Comparison of clinical outcome between day 5 and day 6 single blastocyst transfers in cycles undergoing preimplantation genetic testing for aneuploidy.

OBJECTIVE: In vitro fertilization (IVF) treatment has gradually adopted the practice of culturing embryos until the blastocyst stage on the D5 or D6 as the standard approach. PGT-A is commonly used in vitro fertilization (IVF). This study aimed to evaluate the clinical outcomes of frozen embryo transfers (FETs) using single blastocyst transfers (SBTs) on the fifth (D5) or sixth (D6) day of development in cycles that underwent preimplantation genetic testing for aneuploidy (PGT-A). MATERIALS AND METHODS: The patients who had at least one euploid or mosaic blastocyst of good quality determined by PGT-A results and received single embryo transfer (SET) cycles were included in the study. In this study, the live birth rate (LBR) and neonatal outcomes were compared after the transfer of single biopsied D5 and D6 blastocysts in frozen embryo transfer (FET) cycles. RESULTS: A total of 527 frozen-thawed blastocyst transfer (FET) cycles (8449 biopsied embryos were analyzed). No significant difference in the implantation rate (IR), clinical pregnancy rate (CPR), and live birth rate (LBR) between the transfers of D5 and D6 blastocysts. Birth weight was the only perinatal outcome that showed a significant difference between the D5 and D6 groups. CONCLUSION: The study confirmed that the transfer of a single euploid or mosaic blastocyst, regardless of whether it was on the fifth (D5) or sixth (D6) day of development, can lead to promising clinical results.

Uncovering the Inhibitory Molecular Mechanism of Pomegranate Peel to Urinary Bladder Urothelial Carcinoma Using Proteomics Techniques.

Pomegranate (Punica granatum L.) fruit demonstrates the repressive effectiveness of many tumors. Our previous studies showed that the PEP (pomegranate peel extract) E2 fraction obtained from the ethyl acetate layer of the pomegranate peel's ethanol extract exhibited the highest inhibitory activities to induce Urinary bladder urothelial carcinoma (UBUC) cell apoptosis. The ethyl acetate layer could lower the volume and weight of T24 tumors and initiate apoptosis in nude mice xenografted bladder tumors. In this study, we intended to clarify the inhibitory molecular process of Taiwanese local pomegranate peel to urinary bladder urothelial carcinoma using a proteomics strategy. Gel-based proteomics (two-dimensional gel electrophoresis coupled with tandem mass spectrometry) was used to get an insight into the molecular mechanisms initiated by PEPE2 to evoke bladder cancer cell apoptosis. We found eleven down-regulated and eight up-regulated proteins in PEPE2-treated T24 cells. Our results implied that these PEPE2-dysregulated proteins belong to cell apoptosis, cell proliferation, death receptor signaling, JAK/STAT signaling, the PPAR pathway, the PPARα/RXR α pathway, Rho family GTPase signaling, and RhoGDI signaling. In addition, HSP90 and PTP1B proteins, associated with apoptosis, were de-regulated in xenografted bladder tumors in nude mice fed with an ethyl acetate layer of ethanol extract. The findings above implied that pomegranate might be a potential chemopreventive resource for UBUC carcinogenesis.

Zirconium-Redox-Shuttled Cross-Electrophile Coupling of Aromatic and Heteroaromatic Halides.

Transition metal-catalyzed cross-electrophile coupling (XEC) is a powerful tool for forging C(sp2)-C(sp2) bonds in biaryl molecules from abundant aromatic halides. While syntheses of unsymmetrical biaryl compounds through multimetallic XEC is of high synthetic value, selective XEC of two heteroaromatic halides remains elusive and challenging. Herein we report a homogeneous XEC method which relies on a zirconaaziridine complex as a shuttle for dual palladium catalyzed processes. The zirconaaziridine-mediated palladium (ZAPd) catalyzed reaction shows excellent compatibility with various functional groups and diverse heteroaromatic scaffolds. In accord with density functional theory (DFT) calculations, a redox-transmetallation between the oxidative addition product and the zirconaaziridine is proposed as the crucial elementary step. Thus, cross-coupling selectivity using a single transition metal catalyst is controlled by the relative rate of oxidative addition of Pd(0) into the aromatic halide. Overall, the concept of a combined reducing and transmetallating agent offers opportunities for development of transition-metal reductive coupling catalysis.

