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BACKGROUND: Our study aimed to investigate the Hub genes and their prognostic value in colorectal cancer (CRC) via bioinformatics analysis. METHODS: The data set of colorectal cancer was downloaded from the GEO database (GSE21510, GSE110224 and GSE74602) for differential expression analysis using the GEO2R tool. Hub genes were screened by protein-protein interaction (PPI) comprehensive analysis. GEPIA was used to verify the expression of Hub genes and evaluate its prognostic value. The protein expression of Hub gene in CRC was analyzed using the Human Protein Atlas database. The cBioPortal was used to analyze the type and frequency of Hub gene mutations, and the effects of mutation on the patients' prognosis. The TIMER database was used to study the correlation between Hub genes and immune infiltration in CRC. Gene set enrichment analysis (GSEA) was used to explore the biological function and signal pathway of the Hub genes and corresponding co-expressed genes. RESULTS: We identified 346 differentially expressed genes (DEGs), including 117 upregulated and 229 downregulated. Four Hub genes (AURKA, CCNB1, EXO1 and CCNA2) were selected by survival analysis and differential expression validation. The protein and mRNA expression levels of AURKA, CCNB1, EXO1 and CCNA2 were higher in CRC tissues than in adjacent tissues. There were varying degrees of immune cell infiltration and gene mutation of Hub genes, especially B cells and CD8 + T cells. The results of GSEA showed that Hub genes and their co-expressed genes mainly participated in chromosome segregation, DNA replication, translational elongation and cell cycle. CONCLUSION: Overexpression of AURKA, CCNB1, CCNA2 and EXO1 had a better prognosis for CRC and this effect was correlation with gene mutation and infiltration of immune cells. [ABSTRACT FROM AUTHOR]

Aim. Cyclophilin A (CypA)/CD147 signaling plays critical roles in the regulation of inflammation and bone metabolism. This study aimed to investigate the participation of CypA/CD147 in mice periapical lesions progression and its relationship with bone resorption. Methodology. Periapical lesions were induced by pulp exposure in the first lower molars of 40 C57BL/6J mice. The mice were sacrificed on days 0, 7, 14, 21, 28, 35, 42, and 49. Mandibles were harvested for X-ray imaging, microcomputed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. Western blot was employed to further detect the related molecular signaling pathways in LPS-stimulated RAW 264.7 cells treated with CypA inhibitor. Results. The volume and area of the periapical lesions increased from day 0 to day 35 and remained comparably stable until day 49. Immunohistochemistry demonstrated that the CypA expression levels also increased from day 0 to day 35 and decreased until day 49, similar to CD147 expression (R2 = 0.4423, P < 0.05), osteoclast number (R2 = 0.5101, P < 0.01), and the expression of osteoclastogenesis-related matrix metalloproteinase 9 (MMP-9) (R2 = 0.4715, P < 0.05). Serial sections further confirmed the colocalization of CypA and CD147 on osteoclasts with immunohistochemistry. And the distribution of CypA-positive or CD147-positive cells was positively correlated with the dynamics of MMP-9-positive cells by using immunofluorescence analysis. Furthermore, CD147 and MMP-9 expression in RAW 264.7 cells were both downregulated with CypA inhibitor treatment (P < 0.05). Conclusions. The present study reveals the positive correlation of CypA/CD147 signaling and osteoclast-related MMP-9 expression in mice inflammatory periapical lesions progression. Therefore, intervention of CypA/CD147 signaling could probably provide a potential therapeutic target for attenuating inflammatory bone resorption. [ABSTRACT FROM AUTHOR]

This study investigates the effect of hyperoxic storage on the antioxidant properties and bioactive substance metabolism of post-harvest Ganoderma lucidum fruiting bodies. During storage, the fruiting bodies experienced weight loss, which positively correlated with oxygen content. Oxygen treatment facilitated the increase of antioxidant enzyme activity and the decrease of membrane peroxidation. The enzyme activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were increased by 58 %, 149 %, and 64 %, respectively; malondialdehyde (MDA) content was reduced by 39.91 % compared to the control group when the fruiting bodies were stored in 80% oxygen for 5 d. Meanwhile, the contents of Ganoderma polysaccharides and ganoderic acid increased with time, attaining maximum values of 121 g kg−1 and 8.23 g kg−1 on 4th d and 5th d, respectively, which were 36 % and 32 % higher than the control group. When the fruiting bodies were stored in 40 % and 80 % oxygen, the expression of key enzyme genes involved in the metabolism of Ganoderma polysaccharides and ganoderic acid, such as phosphoglucose metatase (PGM), uridinediphosphate glucose pyrophosphorylase (UGP), β-1,3-glucan synthase (GLS), hydroxymethylglutaryl-CoA reductase (HMGR), squalene synthase (SQS), lanosterol synthase (LS) and squalene epoxidase (SE), was found to be upregulated. • High oxygen promotes the accumulation of Ganoderma polysaccharides. • Storage time and hyperoxia facilitated the accumulation of ganoderic acid. • Gene expression levels of key enzymes were regulated by oxygen storage conditions. • Oxygen treatment enhanced the antioxidant capacity of Ganoderma lucidum. [ABSTRACT FROM AUTHOR]

