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1.  Comparing endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) versus fine needle biopsy (FNB) in the diagnosis of solid lesions: study protocol for a randomized controlled trial 
Trials  2016;17:198.
Linear endoscopic ultrasonography (EUS) allows the visualization, identification, and characterization of the extent of lesions of the gastrointestinal (GI) tract and adjacent structures. EUS-guided fine-needle aspiration (EUS-FNA) facilitates a more accurate diagnosis of mediastinal, intra-abdominal, and pancreatic lesions through the collection of the cytological material under direct visualization. Recent reports suggest that histological samples can be obtained by EUS-FNA with a reverse, bevel-tipped needle (the ProCore needle) to collect the core samples (fine needle biopsy, FNB), thereby adding a new dimension to the diagnostic usefulness of this technique. Certain neoplasms, such as lymphoma and stromal tumors, can be assessed by EUS-FNB to confirm the diagnosis. Here, we aimed to carry out a prospective, multicenter, single-blind, randomized, controlled trial to compare EUS-FNB and EUS-FNA.
A total of 408 patients will be enrolled from five endoscopic centers. Patients will be divided into two groups: (1) group A, which is the EUS regular needle group (EUS-FNA) and (2) group B, which is the EUS ProCore needle group (EUS-FNB). Patients in group A will be examined with a 22G EchoTip Ultra needle, and patients in group B, with a 22G EchoTip ProCore needle. For all included patients, four EUS-guided passes will be made in each lesion. In the first and second pass, a slow-pull suction method of the stylet will be done. The third and fourth pass will use manual suction of 5 cc. The primary objective is to compare the diagnostic yield of malignancy by EUS-FNA versus EUS-FNB.
The trial will compare samples obtained by EUS-FNA versus EUS-FNB for the diagnostic yield of solid lesions. The efficacy of these two sampling methods will be assessed on various lesions, which may provide insights into developing practice guidelines for their future indications.
Trial registration
Clinical, NCT02327065.
PMCID: PMC4830051  PMID: 27071386
EUS-FNA; EUS-FNB; Slow-pull; Suction; Solid lesions; Diagnostic yield
2.  Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway 
Autophagy  2015;11(2):344-354.
Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.
PMCID: PMC4502731  PMID: 25831014
autophagic cell death; autophagy; E. coli O157:H7; ER stress; Shiga toxins; 3-MA, 3-methyladenine; Δ, knockout; AO, acridine orange; ATF4, activating transcription factor 4; ATG, autophagy-related; Baf A1, bafilomycin A1; BECN1, Beclin 1, autophagy-related; CASP3, caspase 3, apoptosis-related cysteine peptidase; DDIT3, DNA-damage-inducible transcript 3; EHEC O157, Escherichia coli O157:H7; FACS, fluorescence activated cell sorting; MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; MAPK, mitogen-activated protein kinase; MDC, monodansylcadaverine; NUPR1, nuclear protein, transcriptional regulator, 1; PBS, phosphate-buffered saline; PARP1, poly (ADP-ribose) polymerase 1; PI, propidium iodide; Stxs, Shiga toxins; Thap, thapsigargin; TEM, transmission electron microscopy; TRIB3, tribbles pseudokinase 3; WT, wild type; Z-VAD, Z-VAD-FMK
3.  Untargeted Metabolomics Reveals Dose-Response Characteristics for Effect of Rhubarb in a Rat Model of Cholestasis 
Cholestasis is a serious manifestation of liver diseases with limited therapies. Rhubarb, a widely used herbal medicine, has been frequently used at a relatively large dose for treating cholestasis. However, whether large doses are optimal and the therapeutic mechanism remain unclear. To explore these questions, the anti-cholestatic effect of five doses of rhubarb (0.21, 0.66, 2.10, 6.60, and 21.0 g/kg) in an alpha-naphthylisothiocyanate (ANIT)-induced rat model of cholestasis was examined by histopathology and serum biochemistry. A dose-dependent anti-cholestatic effect of rhubarb (0.21–6.6 g/kg) was observed, and an overdose of 21.0 g/kg showed a poor effect. LC-MS-based untargeted metabolomics together with pathway analysis were further applied to characterize the metabolic alterations induced by the different rhubarb doses. Altogether, 13 biomarkers were identified. The dose-response curve based on nine important biomarkers indicated that doses in the 0.42–6.61 g/kg range (EC20–EC80 range, corresponding to 4.00–62.95 g in the clinic) were effective for cholestasis treatment. The pathway analysis showed that bile acid metabolism and excretion, inflammation and amino acid metabolism were altered by rhubarb in a dose-dependent manner and might be involved in the dose-response relationship and therapeutic mechanism of rhubarb for cholestasis treatment.
PMCID: PMC4814850  PMID: 27065293
metabolomics; cholestasis; rhubarb; dose response; pathway analysis
4.  The pro-apoptotic effects of TIPE2 on AA rat fibroblast-like synoviocytes via regulation of the DR5–caspase–NF-κB pathway in vitro 
OncoTargets and therapy  2016;9:993-1000.