Early postpartum biofeedback assisted pelvic floor muscle training in primiparous women with second degree perineal laceration: Effect on sexual function and lower urinary tract symptoms.

OBJECTIVE: To evaluate the short-term effect of routine early postpartum electromyographic biofeedback assisted pelvic floor muscle training on sexual function and lower urinary tract symptoms. MATERIALS AND METHODS: From December 2016 to November 2017, primiparous women with vaginal delivery, who experienced non-extended second-degree perineal laceration were invited to participate. Seventy-five participants were assigned into a pelvic floor muscle training (PFMT) group or control group. Women in the PFMT group received supervised biofeedback-assisted pelvic floor muscle training at the 1st week and 4th week postpartum. Exercises were performed at home with the same protocol until 6 weeks postpartum. The Pelvic Organ Prolapse Urinary Incontinence Sexual Questionnaire (PISQ-12) and the Urinary Distress Inventory short form questionnaire (UDI-6) were used to evaluate sexual function and lower urinary tract symptoms respectively at immediate postpartum, 6 weeks, 3 months, and 6 months postpartum. RESULTS: Forty-five women (23 in PFMT group,22 in control group) completed all questionnaires at 6 months postpartum. For overall sexual function and the three sexual functional domains, no statistically significant difference was found in PISQ scores from baseline to 6 weeks, 3 months, and 6 months postpartum between the PFMT and control groups. For postpartum lower urinary tract symptoms, all symptoms gradually improved over time for both groups without a statistically significant difference between groups. CONCLUSION: Our study showed that supervised biofeedback-assisted pelvic floor muscle training started routinely at one week postpartum did not provide additional improvement in postpartum sexual function and lower urinary tract symptoms.

Investigation of the molecular mechanism underlying the inhibitory activities of ethanol extract of Bombyx mori pupa-incubated Cordyceps militaris fruiting bodies toward allergic rhinitis.

Cordyceps militaris has been widely studied for its various pharmacological activities such as antitumor, anti-inflammation, and immune regulation. The binding of an allergen to IgE-sensitized mast cells in nasal mucosa triggers allergic rhinitis. We found that oral administration of 300 mg/kg of the ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruiting bodies significantly alleviated the symptoms of ovalbumin-induced allergic rhinitis in mice, including sneeze/scratch, mast cell activation, eosinophil infiltration, and Syk activation. The treatment of ethanol extract significantly suppressed the release of ß-hexosaminidase (a degranulation marker) and mRNA expression levels of various cytokines, including IL-3, IL-10, and IL-13 in activated RBL2H3 cells. The ethanol extract and ß-sitostenone, which was purified from the extract, could respectively reduce the Ca2+ ion mobilization in activated RBL-2H3 cells. Furthermore, results collected from western immunoblotting demonstrated that ethanol extract significantly retarded Ca2+ ion mobilization-initiated signaling cascade, which provoked the expression of various allergic cytokines. Also, the extract incubation interfered with P38 as well as NF-kB activation and Nrf-2 translocation. Our study suggested that ethanol extract possessed some natural constituents which could inhibit immediate degranulation and de novo synthesis of allergic cytokines via inhibition of Ca2+ ion mobilization in mast cells in the nasal mucosa of allergic rhinitis mice.

Label free Impedimetric Immunosensor for effective bladder Cancer detection in clinical urine samples.