Objective To screen and analyze small molecule therapeutic compounds dissociated with colorectal cancer metastasis. Methods First statistical comparison was made for the expression profile data of early metastasizing colorectal cancer tissues wíth the data of normal colorectum tissues to screen the differentially expressed genes. Then the matching patterns between differenfially expressed genes and the small molecules were used to screen small molecule therapeutic tanespimycin compounds associated with early colorectal cancer metastasis. Last, the enrichment status of the small molecule therapeutic compounds target protein were analyzed to screen the colorectal cancellated target protein. We combined the colorectal cancer expresiion profile and the bond strength between small molecule compounds and the target protein to screen the most relevant target protein and build a regulatory network. Results By the small molecule compounds enrichment analysis, tanespimycin had the highest score, the target protein of tanespimycin had the highest score of enrichment in colorectal cancer. It indicated that tanespimycin was closely related to colorectal cancer. By synthesizing the expression profile and the molecular bonding strength, we found that the colorectal cancer-related target proteins named CHEK1, AURKA, GSTP1 and NQO1 might be the key proteins of tanespimycin therapy that inhibhed the metastasis of colorectal cancer. The regulatory network was built by the four target proteins participating in the biological process of drug metaboiism. That further explained the relationship between the four proteins and tanespimycin. Conclusion Tanespimycin may be the small molecule therapeutic compound that can inhibs eariy metastasis of colorectal cancer by interacting wth CHEK1, AURKA, GSTP1 and NQO1 to regulate the signaling network. [ABSTRACT FROM AUTHOR]

Huanglongbing ( HLB) or citrus greening disease is a destructive disease of citrus worldwide, which is associated with Candidatus Liberibacter asiaticus. This phloem-limited fastidious pathogen is transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that, during infection, Ca. L. asiaticus differentially expresses the genes critical for its survival and/or pathogenicity in either host. To test this hypothesis, quantitative reverse transcription-polymerase chain reaction was performed to compare the gene expression of Ca. L. asiaticus in planta and in psyllid. Overall, 381 genes were analysed for their gene expression in planta and in psyllid. Among them, 182 genes were up-regulated in planta compared with in psyllid ( P < 0.05), 16 genes were up-regulated in psyllid ( P < 0.05) and 183 genes showed no statistically significant difference ( P ≥ 0.05) in expression between in planta and in psyllid. Our study indicates that the expression of the Ca. L. asiaticus genes involved in transcriptional regulation, transport system, secretion system, flagella assembly, metabolic pathway and stress resistance are changed significantly in a host-specific manner to adapt to the distinct environments of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Ca. L. asiaticus genes in a plant host and its insect vector. [ABSTRACT FROM AUTHOR]

Background: RNA-seq and microarray are the two popular methods employed for genome-wide transcriptome profiling. Current comparison studies have shown that transcriptome quantified by these two methods correlated well. However, none of them have addressed if they complement each other, considering the strengths and the limitations inherent with them. The pivotal requirement to address this question is the knowledge of a well known data set. In this regard, HrpX regulome from pathogenic bacteria serves as an ideal choice as the target genes of HrpX transcription factor are well studied due to their central role in pathogenicity. Results: We compared the performance of RNA-seq and microarray in their ability to detect known HrpX target genes by profiling the transcriptome from the wild-type and the hrpX mutant strains of γ-Proteobacterium Xanthomonas citri subsp. citri. Our comparative analysis indicated that gene expression levels quantified by RNA-seq and microarray well-correlated both at absolute as well as relative levels (Spearman correlation-coefficient, rs > 0.76). Further, the expression levels quantified by RNA-seq and microarray for the significantly differentially expressed genes (DEGs) also well-correlated with qRT-PCR based quantification (rs = 0.58 to 0.94). Finally, in addition to the 55 newly identified DEGs, 72% of the already known HrpX target genes were detected by both RNA-seq and microarray, while, the remaining 28% could only be detected by either one of the methods. Conclusions: This study has significantly advanced our understanding of the regulome of the critical transcriptional factor HrpX. RNA-seq and microarray together provide a more comprehensive picture of HrpX regulome by uniquely identifying new DEGs. Our study demonstrated that RNA-seq and microarray complement each other in transcriptome profiling. [ABSTRACT FROM AUTHOR]

Abstract: The reasons underlying the occurrence of multiple revertant genotypes in Wiskott–Aldrich syndrome (WAS) patients remain unclear. We have identified more than 30 revertant genotypes in a C995T WAS patient having 10–15% revertant, WAS protein (WASp)-expressing circulating lymphocytes [1]. Of 497 allospecific T-cell clones generated from the peripheral blood, 47.1% carried a revertant sequence. All revertant T-cell clones exhibited restoration of WASp expression. However, anti-CD3-induced proliferative responses varied greatly amongst revertants. Several revertant T-cell clones expressed an internally deleted WASp mutant lacking much of the proline-rich region. This potentially accounts for the reduced anti-CD3 proliferative responses of these T-cell clones. We found no evidence for an increased DNA mutation rate in this patient. We conclude that the diversity of revertant genotypes in our patient does not result from an extraordinary mutation rate and that the amino acid sequence space explored by WASp in revertant T-cells is significantly smaller than might have been predicted from the diversity of revertant genotypes. [Copyright &y& Elsevier]

