocytoma (PA). Hazard ratios (HRs) and 95% confidence intervals (CIs) for cancer-specific death (CSD) of patients with these four CNS tumors were analyzed using multivariate Cox regression. Results: In patients with primary CNS embryonal tumors, the metastatic rates of patients with MB and ATRT were 14.51% and 19.25%, respectively. The metastatic rate for MB patients aged 0 to 18 years was 16.69%. In the patients with glioma, the metastatic rates of patients with PA and GBM were 1.55% and 1.39%, respectively. On multivariate logistic regression among patients with glioma, GBM (vs PA; OR, 2.12; 95% CI, 1.37 to 3.30; P=0.001) was associated with greater odds of having metastatic disease at diagnosis. On multivariate logistic regression among patients with GBM, MB, or ATRT, MB (vs GBM; OR, 4.66; 95% CI, 2.81 to 7.72; P<0.001) and ATRT (vs GBM; OR, 5.65; 95% CI, 3.27 to 9.75; P<0.001) were associated with greater odds of having metastatic disease at diagnosis. In the multivariate Cox proportional hazards model for CSD among patients with metastatic GBM or MB at diagnosis, gross total resection/total lobectomy (vs partial resection/partial lobectomy) was not related to a decreased or an increased risk of CSD. In patients with metastatic ATRT, compared to no surgery, gross total resection/total lobectomy or partial resection/partial lobectomy was not associated with a decreased risk of CSD. Conclusions: The findings in this study provide a population-based estimate of the incidence and prognosis of metastatic disease at the initial diagnosis of primary CNS tumors. These survival outcomes are relevant because they will help to prioritize future research directions to improve the treatment strategies of these metastatic CNS tumors.

Integrative analysis of gene expression and DNA methylation through one-class logistic regression machine learning identifies stemness features in medulloblastoma.

Most human cancers develop from stem and progenitor cell populations through the sequential accumulation of various genetic and epigenetic alterations. Cancer stem cells have been identified from medulloblastoma (MB), but a comprehensive understanding of MB stemness, including the interactions between the tumor immune microenvironment and MB stemness, is lacking. Here, we employed a trained stemness index model based on an existent one-class logistic regression (OCLR) machine-learning method to score MB samples; we then obtained two stemness indices, a gene expression-based stemness index (mRNAsi) and a DNA methylation-based stemness index (mDNAsi), to perform an integrated analysis of MB stemness in a cohort of primary cancer samples (n = 763). We observed an inverse trend between mRNAsi and mDNAsi for MB subgroup and metastatic status. By applying the univariable Cox regression analysis, we found that mRNAsi significantly correlated with overall survival (OS) for all MB patients, whereas mDNAsi had no significant association with OS for all MB patients. In addition, by combining the Lasso-penalized Cox regression machine-learning approach with univariate and multivariate Cox regression analyses, we identified a stemness-related gene expression signature that accurately predicted survival in patients with Sonic hedgehog (SHH) MB. Furthermore, positive correlations between mRNAsi and prognostic copy number aberrations in SHH MB, including MYCN amplifications and GLI2 amplifications, were detected. Analyses of the immune microenvironment revealed unanticipated correlations of MB stemness with infiltrating immune cells. Lastly, using the Connectivity Map, we identified potential drugs targeting the MB stemness signature. Our findings based on stemness indices might advance the development of objective diagnostic tools for quantitating MB stemness and lead to novel biomarkers that predict the survival of patients with MB or the efficacy of strategies targeting MB stem cells.

Four new aromatic allenic ethers from the fungus Xylaria sp. No. 2508.

Four new aromatic allenic ethers, (7E)-3-(4-buta-2,3-dienyloxy-3-methoxy-phenyl)-acrylic acid methyl ester (1), (7E)-3-[4-(4-buta-2,3-dienyloxy-benzyloxy)-phenyl]-acrylic acid methyl ester (2), 4-(4-buta-2,3-dienyloxy-benzyloxy)-benzoic acid methyl ester (3), (7E)-3-[4-(4-buta-2,3- dienyloxy-benzyloxy)-3-methoxy-phenyl]-acrylic acid methyl ester (4) were isolated from the fungus Xylaria sp. No. 2508. The structures of those compounds were determined by analysis of spectroscopic data, mainly 2D NMR experiments.

A new species of Onryza Watson, 1893 (Lepidoptera: Hesperiidae) from China.

A new species, Onryza pesudomaga is described from Zhejiang Province, S. China. The new taxon resembles O. maga which is widely distributed in S. China and Taiwan. Differences and some biological information of the two allied species are given. A key to species of the genus Onryza Watson, 1893 and a distribution map of all the members of this genus are provided.