TIPE2, also known as TNFAIP8L2, a member of the tumor necrosis factor-alpha-induced protein-8 (TNFAIP8) family, is known as an inhibitor in inflammation and cancer, and its overexpression induces cell death. We examined the role of TIPE2 with respect to adjuvant arthritis (AA)-associated pathogenesis by analyzing the TIPE2 regulation of death receptor (DR5)-mediated apoptosis in vitro. The results showed that TIPE2 was detected in normal fibroblast-like synoviocytes (FLSs), but scarcely observed in AA-FLSs. Therefore, recombinant MIGR1/TIPE2+/+ and control MIGR1 lentivirus vectors were transfected to AA-FLSs, which were denoted as TIPE2+/+-FLSs and MIGR1-FLSs, respectively. Our results showed that TIPE2+/+-FLSs were highly susceptible to ZF1-mediated apoptosis, and ZF1 was our own purification of an anti-DR5 single chain variable fragment antibody. Under the presence of TIPE2, the expression of DR5 was significantly increased compared with that of the MIGR1-FLS group. In contrast, the level of phosphorylated nuclear factor-kappa B (pNF-κB) was lower in the TIPE2+/+-FLS group treated with ZF1, whereas the activity of caspase was higher. Moreover, the rate of apoptosis in the TIPE2+/+-FLS group, which was pretreated with caspase inhibitor Z-VAD-FMK, was significantly decreased. In contrast, the apoptosis occurrence in the MIGR1-FLS group increased significantly with the pretreatment of the NF-κB inhibitor Bay. These results indicated that TIPE2 increased the apoptosis of AA-FLSs by enhancing DR5 expression levels, thereby promoting the activation of caspase and inhibiting the activation of NF-κB in AA-FLSs. TIPE2 might potentially act as a therapeutic target for rheumatoid arthritis.
PMCID: PMC4778775  PMID: 27013892
rheumatoid arthritis; adjuvant arthritis; fibroblast-like synoviocytes; DR5; TIPE2
5.  CD24 genetic variants contribute to overall survival in patients with gastric cancer 
World Journal of Gastroenterology  2016;22(7):2373-2382.
AIM: To investigate the role of single nucleotide polymorphisms (SNPs) in CD24 gene in susceptibility and overall survival of gastric cancer (GC).
METHODS: We genotyped 3 tagging SNPs of CD24-P-534 in the promoter region, P170 in the coding region of exon 2 and P1527 in the 3′ untranslated region - using polymerase chain reaction-restriction fragment length polymorphism in specimens from 679 histologically-confirmed GC cases, 111 gastric atrophy (GA) cases and 976 tumor-free controls. Serum immunoglobulin G antibodies to Helicobacter pylori (H. pylori) of all subjects were detected by enzyme-linked immunosorbent assay. CD24 expression was evaluated by immunohistochemistry in 131 GC specimens. Correlations between SNPs and risk of GC or GA were shown by P values and odd ratios (ORs) with 95% confidence intervals (95%CI) compared with the most common genotype of each SNP using the unconditional logistic regression model after adjusting for age, sex and H. pylori infection. Survival within each SNP group was plotted by Kaplan-Meier method and compared by log-rank test (recessive model). Hazard ratios with 95%CIs were computed by Cox regression model after adjusting for age, sex, histological type, tumor differentiation, clinical stage and post-operational chemotherapy.
RESULTS: All of the three loci were in Hardy-Weinberg equilibrium in the control group. Median follow-up time for the 600 GC patients included in the survival analysis was 36.2 mo (range, 2.1-66.7 mo; 95%CI: 34.3-36.5 mo). Patients with the P-534 A/A genotype had significantly shorter survival (HR = 1.38, 95%CI: 1.01-1.88, P = 0.042) than did the C/C or C/A genotype carriers after adjusting for age, sex, histological type, tumor differentiation, clinical stage and post-operational chemotherapy. This trend was more evident in patients who lived longer than 2.5 years (HR = 7.55, 95%CI: 2.16-26.32, P = 0.001). The P170 T/T genotype was associated with a shorter lifespan than the non-T/T genotypes, but not significantly so. None of the three genetic variants was found to be associated with risk of GC (including tumor stage, grade and distant metastasis) or with risk of gastric atrophy. Furthermore, no difference of CD24 expression was found among the genotypes.
CONCLUSION: The P-534 site in CD24 gene affects the overall survival of gastric cancer and may serve as a prognostic marker for gastric cancer.
PMCID: PMC4735012  PMID: 26900300
Gastric cancer; CD24; Single nucleotide polymorphisms; Gastric atrophy; Overall survival
6.  Three-dimensional spherical spatial boundary conditions differentially regulate osteogenic differentiation of mesenchymal stromal cells 
Scientific Reports  2016;6:21253.
The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 μm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs’ ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues.
PMCID: PMC4756701  PMID: 26884253
7.  Factors associated with diet barriers in patients with poorly controlled type 2 diabetes 
The study was conducted to investigate the diet barriers perceived by patients with poorly controlled type 2 diabetes and examine the associations between diet barriers and sociodemographic characteristics, medical condition, and patient-centered variables.