Galectin-1 protein has been recently recognized as a valuable urinary biomarker for the diagnosis and prognosis of bladder cancer. Herein, we present a sensitive and specific impedimetric immunosensor for the quantitative and label free detection of Galectin-1 protein in clinical urine samples. The immunosensor consists of nine gold interdigitated microelectrodes (3 × 3 array), which can simultaneously monitor multiple immunoreactions by analyzing the normalized impedance variations at each microelectrode during immunosensing. To obtain enhanced sensitivities, we have utilized Galectin-1/Al2O3 nanoprobes (Galectin-1 antibody conjugated to alumina nanoparticles) that can be selectively trapped on the microelectrode surface using positive dielectrophoresis (p-DEP). Preliminary studies highlight the feasibility of the proposed immunosensor for Gal -1 detection in T24 cell lysate spiked phosphate buffer saline and artificial urine samples with a limit of detection that is estimated to be in the pg/ml range. To verify its practical feasibility, we have tested the immunosensor for Galectin-1 detection in clinical urine samples obtained from normal patients and those diagnosed with bladder cancer. Analysis of the clinical tests shows that the median normalized impedance variation during immunosensing for 22 cancer patients and 26 normal patients is 27% and 10%, respectively, with an identified cutoff point of 19.5% above which the sensitivity and specificity of bladder cancer detection was 82.1% and 80.8%, respectively. Based on these results, the proposed immunosensor shows promise for bladder cancer diagnosis and prognosis in a point of care format, thus enabling improved public health monitoring.

[Wind Field Influences on the Spatial Distribution of Cyanobacterial Blooms and Nutrients in Meiliang Bay of Lake Taihu, China].

Wind field is a very important physical factor controlling the formation of cyanobacteria blooms. A surface particle tracking drift experiment was carried out to study the influence of wind field on the surface current in Meiliang Bay of Lake Taihu during the algal bloom season. For this, chlorophyll-a, nitrogen, phosphorus, the permanganate index, dissolved organic carbon (DOC), and dissolved oxygen (DO) were measured in surface, middle, and bottom waters of the Meiliang Bay during the cyanobacteria bloom period to test how wind field affects the temporal and spatial distribution of cyanobacterial blooms and biomass stock in the water column. The results showed that the average drift velocities of surface particles were 3.0 cm·s-1 and 5.0 cm·s-1 when wind speed averaged 1.9 m·s-1 and 2.3 m·s-1, respectively. The wind field determined the spatial distribution of cyanobacterial blooms in surface waters and led to a high spatial heterogeneity of cyanobacterial blooms. The spatial redistribution of cyanobacterial blooms exerted an important influence on water quality indexes such as particulate nitrogen, phosphorus, organic matter, and dissolved oxygen. The concentrations of particulate nitrogen, phosphorus, the permanganate index, and chlorophyll-a showed a similar vertical distribution pattern. Cyanobacterial blooms were less influenced by the distribution of dissolved nitrogen and dissolved organic carbon from external pollution, while long-term legacy loading played a more important role. This meant that the spatial distributions of dissolved nitrogen and dissolved organic carbon were different from that of chlorophyll-a. Because the redistribution of cyanobacterial blooms, as affected by wind fields, has a complex effect on the dissolved oxygen in the water column, the dissolved oxygen concentration decreased with depth, which may affect the release of soluble nutrients from the sediment. The cyanobacterial biomass stock in the surface water was estimated according to the survey of high-density sites. The dry matter of cyanobacteria in the surface 20 cm of Meiliang Bay was approximately 396 tons on the day of sampling. The results from the present study indicated that the factors influencing cyanobacterial blooms should be considered in sampling methods and the analysis of lake water quality due to the significant influence of wind fields on bloom drift. The collection of cyanobacteria has limited effect on the removal of the algal bloom biomass in whole lake, only being effective at prevention of the event of black spots in lake shore.

Proteomic Profile of Sorafenib Resistance in Hepatocellular Carcinoma; GRP78 Expression Is Associated With Inferior Response to Sorafenib.