The role of R-spondins and their receptors in bone metabolism.

R-spondins are a family of four secreted proteins and act as agonists of the canonical Wnt/ß-catenin signaling pathway. They are broadly expressed in different phases of skeleton tissues. Recently, three closely related leucine-rich repeat containing G-protein-coupled receptors (Lgr4/5/6) have been identified as the new and exact receptors of R-spondins. On the cell surface, R-spondins binding with Lgr4/5/6 and Znrf3/Rnf43 lead to reduced turnover of Wnts-receptors and potentiate Wnt/ß-catenin signaling pathway which is critical for the control of bone development and remodeling. There has been a growing interest in understanding the role and mechanism of R-spondins and their receptors in multiple biological processes, including bone development and metabolism. Recent advances in the R-spondins revealed the potential modulatory effect on osteoblastogenesis and bone formation and provided a new avenue for the investigation of adult bone metabolism. The receptors of Lgr4/5/6 and stabilized ß-catenin are essential to the regulatory effect of Rspos on skeleton. The findings on Rspo/Lgr signaling might have clinical potentials for the treatment of bone loss related diseases.

Screening of candidate genes and fine mapping of drought tolerance quantitative trait loci on chromosome 4 in rice (Oryza sativa L.) under drought stress.

Due to severe water resource shortage, genetics of and breeding for DT (drought tolerance) in rice (Oryza sativa L.) have become one of the hot research topics. Identification of grain yield QTLs (quantitative trait loci) directly related to the DT trait of rice can provide useful information for breeding new drought-resistant and water-saving rice varieties via marker-assisted selection. A population of 105 advanced BILs (backcross introgression lines) derived from a cross between Zhenshan97B and IRAT109 in Zhenshan97B background were grown under drought stress in a field experiment and phenotypic traits were investigated. The results showed that in the target interval of RM273-RM255 on chromosome 4, three main-effect QTLs related to panicle length, panicle number, and spikelet number per panicle were identified (LOD [logarithm of the odds] > 2.0). The panicle length-related QTL had two loci located in the neighboring intervals of RM17308-RM17305 and RM17349-RM17190, which explained 18.80% and 20.42%, respectively, of the phenotypic variation, while the panicle number-related QTL was identified in the interval of RM1354-RM17308, explaining 11.47% of the phenotypic variation. As far as the spikelet number per panicle-related QTL was concerned, it was found to be located in the interval of RM17308-RM17305, which explained 28.08% of the phenotypic variation. Using the online Plant-GE query system, a total of 13 matched ESTs (expressed sequence tags) were found in the target region, and of the 13 ESTs, 12 had corresponding predicted genes. For instance, the two ESTs CB096766 and CA765747 were corresponded to the same predicted gene LOC_Os04g46370, while the other four ESTs, CA754286, CB000011, CX056247, and CX056240, were corresponded to the same predicted gene LOC_Os04g46390.

[Construction and identification of small interfering RNA expression vector targeting ATF-2 gene].

AIM: To construct an eukaryotic expression vector for RNA interference targeting activating transcription factor 2 (ATF-2) gene, and explore its effect on proliferation and apoptosis of HepG2 cells. METHODS: Two complementary oligonucleotides were synthesized based on ATF-2 mRNA sequence. The annealed fragment was inserted into the vector PBA-siU6. The recombinant plasmid PBA-siATF-2 was confirmed by DNA sequencing and transfected into HepG2 cells mediated by liposome. After transfection, ATF-2 protein was detected by Western blotting. The cellular growth activity and apoptosis rate were measured by MTT assay and flow cytometry, respectively. RESULTS: Recombinant plasmid expressing siRNA targeting ATF-2 gene was confirmed by DNA sequencing. Plasmid transfection down-regulated the level of ATF-2 protein in HepG2 cells, which blocked cellular growth and induced cell apoptosis. CONCLUSION: The eukaryotic expression vector for RNA interference targeting ATF-2 gene was constructed successfully, which inhibits HepG2 cell proliferation and induces cell apoptosis.

Synthesis and biological evaluation of 12 allenic aromatic ethers.

Twelve allenic aromatic ethers, some of them are natural products isolated from the mangrove fungus Xylaria sp. 2508 in the South China Sea, were synthesized. Their antitumor activities against KB and KBv200 cells were determined. All these compounds demonstrated cytotoxic potential, ranging from weak to strong activity. The analysis of structure-activity relationships suggested that the introduction of allenic moiety could generate or enhance cytotoxicity of these phenol compounds.