Secondary subgroup analyses were conducted based on the responses of 246 adults with poorly controlled type 2 diabetes from a multicenter, cross-sectional study. Diet barriers were captured by the Diet Barriers subscale of the Personal Diabetes Questionnaire. Participants also completed validated measures of diet knowledge, empowerment level, and appraisal of diabetes. Multiple regression techniques were used for model building, with a hierarchical block design to determine the separate contribution of sociodemographic characteristics, medical condition, and patient-centered variables to diet barriers.
Diet barriers were moderately evident (2.23±0.86) among Chinese patients with poorly controlled type 2 diabetes. The feeling of deprivation as a result of complying with a diet was the most recognized diet barrier (3.24±1.98), followed by “eating away from home” (2.79±1.82). Significantly higher levels of diet barriers were observed among those with lower levels of diet knowledge (β=−0.282, P<0.001) and empowerment (β=−0.190, P=0.015), and more negative appraisal (β=0.225, P=0.003).
Culturally tailored, patient-centered intervention programs that acknowledge individuals’ preferences and allow for flexibility in diet management should be launched. Interventions programs that could enhance diet knowledge, promote positive appraisal, and improve empowerment level might effectively address diet barriers perceived by patients with poorly controlled type 2 diabetes.
PMCID: PMC4716765  PMID: 26834464
diabetes; diet barriers; knowledge; appraisal; empowerment
8.  Ovarian steroids, stem cells and uterine leiomyoma: therapeutic implications 
Human Reproduction Update  2014;21(1):1-12.
Uterine leiomyoma is the most common benign tumor in women and is thought to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a cellular insult. Leiomyomas cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are the most common indication for hysterectomy in the USA. A slow rate of cell proliferation, combined with the production of copious amounts of extracellular matrix, accounts for tumor expansion. A common salient feature of leiomyomas is their responsiveness to steroid hormones, thus providing an opportunity for intervention.
A comprehensive search of PUBMED was conducted to identify peer-reviewed literature published since 1980 pertinent to the roles of steroid hormones and somatic stem cells in leiomyoma, including literature on therapeutics that target steroid hormone action in leiomyoma. Reviewed articles were restricted to English language only. Studies in both animals and humans were reviewed for the manuscript.
Estrogen stimulates the growth of leiomyomas, which are exposed to this hormone not only through ovarian steroidogenesis, but also through local conversion of androgens by aromatase within the tumors themselves. The primary action of estrogen, together with its receptor estrogen receptor α (ERα), is likely mediated via induction of progesterone receptor (PR) expression, thereby allowing leiomyoma responsiveness to progesterone. Progesterone has been shown to stimulate the growth of leiomyoma through a set of key genes that regulate both apoptosis and proliferation. Given these findings, aromatase inhibitors and antiprogestins have been developed for the treatment of leiomyoma, but neither treatment results in complete regression of leiomyoma, and tumors recur after treatment is stopped. Recently, distinct cell populations were discovered in leiomyomas; a small population showed stem-progenitor cell properties, and was found to be essential for ovarian steroid-dependent growth of leiomyomas. Interestingly, these stem-progenitor cells were deficient in ERα and PR and instead relied on the strikingly higher levels of these receptors in surrounding differentiated cells to mediate estrogen and progesterone action via paracrine signaling.
It has been well established that estrogen and progesterone are involved in the proliferation and maintenance of uterine leiomyoma, and the majority of medical treatments currently available for leiomyoma work by inhibiting steroid hormone production or action. A pitfall of these therapeutics is that they decrease leiomyoma size, but do not completely eradicate them, and tumors tend to regrow once treatment is stopped. The recent discovery of stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma has the potential to provide the missing link between developing therapeutics that temper leiomyoma growth and those that eradicate them.
PMCID: PMC4255606  PMID: 25205766
leiomyoma; estrogen; progesterone; aromatase; stem cells
9.  Mutational Landscapes of Sequential Prostate Metastases and Matched Patient Derived Xenografts during Enzalutamide Therapy 
PLoS ONE  2015;10(12):e0145176.
Developing patient derived models from individual tumors that capture the biological heterogeneity and mutation landscape in advanced prostate cancer is challenging, but essential for understanding tumor progression and delivery of personalized therapy in metastatic castrate resistant prostate cancer stage. To demonstrate the feasibility of developing patient derived xenograft models in this stage, we present a case study wherein xenografts were derived from cancer metastases in a patient progressing on androgen deprivation therapy and prior to initiating pre-chemotherapy enzalutamide treatment. Tissue biopsies from a metastatic rib lesion were obtained for sequencing before and after initiating enzalutamide treatment over a twelve-week period and also implanted subcutaneously as well as under the renal capsule in immuno-deficient mice. The genome and transcriptome landscapes of xenografts and the original patient tumor tissues were compared by performing whole exome and transcriptome sequencing of the metastatic tumor tissues and the xenografts at both time points. After comparing the somatic mutations, copy number variations, gene fusions and gene expression we found that the patient’s genomic and transcriptomic alterations were preserved in the patient derived xenografts with high fidelity. These xenograft models provide an opportunity for predicting efficacy of existing and potentially novel drugs that is based on individual metastatic tumor expression signature and molecular pharmacology for delivery of precision medicine.