BACKGROUND/AIM: The outcome of patients with advanced hepatocellular carcinoma (HCC) remains poor and therapeutic options, including sorafenib, the first anti-cancer drug proved to prolong survival in patients with advanced HCC, are limited. However, no clinically useful predictive biomarker for sorafenib has been reported. MATERIALS AND METHODS: We exploited two-dimensional gel electrophoresis coupled with mass spectrometry to find de-regulated proteins by using conditioning of a sorafenib-resistant HCC cell line, Huh7. Tumor samples from 60 patients with HCC treated with sorafenib were analyzed and correlated with survival outcome. RESULTS: Comparative proteomics indicated three proteins including, 78 kDa glucose related protein (GRP78), 14-3-3ε, and heat shock protein 90ß (HSP90ß). The three proteins were over-expressed in sorafenib-resistant Huh7 cells. In HCC tumor samples from patients treated with sorafenib, 73% of tumor samples had a high expression of GRP78, 18% had high 14-3-3ε expression and 85% had high HSP90ß expression. Among these, GRP78 was associated with the shortest progression-free survival of HCC patients treated with sorafenib. CONCLUSION: GRP78 can be a predictive biomarker in HCC patients treated with sorafenib. Strategies designed to inhibit the GRP78-related pathway may overcome sorafenib resistance.

Involvement of endothelial nitric oxide synthase gene variants in the aggressiveness of uterine cervical cancer.

To date, few studies explore the involvement of endothelial nitric oxide synthase (eNOS) gene variants in uterine cervical cancer. Therefore, we conducted this study to assess the clinical implication of eNOS in cervical carcinogenesis, clinicopathological characteristics and patient survival. One hundred and seventeen patients with cervical invasive cancer and 95 with preinvasive lesions and 330 control women were consecutively enrolled. Real time polymerase chain reaction was used to examine the genotypic distributions of eNOS single nucleotide polymorphisms (SNPs) rs1799983 (894G>T) at the exon 7 region and rs2070744 (-786T>C) at the promoter region. Our results indicated no significant associations among genotypic distributions of eNOS SNPs and patients with cervical invasive cancer and those with preinvasive lesions as well as normal controls. However, cervical cancer patients with genotypes TC/CC in eNOS SNP rs2070744 carried less risk of advanced stage [odds ratios (OR) = 0.30, 95% confidence interval (CI)=0.09-0.97, p=0.036], parametrium invasion (OR=0.16, 95% CI=0.02-0.75, p=0.009) and pelvic lymph node metastasis (OR=0.12, 95% CI=0.01-0.89, p=0.016). In conclusion, although eNOS SNPs rs2070744 and rs1799983 do not display significant associations with cervical carcinogenesis and patient survival, cervical cancer patients with genotypes TC/CC in rs2070744 carry less risk of advanced stage, parametrium invasion and pelvic lymph node metastasis in Taiwan.

Paper-based immunosensor utilizing dielectrophoretic trapping of microprobes for quantitative and label free detection using electrochemical impedance spectroscopy.

In this study, we have developed a novel paper based immunoassay for the quantitative detection of immunoreactions using electrochemical impedance spectroscopy. Paper provides an attractive platform for fabrication of simple, low cost, and portable diagnostic devices as it allows passive liquid transport, is biocompatible, and has tunable properties such as hydrophilicity, flexibility, permeability, and reactivity. We have used screen-printing to fabricate interdigitated electrodes (finger width and gap of 200 µm) on the paper substrate, while UV-lithography enables patterning of the paper into hydrophobic/hydrophilic regions. As a proof of concept, we have used this immunosensor to detect the immune response of Human Serum Albumin (HSA) antibody-antigen complex formation. To enable efficient immobilization of HSA antibodies, we have utilized dielectrophoresis to trap microprobes (MPs) on the electrode surface. The microprobes consist of an alumina nanoparticle core with a well-adhered polyaniline outer coating to which the HSA antibodies are conjugated in an oriented manner via covalent chemistry. The efficacy of the impedance-based immunosensor is compared when MPs are immobilized specifically on the electrode surface using dielectrophoresis (DEP) as opposed to being dropped and immobilized via physical absorption on the entire sensing area. Results show that a more reproducible and sensitive response is observed when DEP is utilized to trap the microprobes. Furthermore, the normalized impedance variation during immunosensing shows a linear dependence on the concentration of HSA with an observed limit of detection of 50 µg/ml, which is lower than conventionally used paper based urine dipsticks used for urinary protein detection. Thus, we have developed a low cost paper based immunoassay platform that can be used for the quantitative point of care detection of a wide range of immunoreactions.

