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Quantitative real-time PCR (qRT-PCR) is currently one of the most reliable and improved tools for analyzing gene expression. Various studies have shown that housekeeping genes vary with cultivars, tissues and treatment. Reliable and stable reference genes were necessarily identified and evaluated according to different experimental requirements. In this study, 10 candidate reference genes were initially screened based on transcriptome sequencing data of four pear fruit development stages for three pear cultivars, including a candidate housekeeping gene PbrTUB. Furthermore, we ranked the expression stability of 10 candidate reference genes using GeNorm, NormFinder, BestKeeper and ReFinder algorithms. The results showed that Pbr028511, Pbr038418 and Pbr041114 were the most stable reference genes in Cuiguan, Housui and Xueqing fruit, respectively. Taken together, these results serve as a useful reference for gene function investigations and molecular mechanism studies in fruit development and ripening for various pear cultivars. [ABSTRACT FROM AUTHOR]

Background: Abnormal glycosylation of proteins has been identified in almost all types of cancers and is closely related to the cancer progression, metastasis, and survival of cancer patients. This study was to explore the values of serum tumor abnormal protein (TAP), an abnormal glycochain protein, in the diagnosis and prognosis of gastric cancer (GC). Methods: A total of 335 GC patients were included as the study group, and another 335 subjects served as the control group. Tumor abnormal protein expression was compared between the 2 groups. Correlation analysis was used to assess the correlations of TAP with clinicopathological factors. Gastric cancer patients were divided into training set and test set at a ratio of 2:1. Univariate and multivariate Cox regression analyses in training set were used to evaluate the prognostic significance of TAP in GC patients and explore the independent risk factors for overall survival (OS) and disease-free survival (DFS) to establish a prognostic model, followed by testing of the model. According to the median of TAP, 335 GC patients were divided into 2 groups to plot the survival curves of OS and DFS. Results: Tumor abnormal protein expression in the study group was significantly higher than in the control group. Taking the best cut-off value of TAP (110.128 μm2) as the diagnostic criteria for GC, the sensitivity and specificity of TAP were 83.58% and 97.61%, respectively, and the area under the receiver operating characteristics (ROC) curve was 0.935, which was not inferior to computed tomography (CT). Tumor abnormal protein expression was an independent risk factor for OS and DFS. The prognostic predictive value of TAP was better than that of pathological stage in GC patients. The model with TAP was effective in predicting prognosis. Conclusion: Tumor abnormal protein is an effective indicator for early screening and prognostic evaluation of GC and can also assist the clinical diagnosis and treatment of GC. [ABSTRACT FROM AUTHOR]

Objective: To investigate the protective effect of Chinese herbal formula Huangqin Decoction (HQD) on ulcerative colitis mouse model induced by dextran sulphate sodium (DSS) and human intestinal epithelial cell injury induced by tumour necrosis factor-α (TNF-α). Methods: In vivo, 30 male C57BL/6 mice were divided into 5 groups using a random number table (n=6 per group), including control, DSS, 5-aminosalicylic acid (5-ASA), HQD low- (HQD-L) and high-dose (HQD-H) groups. The colitis mouse model was established by 3% (w/v) DSS water for 5 days. Meanwhile, mice in the HQD-L, HQD-H and 5-ASA groups were administrated with 100, 200 mg/kg HQD or 100 mg/kg 5-ASA, respectively, once daily by gavage. After 9 days of administration, the body weight, disease activity index (DAI) score and colon length of mice were measured, the pathological changes of colons were analyzed by hematoxylin-eosin staining (HE) staining, and the levels of serum interleukin (IL)-6, IL-1β and TNF-α were measured by enzyme linked immunosorbent assay. In vitro, the human colon epithelial normal cells (FHC cells) were exposed to HQD (0.6 mg/mL) for 12 h and then treated with TNF-α (10 ng/mL) for 24 h. The tight junction (TJ) protein expression levels of Claudin-4 and Occludin, and the protein phosphorylation levels of p65 and inhibitor of nuclear factor kappaB (NF-κB)-α (IκBα) were measured by Western blot. Results: In vivo, compared with the DSS group, HQD-H treatment attenuated the weight loss and reduced DAI score of mice on the 8th day (P<0.05). Moreover, HQD-H treatment ameliorated the colon shortening in the DSS-induced colitis mice (P<0.05). HE staining showed HQD attenuated the pathological changes of colitis mice, and the histological scores of HQD-H and 5-ASA groups were significantly decreased compared with the DSS group (P<0.05). Meanwhile, HQD-H and 5-ASA significantly decreased the serum IL-1β, IL-6 and TNF-α levels of mice (P<0.05). In vitro experiments showed that HQD up-regulated Occludin and Claudin-4 protein expressions and inhibited p-p65 and p-IκBα levels in FHC cells compared with the TNF-α group (P<0.05). Conclusion: HQD significantly relieved the symptoms in DSS-induced colitis mice by inhibiting pro-inflammatory cytokines expression and maintained the homeostasis of TJ protein in FHC cells by suppressing TNF-α-induced NF-κB activation. [ABSTRACT FROM AUTHOR]