PMCID: PMC4687867  PMID: 26695660
10.  Anti-DR5 mAb inhibits proliferation of human fibroblast-like synovial cells and reduces their cytokine secretion in vitro 
OncoTargets and therapy  2015;8:2745-2755.
We have previously reported that anti-death receptor 5 (DR5) monoclonal antibody (mAb) is therapeutically effective in the treatment of rheumatoid arthritis (RA) in a collagen-induced arthritis rat model. However, the molecular mechanism and the effect of anti-DR5 mAb on proapoptotic genes and cytokine secretion in the human fibroblast-like synovial cells (FLS) requires further clarification. This study may provide new evidence for the application of anti-DR5 mAb as a treatment for RA.
Human FLS were isolated from patients with RA and were treated with anti-DR5 mAb. An MTT assay and flow cytometry were used to detect the induction of apoptosis in vitro. Cytokine secretion by the FLS was detected using the enzyme-linked immunosorbent assay. The mRNA expression was assessed by reverse transcription polymerase chain reaction, and the protein expression was analyzed by Western blot. The apoptotic pathway was investigated further using a caspase inhibition assay.
Anti-DR5 mAb-induced apoptosis in human RA FLS in vitro. The protein expressions of caspase-8, -3, and -9 were decreased in human anti-DR5 mAb-treated FLS in a dose-dependent manner through exposure to a caspase inhibitor, indicating that anti-DR5 mAb induction of apoptosis is through the caspase pathway. Decreased levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were detected after treatment with anti-DR5 mAb in vitro.
Anti-DR5 mAb may induce apoptosis in human FLS through the caspase pathway and through decreased secretions of TNF-α and IFN-γ.
PMCID: PMC4599060  PMID: 26491348
death receptor 5; rheumatoid arthritis; apoptosis
11.  Nonlinear optical properties of near-infrared region Ag2S quantum dots pumped by nanosecond laser pulses 
This study investigates near-infrared region Ag2S quantum dots (QDs) and their nonlinear optical response under 532 nm nanosecond laser pulses. Our experimental result shows that nonlinear transmission is reduced from 0.084 to 0.04. The observed narrowing behavior of the output pulse width shows superior optical limiting. We discuss the physical mechanisms responsible for the nonlinear optical response of the QDs. The average size of the nanocrystals was 5.5 nm. Our results suggest the possibility of using these Ag2S QDs for photoelectric, biosensor, optical ranging, and self-adaptive technologies.
PMCID: PMC4578410  PMID: 26425430
nonlinear optics; quantum dots; silver sulfide (Ag2S); strong absorption
12.  Serum markers of CYFRA 21-1 and C-reactive proteins in oral squamous cell carcinoma 
CYFRA 21-1 (cytokeratin 19 fragment) and C-reactive proteins (CRP) were separately reported to be associated with prognosis of head and neck squamous cell carcinoma. The combined roles of CYFRA 21-1 and CRP levels were rarely investigated in oral squamous cell carcinoma (OSCC). The purpose of the present study was to analyze the relationship between preoperative levels of both CYFRA 21-1 and CRP, with clinicopathological factors and prognosis in OSCC patients.
A retrospective study was performed on 130 OSCC patients between December 2010 and June 2013. Their serum CYFRA 21-1 and CRP levels were measured preoperatively.
CYFRA 21-1 level of ≥3.3 ng/mL and CRP level of ≥5.0 mg/L were significantly associated with pathological tumor status (P < 0.001), tumor depth (>10 vs. ≤10 mm, P = 0.001), bone invasion (P = 0.001), skin invasion (P = 0.006), pathologic nodal metastasis (P = 0.012), and disease-free survival (P = 0.009). Higher CYPFRA 21-1 and CRP levels were also associated with higher risks of distant metastasis (log-rank test, P = 0.013, (HR [95 % CI]) 1.692 [1.097–2.414]).
Preoperative CYFRA 21-1 and CRP levels are probable candidates as biomarkers for risk stratification in OSCC.
PMCID: PMC4546149  PMID: 26292957
13.  Systematic Review of Adverse Effects: A Further Step towards Modernization of Acupuncture in China 
As a further step towards the modernization of acupuncture, the objective of this review was to figure out the frequency and severity of adverse complications and events in acupuncture treatment reported from 1980 to 2013 in China. All first-hand case reports of acupuncture-related complications and adverse events that could be identified in the scientific literature were reviewed and classified according to the type of complication and adverse event, circumstance of the event, and long-term patient outcome. The selected case reports were published between 1980 and 2013 in 3 databases. Relevant papers were collected and analyzed by 2 reviewers. Over the 33 years, 182 incidents were identified in 133 relevant papers. Internal organ, tissue, or nerve injury is the main complications of acupuncture especially for pneumothorax and central nervous system injury. Adverse effects also included syncope, infections, hemorrhage, allergy, burn, aphonia, hysteria, cough, thirst, fever, somnolence, and broken needles. Qualifying training of acupuncturists should be systemized and the clinical acupuncture operations should be standardized in order to effectively prevent the occurrence of acupuncture accidents, enhance the influence of acupuncture, and further popularize acupuncture to the rest of the world.