OBJECTIVE: The expression of S100A6 (calcyclin), a member of the S100 calcium binding protein family, is elevated in a number of malignant tumors, but there have been few reports about its expression in gastric cancer. The aim of this study was to investigate its expression regulations in human gastric cancer and noncancerous mucosa, and the response to chemotherapeutic drugs in the gastric cancer cell line. MATERIALS AND METHODS: In one matched gastric cancer sample pair, the serial analysis of gene expression (SAGE) experiment was conducted to compare the gene expression profiles between cancerous and adjacent tissues. To detect the expression regulations among more cancerous tissues, microarrays were carried out and real-time RT-PCR was conducted to validate the results. At the protein level, Western blot and tissue microarray (TMA) examination were further used to verify S100A6 expression. The regulation detection of S100A6 with flurouracil and doxorubicin at the mRNA and protein level was performed in the SGC7901 cell line. RESULTS: With the SAGE strategy, five times more S100A6 tags were identified in cancer tissues than in normal tissues. With the cDNA microarray, S100A6 was found to be significantly upregulated in 21 of 42 (50%) nonselective gastric cancers. In 10 other paired samples, the upregulation of S100A6 was consolidated with RT-PCR and Western blot analysis as well. A total of 14 endoscopy-sectioned gastric noncancerous lesions and corresponding normal gastric mucosa were also applied to profile the gene expression; both cDNA microarray and RT-PCR demonstrated no significant alterations of S100A6 at the mRNA level. TMA examination showed that 34 of 52 (65.4%) cancer samples were positively stained, while only 17 of 80 (21.3%) noncancerous lesions were positively detected and all nine normal mucosae were detected to be negative. An in vitro experiment showed that in the gastric cell line SGC-7901, S100A6 mRNA was detected to be upregulated from 24 to 72 h after treatment with 5 mg/L 5-flurouracil or 0.3 mg/L doxorubicin, and there were two wave upregulations of the S100A6 protein. CONCLUSION: The observed regulated expression of S100A6 suggests that it is associated with gastric cancer tumorigenesis and quantitation of S100A6 is a promising tool for diagnosis of gastric cancer. [ABSTRACT FROM AUTHOR]

Abstract: ERCC1 is a critical gene within the nucleotide excision repair pathway. Overexpression of ERCC1 through promoter-mediating transcriptional regulation is associated with repair of cisplatin-induced DNA damage and clinical resistance to platinum-chemotherapy. Several transcriptional repressors and activators within the 5′-flanking region of the ERCC1 gene may be involved in the up-regulation of this gene. Minimal sequence within the promoter region required for ERCC1 transcription was analyzed by CAT assay and demonstrated that the region of −220 to −110 is essential to constitutive expression of ERCC1 gene in ovarian cancer cell line A2780/CP70. A more forward upstream region seems to be responsible for cisplatin-induced expression. Study of the functional cis-element in this region by electrophoretic mobility shift assay indicates that a MZF1-like site as well as an AP1-like site responded in a time-dependent manner to cisplatin stimulation with altered binding activities. EMSA with MZF1 ZN1–4 consensus oligonucleotides suggests that the MZF1 N-terminal domain of zinc finger cluster may bind to the MZF1-like site of the ERCC1 promoter region. MZF1 mRNA in A2780/CP70 cells decreased upon cisplatin exposure as analyzed by quantitative PCR, suggesting that MZF1 may mediate cisplatin-invoked gene expression in these cells. Overexpression of MZF1 repressed the ERCC1 promoter activity as determined in co-transfection assay, suggesting that MZF1 might be a repressor of ERCC1 transcription upon cisplatin exposure. In summary, our studies revealed a core promoter region and adjacent drug-responsible region within the ERCC1 promoter. The drug-responsible region contains cis-elements of activator, AP1 and repressor, MZF1. In response to cisplatin treatment, decreased MZF1 and increased AP1 binding activities appear to be the leading mechanism of up-regulation of ERCC1 expression. Our findings imply potential therapeutic strategies to antagonize drug resistant mechanisms in treatment of human ovarian cancer. [Copyright &y& Elsevier]

Background: Single-cell RNA sequencing (scRNA-seq) measurements of gene expression show great promise for studying the cellular heterogeneity of rice roots. How precisely annotating cell identity is a major unresolved problem in plant scRNA-seq analysis due to the inherent high dimensionality and sparsity. Results: To address this challenge, we present NRTPredictor, an ensemble-learning system, to predict rice root cell stage and mine biomarkers through complete model interpretability. The performance of NRTPredictor was evaluated using a test dataset, with 98.01% accuracy and 95.45% recall. With the power of interpretability provided by NRTPredictor, our model recognizes 110 marker genes partially involved in phenylpropanoid biosynthesis. Expression patterns of rice root could be mapped by the above-mentioned candidate genes, showing the superiority of NRTPredictor. Integrated analysis of scRNA and bulk RNA-seq data revealed aberrant expression of Epidermis cell subpopulations in flooding, Pi, and salt stresses. Conclusion: Taken together, our results demonstrate that NRTPredictor is a useful tool for automated prediction of rice root cell stage and provides a valuable resource for deciphering the rice root cellular heterogeneity and the molecular mechanisms of flooding, Pi, and salt stresses. Based on the proposed model, a free webserver has been established, which is available at https://www.cgris.net/nrtp. [ABSTRACT FROM AUTHOR]

Abstract Background Androgen has been implicated in aging-related cardiac remodeling, but its precise role in aging heart remains controversial. We aimed to investigate the role of testosterone in the development of aging-related cardiac remodeling and the mechanisms involved. Methods Wild type and Axl knockout mice (Axl−/−) were randomized into three groups: the young group (n = 30, 3 months old), the aging group (n = 30, 18 months old), the testosterone undecanoate treatment group (TU, n = 30, 18 months old). Mice in the TU group were given testosterone undecanoate (39 mg/kg) by subcutaneous injection on the back at fifteen-months-old, once a month, a total of three times. The old group received solvent reagent (corn oil) by the same method. Results The aging mice exhibited a decrease in serum testosterone, and Gas6 levels and an increase in apoptosis, and manifested cardiac fibrosis. Testosterone injection to wild type mice increased the levels of testosterone and Gas6 in serum and decreased cardiac apoptosis and fibrosis. Axl−/−mice receiving testosterone injection exhibited no obvious improvement in cardiac remodeling although the levels of testosterone and Gas6 in serum elevated. Conclusions These data indicated that testosterone replacement therapy (TRT) alleviates cardiac fibrosis and apoptosis, at least in part by enhancing Gas6 expression. Moreover, deletion of Axl disables testosterone, which indicated that Axl is an important downstream regulator of testosterone. TRT would improve aging-related cardiac remolding via Gas6/Axl signaling pathway, implicating its therapeutic potential to treat aging-related heart disease. Highlights • Testosterone decreases cardiomyocyte apoptosis in aging mice. • Testosterone ameliorates cardiac fibrosis in aging mice. • Testosterone increases the level of Gas6. • Testosterone protects from cardiac aging in a Gas6/Axl dependent manner. [ABSTRACT FROM AUTHOR]