Seed coat-induced dormancy protects embryos from environmental conditions, and delays seed germination. In this study, we developed a simple method to accelerate embryo germination. That is, pear seeds were peeled the coats and then put on the humid gauze under 16 h day-light (75 μmol m−2 s−1), the embryo was germinated at 36 h after treatment. This germination results from the reduction of ABA concentration and unrelated to IAA, GA3, and ZR concentrations. Key genes for ABA biosynthesis were isolated and phylogenetically classified for pear. Only PbZEP2, PbZEP3, and PbNECD.C1 were found to be down-regulated in the germinated embryos, while the other genes, such as SDR and AO genes, are not correlated with ABA biosynthesis. Thus, the reduction of ABA concentration in the germinated embryos resulted from the down-regulations of PbZEP2, PbZEP3, and PbNECD.C1 genes. We speculate that the embryo in the pear cultivar Cuiguang is stressed by the seed coat, which leads to an accumulation of ABA in the embryo. We suggest that when abiotic stress of the seed coat is removed, signal transduction of the stresses is terminated and key genes for ABA biosynthesis are down-regulated. Abbreviations: ABA: Abscisic acid; GA: Gibberellic; ZR: Ribosylzeatin; IAA: auxin; ZEP: Zeaxanthin epoxidase; NCED: 9-cis-epoxycarotenoid dioxygenase; SDR: Short-chain dehydrogenase/reductase; AO: Aldehyde oxidase; MS: Mass spectrometer; ESI: Electrospray ionization; HPLC: High-performance liquid chromatography; qRT-PCR: Quantitative real-time PCR. [ABSTRACT FROM AUTHOR]

There is accumulating evidence that cancer stem cells (CSCs) play an important role in tumor progression. Novel strategies targeting CSCs have been widely researched. In the present study, we explored whether such CSCs existed in human ovarian cancer (OVCA) cell line and whether anti-CD44 antibody had effects on such subpopulation. We isolated and identified spheroid cells from SKOV-3. Then we used A3D8, an anti-CD44 mAb to treat spheroid cells with so-called 'stemness'. Effects of A3D8 on spheroid cells' biological behaviors were examined. Our findings showed that there was a small subpopulation that had so-called 'stemness' in SKOV-3 cell line. Against spheroid cells, A3D8 can (1) inhibit cell proliferation; (2) change cell cycle distribution and expression of p21, CDK2 and cyclinA; (3) enhance cisplatin (DDP)-induced apoptosis; (4) promote cell differentiation; (5) inhibit clone formation efficiency; (6) reduce invasive efficacy; (7) inhibit tumorigenicity. Thus, to sum up points which we have just showed, spheroid cells isolated from SKOV-3 can be used as an appropriate in vitro model for relevant study of human ovarian CSCs. And our results reasoned that anti-CD44 therapy may become a potential promising strategy for OVCA treatment. [ABSTRACT FROM AUTHOR]