PMCID: PMC4538973  PMID: 26339265
14.  17β-Hydroxysteroid Dehydrogenase-2 Deficiency and Progesterone Resistance in Endometriosis 
Estradiol (E2) stimulates the growth and inflammation in the ectopic endometriotic tissue that commonly resides on the pelvic organs. Several clinical and laboratory-based observations are indicative of resistance to progesterone action in endometriosis. The molecular basis of progesterone resistance in endometriosis may be related to an overall reduction in the levels of progesterone receptor (PR). In normal endometrium, progesterone acts via PR on stromal cells to induce secretion of paracrine factor(s) that in turn stimulate neighboring epithelial cells to express the enzyme 17β-hydroxysteroid dehydrogenase type 2 (HSD17B2). HSD17B2 is an extremely efficient enzyme and rapidly metabolizes the biologically potent estrogen E2 to weakly estrogenic estrone. In endometriotic tissue, progesterone is incapable of inducing epithelial HSD17B2 expression due to a defect in stromal cells. The inability of endometriotic stromal cells to produce progesterone-induced paracrine factors that stimulate HSD17B2 may be due to the very low levels of PR observed in vivo in endometriotic tissue. The end result is deficient metabolism of E2 in endometriosis giving rise to high local concentrations of this mitogen. The molecular details of this physiological paracrine interaction between the stroma and epithelium in normal endometrium and its lack thereof in endometriosis are discussed.
PMCID: PMC4511594  PMID: 20108182
17β-HSD-2; HSD17B2; endometriosis; endometrium; estradiol; progesterone; receptor; resistance; retinoic acid; paracrine; epithelial-stromal
15.  Retinoic acid inhibits endometrial cancer cell growth via multiple genomic mechanisms 
Previous studies have indicated that retinoic acid (RA) may be therapeutic for endometrial cancer. However, the downstream target genes and pathways triggered by ligand-activated RA receptor α (RARα) in endometrial cancer cells are largely unknown. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and immunoblotting assays were used to assess the roles of RA and the RA agonist (AM580) in the growth of endometrial cancer cells. Illumina-based microarray expression profiling of endometrial Ishikawa cells incubated with and without AM580 for 1, 3, and 6 h was performed. We found that both RA and AM580 markedly inhibited endometrial cancer cell proliferation, while knockdown of RARα could block AM580 inhibition. Knockdown of RARα significantly increased proliferating cell nuclear antigen and BCL2 protein levels. Incubation of Ishikawa cells with or without AM580 followed by microarray expression profiling showed that 12 768 genes out of 47 296 gene probes were differentially expressed with significant P values. We found that 90 genes were the most regulated genes with the most significant P value (P<0.0001) using F-test. We selected four highly regulated genes with diverse functions, namely G0S2, TNFAIP2, SMAD3, and NRIP1. Real-time PCR verified that AM580 highly regulated these genes, whereas chromatin immunoprecipitation-PCR assay demonstrated that ligand-activated RARα interacted with the promoter of these genes in intact endometrial cancer cells. AM580 also significantly altered 18 pathways including those related to cell growth, differentiation, and apoptosis. In conclusion, AM580 treatment of Ishikawa cells causes the differential expression of a number of RARα target genes and activation of signaling pathways. These pathways could, therefore, mediate the carcinogenesis of human endometrial cancer.
PMCID: PMC4511599  PMID: 21310893
16.  Involvement of Tetraspanin C189 in Cell-to-Cell Spreading of the Dengue Virus in C6/36 Cells 
PLoS Neglected Tropical Diseases  2015;9(7):e0003885.
Dengue virus (DENV) is naturally transmitted by mosquitoes to humans, infecting cells of both hosts. Unlike in mammalian cells, DENV usually does not cause extremely deleterious effects on cells of mosquitoes. Despite this, clustered progeny virions were found to form infection foci in a high density cell culture. It is thus interesting to know how the virus spreads among cells in tissues such as the midgut within live mosquitoes. This report demonstrates that cell-to-cell spread is one way for DENV to infect neighboring cells without depending on the “release and entry” mode. In the meantime, a membrane-bound vacuole incorporating tetraspanin C189 was formed in response to DENV infection in the C6/36 cell and was subsequently transported along with the contained virus from one cell to another. Knockdown of C189 in DENV-infected C6/36 cells is shown herein to reduce cell-to-cell transmission of the virus, which may be recovered by co-transfection with a C189-expressing vector in DENV-infected C6/36 cells. Moreover, cell-to-cell transmission usually occurred at the site where the donor cell directly contacts the recipient cell. It suggested that C189 is crucially involved in the intercellular spread of progeny viral particles between mosquito cells. This novel finding presumably accounts for the rapid and efficient infection of DENV after its initial replication within tissues of the mosquito.