The greenfin horse-faced filefish (Thamnaconus septentrionalis) is susceptible to recurrent Amyloodinium ocellatum (AO) infestation over the grow-out production cycle. This parasite breeds mainly on the gills, causing hypoxia in the fish body, and leading to many deaths. The host-parasite response drives a complex immune reaction, which is poorly understood. To generate a model for host-parasite interaction and the pathogenesis of AO in greenfin horse-faced filefish, an RNA-seq approach, differential gene expression, GO, and KEGG analyses were employed. Overall, 624 new genes and 2076 differentially expressed genes (DEGs) were detected, including 942 upregulated and 1134 downregulated genes in the gills. Compared with the control group, the expression of leptin a, GTPase IMAP family member 4, and NLR family CARD domain-containing protein 3 was significantly higher in the AO-infected group. Conversely, cell wall integrity and stress response component 1-like, and hepcidin-like were significantly downregulated in the gills of AO-infected fish. GO and KEGG enrichment analysis indicated that DEGs were significantly enriched in signaling pathways associated with viral protein interaction with cytokine and cytokine receptor and cytokine–cytokine receptor interaction. Collectively, this transcriptomic study provides novel molecular insights into the pathology caused by AO infestation and alternative theories for future research implementing strategies to control and manage AO. [ABSTRACT FROM AUTHOR]

Fructose-1,6-bisphosphatase 1 (FBP1) is a rate-limiting enzyme in gluconeogenesis. Recently, the catalytic activity-independent function of FBP1, hypoxia-induced factor (HIF) repression in the nucleus, was identified. The aim of the present study was to investigate the association between FBP1 and hypoxia-related gene expression in clear cell renal cell carcinoma (ccRCC). The protein expression levels of FBP1, HIF-1α, HIF-2α, erythropoietin (EPO) and carbonic anhydrase IX (CA9) were assessed by immunohistochemical staining of ccRCC paraffin blocks from 123 patients using the tissue microarray technique. The expression level of FBP1 was then correlated with various clinicopathological factors, and the protein expression levels of HIF-1α, HIF-2α, EPO and CA9. Clinicopathological factors, including age, gender, T stage and Fuhrman grade, were not significantly different between patients with low and high FBP1 expression in ccRCC (P>0.05). FBP1 protein expression level was significantly correlated with the expression levels of HIF-1α (P=0.005) and EPO (P=0.010), but not significantly correlated with the expression levels of HIF-2α (P=0.123) and CA9 (P=0.513) in ccRCC tissues. The current findings confirm the association between FBP1 and hypoxia-related gene expression, and may facilitate understanding of the mechanisms of ccRCC tumorigenesis. [ABSTRACT FROM AUTHOR]

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been described as a possible prognostic marker in patients with acute myeloid leukemia (AML). However, the results in this field are not reproducible in different cohorts. In this study, we investigated WT1 mutations, expression levels and SNP rs16754 in a cohort of 122 adult patients with AML. As the major allele (65.6%) in a Chinese population, WT1GG was associated with younger age (≤ 60) and lower percentage of blasts than WT1GA/AA. Meanwhile, improved overall survival (OS, p = 0.035) and disease-free survival (DFS, p = 0.021) were observed in WT1GG compared with WT1GA/AA. We then found that WT1 mutation, occurring in 8% of patients with AML, did not predict clinical outcome. Finally, WT1 levels were higher in patients with WT1GG than in those with WT1GA/AA. However, high levels of WT1 (> median) predicted worse OS ( p = 0.015) and DFS ( p = 0.034) than low levels of WT1 (≤ median). However, further studies are required to elucidate the mechanism of why WT1GG, which was associated with higher median expression of WT1 that predicts worse OS and DFS compared to low expression of WT1, predicted better OS and DFS compared with WT1GA/AA. In summary, WT1 rs16754 and WT1 expression have a significant impact on clinical outcome in patients with AML. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) is a malignant tumor with complex pathogenesis. In HCC, the possible roles of transcriptional factor WD repeat and HMG-box DNA binding protein 1 (WDHD1) remain unclear. Hence, our study is aimed at verifying the prognosis prediction ability and potential biological mechanisms of WDHD1 in HCC. Results: In this study, a total of 7171 clinical samples were obtained to quantitatively analyze the protein and mRNA expression levels of WDHD1 by using immunohistochemistry, gene microarrays, and highthroughput sequencing technologies. The result of in-house immunohistochemistry assay indicated that WDHD1 protein was remarkably overexpressed in HCC tissues compared with the non-HCC tissues (AUC > 0.99, the single Standardized Mean Difference [SMD] = 4.46). The overexpression trend of WDHD1 was validated by the comprehensive analysis based on a total of 4004 HCC tissues and 3167 controls (SMD = 1.333; AUC = 0.91). Moreover, the higher WDHD1 expression resulted in the poorer prognosis of HCC, as assessed by overall survival and relapse-free survival analyses (pooled hazard ratios > 1). WDHD1-coexpressed genes were screened out for enrichment analyses to enquire the prospective signaling pathways of WDHD1 in HCC and to probe the potential transcriptional targets of WDHD1. The WDHD1-coexpressed genes were mainly involved in the division process of chromosome and cell nucleus in HCC. UBA52 was identified as a crucial target of WDHD1. Conclusions: WDHD1 may act as an oncogene in HCC and it has the potential to become a novel marker for predicting the prognosis of HCC patients, which may benefit from the early intervention of HCC. [ABSTRACT FROM AUTHOR]