Proteomic Identification of the Galectin-1-Involved Molecular Pathways in Urinary Bladder Urothelial Carcinoma.

Among various heterogeneous types of bladder tumors, urothelial carcinoma is the most prevalent lesion. Some of the urinary bladder urothelial carcinomas (UBUCs) develop local recurrence and may cause distal invasion. Galectin-1 de-regulation significantly affects cell transformation, cell proliferation, angiogenesis, and cell invasiveness. In continuation of our previous investigation on the role of galectin-1 in UBUC tumorigenesis, in this study, proteomics strategies were implemented in order to find more galectin-1-associated signaling pathways. The results of this study showed that galectin-1 knockdown could induce 15 down-regulated proteins and two up-regulated proteins in T24 cells. These de-regulated proteins might participate in lipid/amino acid/energy metabolism, cytoskeleton, cell proliferation, cell-cell interaction, cell apoptosis, metastasis, and protein degradation. The aforementioned dys-regulated proteins were confirmed by western immunoblotting. Proteomics results were further translated to prognostic markers by analyses of biopsy samples. Results of cohort studies demonstrated that over-expressions of glutamine synthetase, alcohol dehydrogenase (NADP⁺), fatty acid binding protein 4, and toll interacting protein in clinical specimens were all significantly associated with galectin-1 up-regulation. Univariate analyses showed that de-regulations of glutamine synthetase and fatty acid binding protein 4 in clinical samples were respectively linked to disease-specific survival and metastasis-free survival.

Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma.

Pomegranate (Punica granatum L.) fruit has been demonstrated to have the inhibitory activities to various tumors. In this study, we try to uncover the molecular mechanism underlying the inhibitory capability of Taiwanese local pomegranate fruit to urinary bladder urothelial carcinoma. The results collected from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that the ethanol extract of pomegranate peel exhibited better inhibitory activity to human urinary bladder urothelial carcinoma T24 and J82 cells than that of pulp. Furthermore, the ethylacetate layer of peel ethanol extract was observed to have the best inhibitory activity against urinary bladder urothelial carcinoma cells. One of the eight fractions (PEPE2 fraction) collected from the ethylacetate layer with Diaion HP-20 column chromatography demonstrated the highest inhibitory activity in urinary bladder urothelial carcinoma cells. The results of the flow cytometry and apoptotic pathway studies suggested that the inhibitory activity of PEPE2 fraction were attributed to the UBUC cell apoptosis. To confirm the above results, our results of xenograft-induced bladder tumor in nude mice showed that the oral consumption of the ethylacetate layer (2, 5, 10 and 100 mg/kg) could decrease the volume and weight of T24 tumors and caused the apoptosis in the xenografted tumors, which was observed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. This study provided the likelihood that the traditionally non-edible pomegranate peel waste is re-utilized to make an affordable and promising chemopreventive product to prevent UBUC incidence or recurrence.

Butyrate ameliorated-NLRC3 protects the intestinal barrier in a GPR43-dependent manner.

BACKGROUND: Intestinal barrier dysfunctions are related to dysbacteriosis and chronic gut inflammation in type 2 diabetes. Although there is emerging evidence that the chronic gut inflammatory response is stimulated by nucleotide-binding oligomerization domain-like receptors (NLRs), the relationship and precise mechanism between NLRC3 and the colonic epithelial barrier remains largely elusive. METHODS: We investigated the function and mechanism of NLRC3 in the colonic tissues of diabetic mice and colonic epithelial cell lines. The regulatory mechanism between NLRC3, butyrate and tight junctions was elucidated via a transepithelial electrical resistance measurement, transmission electron microscopy, RNA interference and western blotting. RESULTS: In this study, we found that NLRC3 expression was decreased in the colonic tissues of diabetic mice. NLRC3 over-expression ameliorated colonic epithelial barrier integrity and up-regulated tight junction proteins in colonic epithelial cells. Knockdown of TRAF6 diminished NLRC3-induced ZO-1/occludin expression. In addition, we demonstrated that butyrate could stimulate NLRC3 expression in both diabetic mice and colonic epithelial cells. GPR43 on colonic epithelial cells is involved in the activation of NLRC3 induced by butyrate. CONCLUSION: Our findings demonstrated that NLCR3 could ameliorate colonic epithelial barrier integrity in diabetes mellitus in a TRAF6-dependent manner, and NLCR3 was stimulated by butyrate via binding GPR43 on colonic epithelial cells.