Author Summary
Dengue fever is one of the most important mosquito-borne viral infectious diseases in the world. Its etiological agent is naturally transmitted via blood feeding by Aedes mosquitoes. An ingested virus can replicate and be disseminated within and between tissues in mosquitoes. In this study, we found that infection of DENV in C6/36 mosquito cells can stimulate the up-regulation of tetraspanin C189, which usually co-localizes but does not directly interact to form C189-containing membrane-bound vacuoles (C189-VCs). Our results also showed that the virus can be delivered to a neighboring cell along with C189-VCs, frequently through cell contact with filopodia extended by the donor cell that touch the recipient cell. Knockdown of C189 can reduce the efficiency of virus delivery, indicating its crucial role in cell-to-cell transmission of DENV in C6/36 cells. Cell-to-cell transmission may thus be an alternative route for the efficient intercellular spread of progeny viruses within tissues of the mosquito.
PMCID: PMC4488468  PMID: 26132143
17.  Contraception and Unintended Pregnancy among Unmarried Female University Students: A Cross-sectional Study from China 
PLoS ONE  2015;10(6):e0130212.
This study aims to understand the level of contraceptive knowledge and attitudes towards contraception, and then to explore the association between the contraceptive behavior and unintended pregnancy in unmarried female university students in China. A cross-sectional study was conducted of university students in 49 universities across 7 cities in China from September 2007 to January 2008. We distributed 74,800 questionnaires, of which 69,842 were returned. In this paper, the data from 35,383 unmarried female university students were analyzed. The prevalence of sexual intercourse in unmarried female university students was 10.2%. The prevalence of unintended pregnancy in those sexually active female university students, was 31.8%. Among students with pregnancy, 53.5% experienced two or more pregnancies. 28.3% of the students with sexual intercourse reported that they always adopted contraceptive methods, and of those 82.9% chose to use male condoms. The majority (83.9%) of students with unintended pregnancy chose to terminate the latest pregnancy by surgical abortion or medical abortion. The contraceptive knowledge level of students who experienced unintended pregnancy was lower than those who did not. In China, about one third of unmarried female students with sexual intercourse experience unintended pregnancy. A variety of contraceptive methods are adopted, but the frequency of contraceptive use is low. Most of unmarried female students who experienced unintended pregnancy would choose to terminate the pregnancy with surgical or medical abortion. University students, especially the ones who have experienced unintended pregnancy, lack contraceptive and reproductive health knowledge.
PMCID: PMC4474598  PMID: 26091505
18.  Microinvasion of liver metastases from colorectal cancer: predictive factors and application for determining clinical target volume 
This study evaluates the microscopic characteristics of liver metastases from colorectal cancer (LMCRC) invasion and provides a reference for expansion from gross tumor volume (GTV) to clinical targeting volume (CTV).
Data from 129 LMCRC patients treated by surgical resection at our hospital between January 2008 and September 2009 were collected for study. Tissue sections used for pathology and clinical data were reviewed. Patient information used for the study included gender, age, original tumor site, number of tumors, tumor size, levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199), synchronous or metachronous liver metastases, and whether patients received chemotherapy. The distance of liver microinvasion from the tumor boundary was measured microscopically by two senior pathologists.
Of 129 patients evaluated, 81 (62.8 %) presented microinvasion distances from the tumor boundary ranging between 1.0 − 7.0 mm. A GTV-to-CTV expansion of 5, 6.7, or 7.0 mm was required to provide a 95, 99, or 100 % probability, respectively, of obtaining clear resection margins by microscopic observation. The extent of invasion was not related to gender, age, synchronous or metachronous liver metastases, tumor size, CA199 level, or chemotherapy. The extent of invasion was related to original tumor site, CEA level, and number of tumors. A scoring system was established based on the latter three positive predictors. Using this system, an invasion distance less than 3 mm was measured in 93.4 % of patients with a score of ≤1 point, but in only 85.7 % of patients with a score of ≤2 points.
The extent of tumor invasion in our LMCRC patient cohort correlated with original tumor site, CEA level, and number of tumors. These positive predictors may potentially be used as a scoring system for determining GTV-to-CTV expansion.
PMCID: PMC4462112  PMID: 26040515
Liver metastases; Colorectal cancer; Radiotherapy; Pathological characteristics; Tumor invasion
19.  A Comparision of Nalbuphine with Morphine for Analgesic Effects and Safety : Meta-Analysis of Randomized Controlled Trials 
Scientific Reports  2015;5:10927.
Although morphine is the standard opioid analgesic for pain control and has been widely used, certain drug-induced adverse effects have been reported as intolerable and need to be addressed. Nalbuphine may have a few advantages over morphine in this respect. We aimed to describe the effect of nalbuphine as well as its saftey compared to morphine by analyzing published randomized controlled trials (RCTs) with meta-analysis approach. We analysed 15 trials (820 patients). Overall, there was no evidence to show that the effect of pain relief had any difference between nalbuphine and morphine (pooled relative risks [RRs], 1.01; 95% CI, 0.91 to 1.11; P = 0.90). On the other hand, the incidences of pruritus, nausea, vomiting, respiratory depression were significantly lower in nalbuphine group compared with morphine group, and the pooled RRs were 0.78(95%CI, 0.602–0.997; P = 0.048) for nausea, 0.65(95%CI, 0.50–0.85; P = 0.001) for vomiting, 0.17(95%CI, 0.09–0.34; P < 0.0001) for pruritus, and 0.27(95%CI, 0.12–0.57; P = 0.0007) for respiratory depression. The analgesic efficacy of nalbuphine is comparable to morphine, but nalbuphine provides a better safety profile than morphine in the aspect of certain side-effects, especially related to pruritus and respiratory depression.