Highlights: [•] TMZ resistant U251 cells express low LRIG1, but high levels of EGFR/Topo-2/Bcl-2. [•] U251 cells with LRIG1 overexpression are hyper-sensitive to TMZ. [•] U251 cells with LRIG1 knocking-down are resistant to TMZ. [•] EGFR-mTOR activation is important for Topo-2/Bcl-2 expression and TMZ resistance. [•] Topo-2/Bcl-2 inhibition or depletion restores TMZ sensitivity in resistant U251 cells. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have indicated that long-term chemotherapy decreases the sensitivity of oral cancer cells to chemotherapeutics while simultaneously increasing resistance to these drugs. COX-2 inhibitors are known to enhance the toxic action of anti-tumor drugs against cancer cells. Using the MTT method, we investigated the influence of the COX-2 selective inhibitor Celecoxib on the proliferation of KB/VCR oral cancer cell lines and analyzed the effect of Celecoxib on the regulation of P-glycoprotein (P-gp) expression and function. Western blot analysis was employed to detect the expression of P-gp, and flow cytometry was used to evaluate P-gp function by detecting the accumulation of the active P-gp functional fluorescence substrate within KB/VCR cells. The results revealed that a low dose of Celecoxib (10μmol/L) showed no growth inhibitory effects on KB/VCR cell lines. When the concentration of Celecoxib was greater than or equal to 20μmol/L, the inhibitory effect on KB/VCR cells was significantly enhanced in a time- and dose-dependent manner. The lower dose of Celecoxib (10μmol/L) significantly enhanced the toxicity of Vincristine (VCR) against KB/VCR cell lines. After the application of Celecoxib plus VCR (10μmol/L+1.5μmol/L, respectively) treatment for 24, 48 or 72h, the growth inhibition rates of KB/KBV cells were 37.82±1.60%, 47.84±1.29% and 54.43±2.35%, respectively, which were significantly higher than the rates in the cells treated only with Celecoxib (10μmol/L) or VCR (1.5μmol/L) (all P <0.01). P-gp expression levels in KB/KBV cells treated with Celecoxib plus VCR (10μmol/L+1.5μmol/L, respectively) were markedly lower than the levels in control cells and those treated with VCR (1.5μmol/L) (all P <0.01). In addition, the intensity of Rho123 fluorescence of KB/KBV cells in cells treated with Celecoxib plus VCR (10μmol/L+1.5μmol/L, respectively) or Celecoxib alone (10μmol/L) was significantly higher than the intensity observed in control cells and those treated with VCR alone (1.5μmol/L) (all P <0.01). The underlying mechanism of these phenomena is likely correlated with the down-regulation of the expression and function of P-gp due to Celecoxib, thereby increasing the amount of VCR accumulated in KB/VCR cells. [Copyright &y& Elsevier]

C2H2 zinc finger protein (ZFP) genes have been extensively studied in many organisms and can function as transcription factors and be involved in many biological processes including plant growth and development and stress responses. In the current study, a comprehensive genomics analysis of the C2H2-ZFP genes in B. rapa was performed. A total of 301 B. rapa putative C2H2-ZFP (BrC2H2-ZFP) genes were identified from the available Brassica genome databases, and further characterized through analysis of conserved amino acid residues in C2H2-ZF domains and their organization, subcellular localization, phylogeny, additional domain, chromosomal location, synteny relationship, Ka/Ks ratio, and expression pattern. We also analyzed the expression patterns of eight B. rapa C2H2-ZFP genes under salt and drought stress conditions by using qRT-PCR technique. Our results showed that about one-third of these B. rapa C2H2-ZFP genes were originated from segmental duplication caused by the WGT around 13 to 17 MYA, one-third of them were highly and consecutively expressed in all tested tissues, and 92% of them were located in nucleus by prediction supporting then their functional roles as transcription factors, of which some may play important roles in plant growth and development. The Ka/Ks ratios of 264 orthologous C2H2-ZFP gene pairs between A. thaliana and B. rapa were all, except two, inferior to 1 (varied from 0.0116 to 1.4919, with an average value of 0.3082), implying that these genes had mainly experienced purifying selection during species evolution. The estimated divergence times of the same set of gene pairs ranged from 6.23 to 38.60 MY, with an average value of 18.29 MY, indicating that these gene members have undergone different selective pressures resulting in different evolutionary rates during species evolution. In addition, a few of these B. rapa C2H2-ZFPs were shown to be involved in stress responses in a similar way as their orthologs in A. thaliana. Comparison between A. thaliana and B. rapa orthologous C2H2-ZFP genes showed that the majority of these C2H2-ZFP gene members encodes proteins with conserved subcellular localization and functional domains between the two species but differed in their expression patterns in five tissues or organs. Thus, our study provides valuable information for further functional determination of each C2H2-ZFP gene across the Brassica species, and may help to select the appropriate gene targets for further in-depth studies, and genetic engineering and improvement of Brassica crops. [ABSTRACT FROM AUTHOR]