Epigenetic modification of TLE1 induce abnormal differentiation in diabetic mice intestinal epithelium.

The intestinal epithelium cells (IECs) in diabetes mellitus (DM) patients have been proven to be abnormally differentiated. During the differentiation of IECs, epigenetic modification acts as an important regulator. In this study, we aimed to examine the epigenetic alteration of Transducin-like Enhancer of Split 1 (TLE1), a multitask transcriptional co-repressor, contributing to the differentiation homeostasis in IECs of DM mice. The IECs of type 2 diabetic mice model were isolated and collected. Methylation states of whole genomic DNA promoter regions were investigated by microarray. Methylated-specific PCR was used to detect the methylation state of TLE1 promoter in DM mice IECs. The expression of TLE1, Hes1, and differentiated cell markers were measured through real-time PCR, Western blots, and immunohistochemistry; by transfection assay, TLE1 or Hes1 was independently down-regulated in intestinal epithelium cell line, IEC-6. Subsequent modulation on TLE1, Hes1, and differentiated intestinal cell markers were detected. Global gene promoter regions in DM intestinal epithelium were less methylated comparing to normal control. The expression of TLE1 was significantly increased via hypomethylated activation in DM mice IECs. Hes1 was significantly suppressed and the terminal cell markers abnormally expressed in DM mice IECs (P < 0.05). Inhibition or induction on the abundance of TLE1 in IEC-6 cell line resulted in the corresponding dysregulation of Hes1 and intestinal epithelium differentiation (P < 0.05). Demethylation of TLE1 promoter region activates the self-expression in diabetic mice IECs. Subsequently, TLE1, through the transcriptional suppression on expression of Hes1, contributes to the aberrant differentiation of IECs in DM mice.

Helicobacter pylori Infection Is Associated with Type 2 Diabetes, Not Type 1 Diabetes: An Updated Meta-Analysis.

BACKGROUND: Extragastric manifestations of Helicobacter pylori (H. pylori) infection have been reported in many diseases. However, there are still controversies about whether H. pylori infection is associated with diabetes mellitus (DM). This study was aimed at answering the question. METHODS: A systematic search of the literature from January 1996 to January 2016 was conducted in PubMed, Embase databases, Cochrane Library, Google Scholar, Wanfang Data, China national knowledge database, and SinoMed. Published studies reporting H. pylori infection in both DM and non-DM individuals were recruited. RESULTS: 79 studies with 57,397 individuals were included in this meta-analysis. The prevalence of H. pylori infection in DM group (54.9%) was significantly higher than that (47.5%) in non-DM group (OR = 1.69, P < 0.001). The difference was significant in comparison between type 2 DM group and non-DM group (OR = 2.05), but not in that between type 1 DM group and non-DM group (OR = 1.23, 95% CI: 0.77-1.96, P = 0.38). CONCLUSION: Our meta-analysis suggested that there is significantly higher prevalence of H. pylori infection in DM patients as compared to non-DM individuals. And the difference is associated with type 2 DM but not type 1 DM.

Uncovering the Molecular Mechanism of Anti-Allergic Activity of Silkworm Pupa-Grown Cordyceps militaris Fruit Body.