PMCID: PMC4454168  PMID: 26039709
20.  Inhibition of canonical WNT signaling attenuates human leiomyoma cell growth 
Fertility and sterility  2014;101(5):1441-1449.e1.
Dysregulation of WNT signaling plays a central role in tumor cell growth and progression. Our goal was to assess the effect of three WNT/β-catenin pathway inhibitors, Inhibitor of β-Catenin And TCF4 (ICAT), niclosamide, and XAV939 on the proliferation of primary cultures of human uterine leiomyoma cells.
Prospective study of human leiomyoma cells obtained from myomectomy or hysterectomy.
University research laboratory.
Women (n=38) aged 27–53 years undergoing surgery.
Adenoviral ICAT overexpression or treatment with varying concentrations of niclosamide or XAV939.
Main Outcome Measure(s)
Cell proliferation, cell death, WNT/β-catenin target gene expression or reporter gene regulation, β-catenin levels and cellular localization.
ICAT, niclosamide, or XAV939 inhibit WNT/β-catenin pathway activation and exert anti-proliferative effects in primary cultures of human leiomyoma cells.
Three WNT/β-catenin pathway inhibitors specifically block human leiomyoma growth and proliferation, suggesting that the canonical WNT pathway may be a potential therapeutic target for the treatment of uterine leiomyoma. Our findings provide rationale for further preclinical and clinical evaluation of ICAT, niclosamide, and XAV939 as candidate anti-tumor agents for uterine leiomyoma.
PMCID: PMC4008647  PMID: 24534281
Leiomyoma; WNT/β-catenin; niclosamide; XAV939; tumor biology
21.  Primary adenomatoid tumor of the testis: report of a case and review of literature 
Adenomatoid tumor (AT) is an extremely rare benign tumor in the testis of infants. A case of 14-month-old boy with testicular adenomatoid tumor was reported in this study. On physical examination, a smooth solid nodule sized 8 mm could be palpated with little tenderness on the head of the right testis. It could be clearly revealed by B ultrasonic scanning and computerized tomography. The patient underwent right radical orchiectomy. In postoperative histopathological study, the tumor was characterized by diffuse sheets of epithelioid cell and desmo-stroma structures. There was positive immunohistochemical staining of mesothelioma-associated antigens. The tumor should be differentiated from the tumor of the male genital tract including benign and malignant tumors of both epithelial and stromal origin. And we followed the case and no nodule was found in his scrotum by physical examination and scrotal ultrasonography after 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60 months. These findings have important implications that the histogenesis of adenomatoid tumor of the testis is unclear yet. The diagnosis depends on pathologic studies, and should be differentiated from paratesticular malignant mesothelioma and sclerosed lipogranuloma. Radical surgery is the common choice, and as a result of getting a good prognosis.
PMCID: PMC4503189  PMID: 26191318
Adenomatoid tumor; testicle; infant; case report; review
22.  Immunosuppressive mechanisms of human bone marrow derived mesenchymal stromal cells in BALB/c host graft versus host disease murine models 
Mesenchymal stromal cells (MSCs) are proven to have immunosuppressive functions via various mechanisms. These mechanisms were demonstrated by administering bone marrow derived human MSCs (hMSCs) to graft versus host disease (GVHD) murine models.
BALB/c host mice were irradiated prior to receiving C57BL/6 donor T cell depleted bone marrow (TCDBM) cells (negative control) and donor CD4+ T lymphocyte with (treatment group) or without hMSCs (positive control). The presence of hMSCs in target tissues and lymphoid organs was documented by using in vivo imaging and measuring the expression of EphB2 and ephrin-B2 by RTqPCR. Survival rate and GVHD score were also monitored. Tissue sections were obtained for histopathologic analysis. Flow cytometry was used to document donor T cell alloreactivity and expression of CCR5, CXCR3 and CCR7. ELISA was utilized to determine levels of proinflammatory cytokines, RANTES (CCL5) and phosphorylated STAT 5A/B. RTqPCR was performed to quantify expression of CCL3 and CXCL9. Western blotting was performed to qualitatively measure iNOS expression.
Survival rate and GVHD score improved with hMSC treatment. Pathologic changes of GVHD were abrogated. Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs. Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed.
These mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.
Electronic supplementary material
The online version of this article (doi:10.1186/s40164-015-0007-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4440561  PMID: 26000204
GVHD; MSC; Immunosuppression; Bone marrow transplantation
23.  Vitrification of day 3 cleavage-stage embryos yields better clinical outcome in comparison with vitrification of day 2 cleavage-stage embryos 
Zygote (Cambridge, England)  2013;23(2):169-176.