Xingnaojing (XNJ) injection, derived from traditional Chinese medicine formulation, has a protective effect against stroke, but the underlying mechanism is unclear, which severely limited its clinical application. This research aims to elucidate the role and mechanism of XNJ in reducing cerebral ischemic reperfusion (I/R) injury. Rats received 2 h cerebral ischemia followed by reperfusion of 24 h and were intraperitoneally given 5, 10, or 15 ml/kg XNJ 24 h before ischemia and at the onset of reperfusion, respectively. TTC staining, HE staining, and neurological score were implied to evaluate the effectiveness of XNJ. The protein expressions of PI3K/Akt and eNOS signaling were measured. Experiments were further performed in human brain microvascular endothelial cells (HBMECs) to investigate the protective mechanisms of XNJ. HBMECs were subjected to 3 h oxygen and glucose deprivation following 24 h of reoxygenation (OGD) to mimic cerebral I/R in vitro. PI3K inhibitor LY294002 was added with or without the preconditioning of XNJ. Multiple methods including western blot, immunofluorescence, DAPI staining, JC-1, and flow cytometry were carried out to evaluate the effect of XNJ on HBMECs. XNJ could improve rat cerebral ischemic injury and OGD induced HBMECs apoptosis. In vivo and in vitro researches indicated that the mechanism might be relevant to the activation of PI3K/Akt/eNOS signaling. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying endometrial stromal cell proliferation and differentiation (decidualization) are still not fully understood. This study revealed that increased Slp-2 expression is a significant factor modulating endometrial stromal cell proliferation and decidualization in both mice and humans. Our results showed a significant difference in the mRNA and protein levels between the implantation site and inter-implantation site on day 5 and day 6 of pregnancy in mice (all P < 0.05). Strong Slp-2 immunostaining was mainly localized within the decidual zone of mice through the post-implantation period. Mice with artificially induced deciduoma showed significantly higher expression of Slp-2 compared with uninduced controls ( P < 0.005). Human stromal cells in the middle and late-secretory phases demonstrated significantly (all P < 0.05) upregulated SLP-2, compared with cells in the proliferative phase and early secretory phases. Further analyses of the SLP-2 gene knocked down revealed a significant ( P < 0.005) repression of both the decidualization marker gene's expression (decidual/trophoblast prolactin-related protein in mice, insulin-like growth factor binding protein and prolactin in human) and the cell proliferation in in vitro-induced decidualized primary endometrial stromal cells in mice and humans. [ABSTRACT FROM AUTHOR]

We characterised DNA methylation and gene expression of four tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4, DR5, DcR1 and DcR2 in three choriocarcinoma (JAR, JEG-3, BeWo) and two transformed (HTR-8/SVneo and HPT-8) cell lines. DR4 mRNA was detected in JAR, JEG-3, BeWo and HTR-8/SVneo cells, whereas DR5 was present in all detected cells. DcR1 transcripts were expressed only in JAR, JEG-3 and BeWo cells, whereas DcR2 transcripts were detected only in HTR-8/SVneo and HPT-8 cells. Hypermethylated DR4 promoter was observed in JAR, JEG-3, BeWo and HTR-8/SVneo cells, hypermethylated DcR1 promoter in HTR-8/SVneo and HPT-8 cells and hypermethylated DcR2 promoter in JAR, JEG-3 and BeWo cells. Restoration of DR4, DcR1 and DcR2 expression with decreased DNA methylation of these genes was induced by the DNA demethylation agent 5-aza-2'-deoxycytidine (5-aza-CdR) in trophoblast cells, whereas DR5 expression did not exhibit any change. Significant negative correlation between the expression and DNA methylation of these genes was also observed. In all tested cell lines, only HPT-8 demonstrated sensitivity to TRAIL-induced apoptosis. Combined treatment with 5-aza-CdR and TRAIL resulted in apoptosis in JAR, JEG-3, BeWo and HTR-8/SVneo cells but not in HPT-8 cells. The results indicate that DNA methylation is associated with TRAIL receptor expression and might be involved in trophoblast apoptosis. [ABSTRACT FROM AUTHOR]

Background: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/β-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce. Methods: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR. Results: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1. Conclusion: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis. [ABSTRACT FROM AUTHOR]

Hyperlipidemia can be harmful to the pancreas and β 3 -adrenoceptor agonist can improve lipid metabolism disorder. We aimed to study the effects of β 3 -adrenoceptor activation on glucose, insulin and the expression of pancreatic adrenoceptors and angiotensin II receptors. Ten C57BL/6J mice at the age of 10 weeks served as normal control, and forty age-matched apolipoprotein E knockout (ApoE −/− ) mice were randomly divided into hyperlipidaemia model group, low-dose and high-dose β 3 -adrenoceptor agonist group and β 3 -adrenoceptor antagonist group. After 26 weeks of high-fat diet, treatments were given for 12 weeks. Serum glucose and insulin levels in 48 weeks old mice were measured using an automatic biochemical detector. Quantitative rt-PCR and Western blot were used to analyze the mRNA and protein expression of α 1A -, α 2A -, β 2 -, β 3 -adrenoceptors and angiotensin II type 1 and type 2 receptors in pancreas. We found that β 3 -adrenoceptor agonist could decrease serum glucose and insulin levels in aged ApoE −/− mice ( P <0.01) and down-regulate the expression of α 1A -adrenoceptor and angiotensin II type 1 receptor which were significantly increased in model mice ( P <0.05, P <0.01). Compared with the model mice, α 2A -, β 2 -, β 3 -adrenoceptor and angiotensin II type 2 receptor expression were up-regulated in β 3 -adrenoceptor agonist treat mice ( P <0.05, P <0.01). These results suggest that chronic β 3 -adrenoceptor activation regulated the expression of adrenoceptors and angiontensin II receptors towards contrary direction, which indicates that there are interactions between β 3 -adrenoceptor and subtypes of adrenoceptor and angiotensin II receptor, and these interactions may play a protective role in pancreas and improve glucose metabolism disorders. [ABSTRACT FROM AUTHOR]