The main clinical manifestations of eczema include itching, erythema, swelling and pain. Currently, allergies and TH1/TH2 cytokine imbalances are significant causes of eczema. TCM believes that eczema is mainly caused by incongruity between dry and wet. Wenguanmu ointment is a classic Mongolian medicine, which mainly composed of Xanthoceras sorbifolia Bunge , Coptis chinensis Franch and Bezoar. These ingredients can clear heat and dampness, dispel wind and dehumidification, anti-inflammatoryad analgesic. In this study, it was found that Wenguanmu ointment can treat eczema with anti-inflammatory, analgesic and antipruritic. In this study, the content of main components in Wenguanmu ointment was tested. Moreover, the therapeutic effect and mechanism of Wenguanmu ointment on eczema model mice were studied. Kunming mice (25 ± 2 g) were randomly divided into 6 groups: Control group; Model group; Vehicle group; Wenguanmu ointment group; Compound dexamethasone acetate cream group; Chushizhiyang ointment group. The eczema mouse model was established by DNCB. HPLC and TLC tests were used to determine the content of the main components in Wenguanmu ointment. HE staining was used to assess skin damage in mice. In order to detect the anti-inflammatory effect of Wenguanmu ointment on eczema, The levels of IgE, TNF-α, IFN-γ, COX-2 and IL-4 in serum was measured by ELISA. Genecards and Online Mendelian Inheritance in Man databases were used to analyze potential target gene predictions, and it was speculated that Wenguanmu ointment was associated with NF−κB signaling pathway and chemokine signaling pathway. To detect this inference, RT-qPCR and western blotting were used to detect protein and mRNA levels of CKLF-1, IκB-α, and NF-κB. Wenguanmu ointment can repress the symptoms of eczema caused by 2, 4-dinitrochlorobenzene, and inhibit the level of serum immunoglobulin E. Simultaneously it restrain the elevation of miscellaneous pro-inflammatory cytokines and chemokines, as well as reducing the expression of CKLF-1 and NF-κB protein in the nucleus, and increasing the protein expression of IκB to improve eczema. The ameliorating effect of Wenguanmu ointment on eczema lesions can play a importment role by inhibiting the CKLF-1/NF-κB pathway. [Display omitted] [ABSTRACT FROM AUTHOR]

Tripartite motif-containing protein 29 (TRIM29) is a member of TRIM family protein which has been reported to play a role in the progress of inflammatory and cancer diseases. However, its specific role in gastric cancer (GC) has yet to be fully understood. Here, we investigated the expression of TRIM29 in gastric cancer and its functions in the antitumor immunity. TRIM29 expression was lower in tumor tissues than that in paired normal tissues. Lower expression of TRIM29 was related to aberrant hypermethylation of CpG islands in TRIM29 gene. Comprehensive proteomics and immunoprecipitation analyses identified IGF2BP1 as TRIM29 interactors. TRIM29 interacted with IGF2BP1 and induced its ubiquitination at Lys440 and Lys450 site by K48-mediated linkage for protein degradation. IGF2BP1 promoted PD-L1 mRNA stability and expression in a 3′UTR and m6A-dependent manner. Functionally, TRIM29 enhanced antitumor T-cell immunity in gastric cancer dependent on the IGF2BP1/PD-L1 axis in vivo and in vitro. Clinical correlation analysis revealed that TRIM29 expression in patient samples was associated with CD8+ immune cell infiltration in the GC microenvironment and the overall survival rates of GC patients. Our findings revealed a crucial role of TRIM29 in regulating the antitumor T-cell immunity in GC. We also suggested that the TRIM29/IGF2BP1/PD-L1 axis could be used as a diagnostic and prognostic marker of gastric cancer and a promising target for GC immunotherapy. In the study, "TRIM29 promotes antitumor immunity through enhancing IGF2BP1 ubiquitination and subsequent PD-L1 downregulation in gastric cancer," we explored for the first time the role of the E3 ubiquitin ligase TRIM29 in antitumor T-cell immunity and showed that this role is dependent on the IGF2BP1/PD-L1 signaling axis. • Through a gastric cancer cell-T cell co-culture model, we found that TRIM29 enhanced anti-tumor T cell immunity. Further exploring its molecular mechanism, we confirmed that TRIM29 induces K48-linked ubiquitination degradation of the IGF2BP1 K440 and K450 sites through its C-terminal structural domain binding to the KH3/4 structural domain of IGF2BP1, which further mediates the effect of IGF2BP1 in a 3′UTR- and m6A-dependent manner on the PD-L1 mRNA stability and expression. • In animal models with different immune systems, we also found that specific antagonism of TRIM29 could synergize with PD-1 monoclonal antibody to exert anti-tumor effects, and clinical samples also verified that the expression of TRIM29 in gastric cancer was significantly correlated with the infiltration of CD8+ T-cells and the overall survival of gastric cancer patients. • This study provides new ideas for the immunotherapy of gastric cancer, and TRIM29 may be a potential prognostic predictor and therapeutic target for gastric cancer patients. The expression of TRIM29 can improve the prediction accuracy of PD-L1 histochemical scores, a predictive target for the efficacy of immunotherapy in gastric cancer, and the combination of TRIM29 specific antagonist and PD-1 monoclonal antibody can enhance the efficacy of immunotherapy for gastric cancer by increasing the activity of CD8+ T-cells. The combination of TRIM29-specific antagonist and PD-1 monoclonal antibody can improve the immunotherapeutic effect of gastric cancer by enhancing CD8+ T cell activity. [ABSTRACT FROM AUTHOR]