Cordyceps militaris has been widely used as an herbal drug and tonic food in East Asia and has also been recently studied in the West because of its various pharmacological activities such as antitumoral, anti-inflammatory and immunomodulatory effects. In this study, we examined the molecular mechanism underlying the anti-allergic activity of ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruit bodies in activated mast cells. Our results showed that ethanol extract treatment significantly inhibited the release of [Formula: see text]-hexosaminidase (a degranulation marker) and mRNA levels of tumor necrosis factor-[Formula: see text] as well as interleukin-4 in RBL-2H3 cells. The cells were sensitized with 2,4-dinitrophenol specific IgE and then stimulated with human serum albumin conjugated with 2,4-dinitrophenol. Oral administration of 300[Formula: see text]mg/kg ethanol extract significantly ameliorated IgE-induced allergic reaction in mice with passive cutaneous anaphylaxis. Western immunoblotting results demonstrated that ethanol extract incubation significantly inhibited Syk/PI3K/MEKK4/JNK/c-jun biochemical cascade in activated RBL-2H3 cells, which activated the expression of various allergic cytokines. In addition, it suppressed Erk activation and PLC[Formula: see text] evocation, which would respectively evoke the synthesis of lipid mediators and Ca[Formula: see text] mobilization to induce degranulation in stimulated RBL-2H3 cells. A compound, identified as [Formula: see text]-sitostenone, was shown to inhibit [Formula: see text]-hexosaminidase secretion from activated mast cells. Our study demonstrated that ethanol extract contained the ingredients, which could inhibit immediate degranulation and de novo synthesis of allergic lipid mediators and cytokines in activated mast cells.

Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

BACKGROUND: Distinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. Mammalian IESCs and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. However, the association between Sox9 and DNA methylation in this process remains elusive. METHODS: The DNA methylation of small intestinal epithelial crypts in db/db mice was detected via combining methylated DNA immunoprecipitation with microarray hybridization. DNA methylation of Sox9 promoter in crypts and IESCs was validated using bisulfite sequence analysis. The target sequence of the transcription factor Sox9 in IESCs was investigated via chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq). RESULTS: Increased Sox9 expression is accompanied by the loss of methylation in its promoter in IESCs. Sox9 targets the enhancers of the Wnt signaling pathway-related genes. Sox9 predominantly acts as a transcriptional activator at proximal enhancers of Wnt4, Tab2, Sox4, and Fzd8, but also functions as a potential transcriptional inhibitor at a distant enhancer of Cdk1. Lack of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway leads to the loss of intrinsic inhibitory action and ultimately produces overactivation of this pathway in db/db mice. CONCLUSIONS: Our study sheds light on the connections among DNA methylation, transcription factor modulation, and Wnt signaling in IESCs in the diabetic state. Hypomethylation in the Sox9 promoter is correlated to increased Sox9 expression in db/db IESCs. Although there is increased expression of Sox9 in db/db IESCs, the loss of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway might result in abnormalities in this pathway.

Role of galectin-1 in urinary bladder urothelial carcinoma cell invasion through the JNK pathway.

Human galectin-1 is a member of the galectin family, proteins with conserved carbohydrate-recognition domains that bind galactoside. Galectin-1 is highly expressed in various tumors and participates in various oncogenic processes. However, detailed descriptions of the function of galectin-1 in urinary bladder urothelial carcinoma have not been reported. Our previous cohort investigation showed that galectin-1 is associated with tumor invasiveness and is a possible independent prognostic marker of urinary bladder urothelial carcinoma. The present study aimed to clarify the relevance of galectin-1 expression level to tumor progression and invasion. In order to decipher a mechanism for the contribution of galectin-1 to the malignant behavior of urinary bladder urothelial carcinoma, two bladder cancer cell lines (T24 and J82) were established with knockdown of galectin-1 expression by shRNA. Bladder cancer cells with LGALS1 gene silencing showed reduced cell proliferation, lower invasive capability, and lower clonogenicity. Extensive signaling pathway studies indicated that galectin-1 participated in bladder cancer cell invasion by mediating the activity of MMP9 through the Ras-Rac1-MEKK4-JNK-AP1 signaling pathway. Our functional analyses of galectin-1 in urinary bladder urothelial carcinoma provided novel insights into the critical role of galectin-1 in tumor progression and invasion. These results revealed that silencing the galectin-1-mediated MAPK signaling pathway presented a novel strategy for bladder cancer therapy.

CSF2 Overexpression Is Associated with STAT5 Phosphorylation and Poor Prognosis in Patients with Urothelial Carcinoma.