The presence of cancer stem cells (CSCs) has major implications in the choice of cancer treatment strategy and is responsible for tumor relapse. CSCs have been isolated and characterized in several types of cancer; however, studies concerning the CSCs from nasopharyngeal carcinoma (NPC) are limited. Thus, the present study was designed to isolate and characterize the cancer stem-like side population (SP) cells from NPC samples. The fluorescence-activated cell sorting (FACS)-based Hoechst 33342 dye exclusion technique identified that 3.9% of cells from NPC samples were cancer stem-like SP cells. Upon treatment with verapamil (ABC transporter inhibitor), the percentage of SP cells was significantly reduced to 0.7%, which confirms that the ABC transporter protein exhibits a significant role in drug exclusion. Fluorescence microscopy analysis revealed that the FACS purified SP cells showed increased expression of ABCG2 (ATP transporter protein), Oct-4 and CD44 (stem cell surface protein). Furthermore, these SP cells exhibited increased mRNA expression of ABCG2 and anti-apoptotic factor Bmi-1, which contribute to multi-drug resistance and increased cell survival rate. Notably, the Wnt/β-catenin signaling pathways are altered in SP cells. In addition, using reverse transcription-quantitative polymerase chain reaction analysis it was observed that the cells exhibited increased expression of DKK1 and AXIN2. In conclusion, data from the present study clearly demonstrated that the presence of cancer stem-like SP cells from NPC may be responsible for chemotherapeutic drug resistance, tumor recurrence and invasion. [ABSTRACT FROM AUTHOR]

The present study aimed to identify key genes and relevant microRNAs (miRNAs) involved in laryngeal squamous cell carcinoma (LSCC). The gene expression profiles of LSCC tissue samples were analyzed with various bioinformatics tools. A gene expression data set (GSE51985), including ten laryngeal squamous cell carcinoma (LSCC) tissue samples and ten adjacent non-neoplastic tissue samples, was downloaded from the Gene Expression Omnibus. Differential analysis was performed using software package limma of R. Functional enrichment analysis was applied to the differentially expressed genes (DEGs) using the Database for Annotation, Visualization and Integrated Discovery. Protein-protein interaction (PPI) networks were constructed for the protein products using information from the Search Tool for the Retrieval of Interacting Genes/Proteins. Module analysis was performed using ClusterONE (a software plugin from Cytoscape). MicroRNAs (miRNAs) regulating the DEGs were predicted using WebGestalt. A total of 461 DEGs were identified in LSCC, 297 of which were upregulated and 164 of which were downregulated. Cell cycle, proteasome and DNA replication were significantly over-represented in the upregulated genes, while the ribosome was significantly over-represented in the downregulated genes. Two PPI networks were constructed for the up- and downregulated genes. One module from the upregulated gene network was associated with protein kinase. Numerous miRNAs associated with LSCC were predicted, including miRNA (miR)-25, miR-32, miR-92 and miR-29. In conclusion, numerous key genes and pathways involved in LSCC were revealed, which may aid the advancement of current knowledge regarding the pathogenesis of LSCC. In addition, relevant miRNAs were also identified, which may represent potential biomarkers for use in the diagnosis or treatment of the disease. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit the expression of target protein-coding genes, most often at the post-transcriptional level. miRNAs are often found to be misregulated in human cancer and they can act as potent oncogenes or tumor suppressor genes. In this study, we found that a germline mutation in the miR-125a coding region is associated with human gastric cancer. This mutation reduced the expression of mature miR-125a and alleviated its inhibitory effect on erythroblastic leukemia viral oncogene homolog 2 (ERBB2) gene expression and on gastric tumor cell proliferation. Thus, the data of this study suggested that this germline mutation in pri-miR-125a likely contributes to the genetic predisposition to gastric cancer by reducing the production of miR-125a, thereby interfering with the expression of miR-125a target genes. [ABSTRACT FROM AUTHOR]

Abstract: The overexpression of the serine/threonine specific Polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. BI 2536 is the first selective inhibitor of Plk1 that inhibits cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to BI 2536 is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that overexpressing of either ABCB1 or ABCG2 is a novel mechanism of acquired resistance to BI 2536 in human cancer cells. Moreover, BI 2536 stimulates the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner, and inhibits the drug substrate transport mediated by these transporters. More significantly, the reduced chemosensitivity and BI 2536-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor or other chemotherapeutic agents that also interact with ABCB1 and ABCG2, such as tyrosine kinase inhibitors nilotinib and lapatinib. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to BI 2536, a combined regimen of BI 2536 and inhibitors or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic. [Copyright &y& Elsevier]