The objective of this retrospective study was to determine an optimal time point for vitrification of cleavage-stage human embryos. This study included patients who were undergoing day 2 or day 3 vitrified–warmed cleavage-stage embryo transfer at the In Vitro Fertilization (IVF) Programme of the Shanghai First Maternity and Infant Hospital, China, affiliated to the Tongji University School of Medicine, from April 2010 to March 2012. Intervention was made for the entire cohort of vitrified embryos for poor responder patients so as to avoid severe ovarian hyperstimulation syndrome. Embryo survival rate (SR) after vitrification–warming, implantation rate (IR), and clinical pregnancy rate (CPR) were the main outcome measurements. In total, 380 vitrified–warmed cleavage-stage embryo transfer (VWT) cycles were included. We found that the SR after vitrification and warming for day 2 embryos and day 3 embryos were 92.7% and 92.8%, respectively. For poor ovarian responders, the IR of day 2 and day 3 vitrified–warmed embryos was 6.4% and 13.2%, respectively (P = 0.186). The CPR for day 3 vitrified–warmed embryos was significantly higher than that of day 2 vitrified–warmed embryos (17.6 vs. 4.0 % per transfer cycle, P = 0.036). For patients who had their entire cohort of embryos vitrified to prevent severe ovarian hyperstimulation syndrome (OHSS), the IR and CPR were not significantly different for day 2 and day 3 vitrified–warmed embryo transfer. In conclusion, for vitrified–warmed embryo transfer, cryopreservation of the entire cohort of embryos on day 3 resulted in better clinical outcomes compared with cryopreservation on day 2. Therefore, it is highly recommended that cleavage-stage embryos should be vitrified on day 3, but not on day 2, particularly for poor ovarian responder patients.
PMCID: PMC4413873  PMID: 23965660
Clinical pregnancy; Implantation; Vitrification; Vitrified–warmed embryo transfer
24.  Effectiveness of smoking reduction intervention for hardcore smokers 
Tobacco Induced Diseases  2015;13(1):9.
The prevalence and correlates of hardcore smokers, who have high daily cigarette consumption, no quitting history and no intention to quit, have been studied in several western developed countries, but no previous trials of smoking cessation have tested intervention effectiveness for these smokers. The current study examined if hardcore smokers can benefit from smoking reduction intervention to achieve cessation, and explored the underlying reasons.
A posteriori analysis was conducted on data from a randomized controlled trial of smoking reduction intervention on 1,154 smokers who did not want to quit. Odds ratios of 7-day point prevalence of abstinence, smoking reduction by at least 50% and quit attempt at the 6-month follow-up comparing subgroups of smokers were analyzed.
In hardcore smokers, the odds ratio comparing the quit rate between the intervention and control group was 4.18 (95% CI: 0.51-34.65), which was greater than non-hardcore smokers (OR = 1.58, 95% CI: 0.98-2.54). The number needed to treat for hardcore and non-hardcore smokers was 8.33 (95% CI: 5.56-16.67) and 16.67 (95% CI: 8.33-233.64), respectively. In smokers who did not have quit attempt experience and those who smoked more than 15 cigarettes daily, the odds ratio comparing intervention and control group was 3.29 (95% CI: 0.72-14.98) and 1.36 (95% CI: 0.78-2.36), respectively.
The a posteriori analysis provided pilot results that smoking reduction intervention may be effective to help hardcore smokers to quit and reduce smoking. Having no previous quit attempt was identified as more important than having large cigarette consumption in explaining the greater effectiveness of the intervention.
Electronic supplementary material
The online version of this article (doi:10.1186/s12971-015-0034-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4391680  PMID: 25859176
Smoking cessation; Hardcore; Smoking reduction; Cigarette consumption; Quit attempt
25.  Nicotine replacement therapy to aid gradual cessation in smokers with no intention to quit: Association between reduction quantity and later abstinence 
Preventive Medicine Reports  2015;2:196-201.
We examined how quantity and trajectory of smoking reduction influence later abstinence in smokers without intention to quit and being prescribed free nicotine replacement therapy (NRT).
We conducted an a posteriori analysis from a data archive of adult smokers in a randomized controlled trial of smoking reduction using counseling and free NRT (n = 928). Reduction was analyzed as the absolute and percentage decrease in self-reported daily cigarette consumption at three follow-ups (1 week, 1 and 3 months) compared with the baseline. Logistic regression model and multiple imputation were used to examine the association between early reduction and abstinence at 6 months.
Reducing 10% of cigarette consumption at the three follow-ups was associated with 16% (95% CI 5–28%), 23% (95%CI 11–36%) and 27% (95% CI 13–42%) increase in abstinence, respectively. Greater reduction predicted abstinence when the percentage reduction was more than one-third (above 31.4%). Progressive increase in the percentage reduction predicted more abstinence (OR = 1.90, 95%CI 1.01–3.58).
Greater percentage reduction by at least one-third and progressive reduction predicted abstinence in those who reduced smoking. Such new evidence can guide the improvement of clinical service for tobacco dependency treatment and support further studies on smoking reduction and cessation.
PMCID: PMC4721443  PMID: 26844073
Smoking cessation; Cigarette smoking; Nicotine replacement products

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