BACKGROUND: Urothelial carcinoma (UC) commonly occurs in the urinary bladder (UB) and rarely in upper the upper urinary tract (UT). Its molecular pathogenesis, however, remains obscure. Though the constitutive phosphorylation of Signal Transducer and Activator of Transcription 5 (STAT5) is an important part of carcinogenesis generally, researchers have not systematically investigated this process specifically in relation to UC. The present study addresses this gap. Through data mining a published transcriptomic database of UBUCs (GSE32894), it identified Colony Stimulating Factor 2 (CSF2) as the stepwise upregulated gene of much significance among those related to the positive regulation of tyrosine phosphorylation of STAT5 (GO:0042523). Since the phosphorylation of STAT5, a key process in the development of UC, is closely associated with CSF2, we then examine CSF2 transcript and protein expression, justifying their association with clinicopathological features and survival in our well-established cohort of patients with UC. DESIGN: Laser capture microdissection in conjunction with real-time qRT-PCR are used to detect CSF2 transcript levels in 24 UBUCs and 6 non-tumor urothelium samples. We then used the H-score method to evaluate the immunohistochemistry in order to determine CSF2 protein expression in 296 UBUCs and 340 UTUCs, respectively. After correlating protein expression status with key clinicopathological features, the prognostic significance of CSF2 protein expression was determined for disease-specific survival (DSS) and metastasis-free survival (MeFS). RESULTS: We exclusively detected the CSF2 transcript, which was stepwise upregulated in tumor lesions (p=0.010). In both groups of UC we found overexpression of CSF2 significantly related to incremental pT status (UTUC, p=0.011; UBUC, p<0.001), as well as with perineural invasion (UTUC, p=0.002; UBUC, p=0.001). Univariate analysis found a close correlation between CSF2 overexpression and unfavorable prognosis for both DSS (UTUC, p=0.0001; UBUC, p<0.0001) and MeFS (UTUC, p=0.0001; UBUC, p=0.0002). High expression of CSF2 still remained prognostically for DSS (UTUC, p=0.015; UBUC, p=0.004) and MeFS (UTUC, p=0.008; UBUC, p=0.027) in multivariate comparison. CONCLUSION: Our data showed that overexpression of CSF2 was inferred in advanced disease status and poor clinical outcomes for both UTUC and UBUC patients, suggesting that CSF2 may serve as an important prognosticator and a potential therapeutic target of UC.

Clarification of the molecular pathway of Taiwan local pomegranate fruit juice underlying the inhibition of urinary bladder urothelial carcinoma cell by proteomics strategy.

BACKGROUND: Pomegranate fruit has been shown to exhibit the inhibitory activity against prostate cancer and lung cancer in vitro and in vivo, which might be a resource for chemoprevention and chemotherapy of cancer. Our previous documented findings indicated that treatment of urinary bladder urothelial carcinoma cell with the ethanol extract isolated from the juice of pomegranate fruit grown in Taiwan could inhibit tumor cell. In this study we intended to uncover the molecular pathway underlying anti-cancer efficacy of Taiwan pomegranate fruit juice against urinary bladder urothelial carcinoma. METHODS: We exploited two-dimensional gel electrophoresis coupled with tandem mass spectrometry to find the de-regulated proteins. Western immunoblotting was used to confirm the results collected from proteomics study. RESULTS: Comparative proteomics indicated that 20 proteins were differentially expressed in ethanol extract-treated T24 cells with 19 up-regulated and 1 down-regulated proteins. These de-regulated proteins were involved in apoptosis, cytoskeleton regulation, cell proliferation, proteasome activity and aerobic glycolysis. Further studies on signaling pathway demonstrated that ethanol extract treatment might inhibit urinary bladder urothelial carcinoma cell proliferation through restriction of PTEN/AKT/mTORC1 pathway via profilin 1 up-regulation. It also might evoke cell apoptosis through Diablo over-expression. CONCLUSIONS: The results of this study provide a global picture to further investigate the anticancer molecular mechanism of pomegranate fruit.