Highlights • Phylogenetic classification of SOT genes in rosaceae fruit trees. • Characterizing expression patterns of 24 SOT genes in all the pear tissues. • PbSOT6/20 is associated with the accumulation of sorbitol in the pear fruit. Abstract Sorbitol is a primary substrate that is translocated from its source to its sink in pear and apple by sorbitol transporters (SOTs). However, little is known about the expression profiles of SOTs in tree tissues, and their association with sorbitol accumulation. In this study, 64 SOT genes were isolated from the sequenced genomes of six rosaceae trees, which clustered phylogenetically into four groups. Groups I and III contained genes from pome trees, group II those from drupe trees, and group IV those from pome, drupe, and berry trees. A gene duplication analyses showed that the 24 SOT genes of pear arose from tandem, proximal, and dispersed duplications, which differs from their evolution in the other rosaceae fruit trees. A qRT-PCR analysis showed that the PbSOT6/20 gene in group IV is most strongly expressed in the leaf, branch, flower, fruit, and seed of pear. The expression pattern of PbSOT6/20 correlates more strongly with the pattern of sorbitol accumulation in the pear fruits than do those of other SOT genes. A low concentration (100 mg/g) of exogenous sorbitol induced the expression of PbSOT6/20 in the pear fruits, but not in the leaves. These results suggest that PbSOT6/20 is associated with sorbitol accumulation in pear fruits and plays important role in sorbitol translocation from its source to its sink. This study provides new insights into sorbitol translocation mediated by the SOT genes in pear. [ABSTRACT FROM AUTHOR]

• The expression pattern of eight ABF genes was characterized in various tissues at different stages. • RNA-Seq analysis of the self- and cross-pollinated styles revealed the 278 differentially expressed genes (DEGs) responsive to self-incompatibility reaction. • The ABRE-including promoter of six DEGs was activated by PbABF.E.1, PbABF.E.2, and/or PbABF.B. Self-incompatibility (SI) facilitates the rejection of pollen by self S-RNase. It is believed that a large number of genes are responsive to SI reactions; however, little is known about the transcriptional regulation of these genes. Herein, we explored the role of ABRE-binding factor (ABF) in gametophytic self-incompatibility (GSI). Phylogenetic analysis showed that eight ABF genes in pear clustered with five ABF genes in Arabidopsis. Of these ABF genes, PbABF.E.1 and PbABF.E.2 were expressed in all tissues, and PbABF.B was expressed in most tissues. These three ABF genes were also detected in the self- and cross-pollinated styles. Additionally, RNA-Seq analysis revealed 11,476 genes that were differentially expressed between the self- and cross-pollinated styles at 24, 48, and/or 72 h after pollination. Of these differentially expressed genes (DEGs), 278 were differentially expressed between the self- and cross-pollinated styles at any stage and thus are responsive to the GSI reaction. Notably, abscisic acid-responsive elements were detected in the promoter sequences of the 1213 DEGs including the six genes negatively correlated with pollen tube growth. The dual-luciferase assay showed that the promoter of these six genes was activated by PbABF.B, PbABF.E.1 , and/or PbABF.E.2. Therefore, PbABF.B, PbABF.E.1 , and PbABF.E.2 were involved in the GSI reaction by mediating the expression of genes responsive to the GSI reaction. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Luteinizing hormone (LH) plays an important role in the development of ovarian cancer, and has been shown to inhibit apoptosis in ovarian cancer cells. Similarly, survivin is a molecule that has been shown to inhibit apoptosis in other types of cancer. Therefore, the aim of this study was to determine whether survivin can be induced by LH in ovarian cancer, and whether this induction influences the sensitivity of ovarian cancers to chemotherapy. Study design: Survivin expression was monitored using western blot assays, and flow cytometry was used to detect the effects of cisplatin on the induction of apoptosis by LH. MTT assays were also used to analyze rates of cell proliferation. Results: Administration of LH in vitro induced survivin expression in a dose-dependent manner. Moreover, this signaling was dependent on the ERK1/2 signaling pathway. LH also blocked apoptosis induced by cisplatin. Conclusion: These results suggest that LH influences the sensitivity of ovarian cancer cells to chemotherapy via signaling to inhibit apoptosis that also upregulates survivin. [Copyright &y& Elsevier]