Background: T lymphoma invasion and metastasis 1 (Tiam1) is a potential modifier of tumor development and progression. Our previous study in vitro and in nude mice suggested a promotion role of Tiam1 on invasion and metastasis of colorectal cancer (CRC). In the present study, we generated Tiam1/C1199-CopGFP transgenic mice to investigate the tumorigenetic, invasive and metastatic alterations in the colon and rectum of wild-type and Tiam1 transgenic mice under 1,2-dimethylhydrazine (DMH) treatment. Methods: Transgenic mice were produced by the method of pronuclear microinlectlon. Whole-body fluorescence imaging (Lighttools, Edmonton, Alberta, Canada), PCR, and immunohistochemical techniques (IHC) were applied sequentially to identify the transgenic mice. The carcinogen DMH (20 mg/kg) was used to induce colorectal tumors though intraperitoneal (i.p.) injections once a week for 24 weeks from the age of 4 weeks on Tiam1 transgenic or non-transgenic mice. Results: We successfully generated Tiam1/C1199-CopGFP transgenic mice and induced primary tumors in the intestine of both wild type and Tiam1 transgenic mice by DMH treatment. In addition, Tiam1 transgenic mice developed larger and more aggressive neoplasm than wild-type mice. Moreover, immunohistochemical staining revealed that upregulation of Tiam1 was correlated with increased expression of β-Catenin and Vimentin, and downregulation of E-Cadherin in these mice. Conclusions: Our study has provided in vivo evidence supporting that Tiam1 promotes invasion and metastasis of CRC, most probably through activation of Wnt/β-catenin signaling pathway, in a Tiam1 transgenic mouse model. [ABSTRACT FROM AUTHOR]

Background: The integrin α6 subunit is part of the integrin α6β1 and α6β4 complexes, which are known to mediate the invasion of carcinoma cells. However, the precise role of integrin α6 in intrahepatic cholangiocarcinoma (ICC) has not yet been addressed. Methods: Twenty cases of ICCs and matched nontumor samples were used to analyze integrin α6 expression by immunohistochemistry. After the expression of integrin α6 was determined by RT-PCR and Western blot in ICC cells, we regulated the expression of integrin α6 in ICC cells with specific vshRNA-integrin α6, and assessed the role of integrin α6 in the proliferation and metastasis/invasion of ICC cells. Finally, the involved mechanisms and clinical significance were further investigated. Results: The expression of integrin α6 in ICC tissues was much higher than that in nontumor samples, and the high level of integrin α6 was detected in ICC cells compared with normal liver cells and HepG2 cells. After the down-regulation of integrin α6 in HCCC-9810 cells, we showed that the ability of ICC cells to metastasize and invade was much decreased in vitro, and cell proliferation was inhibited significantly. Further study indicated high expression of integrin α6 enhanced the activation of ERK1/2 and AKT signals in ICC cells and the inhibition of ERK1/2 down-regulated ICC cell proliferation, while the inhibition of AKT markedly impaired ICC cell metastasis and invasion. Integrin α6 overexpression was significantly correlated with larger tumors, multiple nodular, microvascular/bile duct invasion, and lymphatic metastasis ( p < 0.05). The postoperative 5-year overall survival (OS) rate in patients with integrin α6 was higher than that of the integrin α6 group. Conclusions: Overexpression of integrin α6 is associated with a migratory and invasive phenotype of ICC, and integrin α6 may be used as molecular target for therapy of ICC. [ABSTRACT FROM AUTHOR]

Abstract: Melanoma is the most serious type of skin cancer with a high potential for metastasis and very low survival rates. The discovery of constitutive activation of the BRAF kinase caused by activating BRAF(V600E) kinase mutation in most melanoma patients led to the discovery of the first potent BRAF(V600E) signaling inhibitor, vemurafenib. Vemurafenib was effective in treating advanced melanoma patients and was proposed for the treatment of other BRAF(V600E) mutant cancers as well. Unfortunately, the success of vemurafenib was hampered by the rapid development of acquired resistance in different types of BRAF(V600E) mutant cancer cells. It becomes important to identify and evaluate all of the potential mechanisms of cellular resistance to vemurafenib. In this study, we characterized the interactions of vemurafenib with three major ATP-binding cassette (ABC) transporters, ABCB1, ABCC1 and ABCG2. We found that vemurafenib stimulated the ATPase activity and potently inhibited drug efflux mediated by ABCB1 and ABCG2. Vemurafenib also restored drug sensitivity in ABCG2-overexpressing cells. Moreover, we revealed that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF(V600E) mutant A375 cells. Taken together, our findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting involvement of this transporter in acquired resistance to vemurafenib. Thus, combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation. [Copyright &y& Elsevier]

Background: Polymorphism of genes encoding drug-metabolizing enzymes is known to play an important role in increased susceptibility of colorectal cancer. UGT1A gene locus has been suggested to define tissue-specific glucuronidation activity. Reduced capacity of glucuronidation is correlated with the development of colorectal cancer. Therefore, we sought to explore polymorphism of UGTlA gene in human colorectal cancer. Methods: Cancerous and healthy tissues were obtained from selectedpatients. Blood samples were collected and UGTlA mRNA transcriptions were analyzed. Genomic DNA was prepared and UGTlA8 exon-1 sequences were amplified, visualized and purified. The extracted DNA was subcloned and sequenced. Two-tailed Fisher's exact test, Odds ratios (ORs), confidence interval (CIs) and Logistics Regression Analysis were used for statistical analysis. Results: UGTlA mRNA expression was reduced in cancerous tissues compared with healthy tissues from the same patient . The UGTlA mRNA expression of healthy tissue in study patients was lower than control . The mRNA expression of cancerous tissue was down-regulated in UGTlAl, 1A3, 1A4, lA6, 1A9 and up-regulated in UGTlA8 and UGTlAl0 UGT1A5 and UGT1A7 were not expressed in colonic tissue of either group. The allele frequency of WT UGTlA8*1 was higher (p = 0.000), frequency of UGTlA8*3 was lowered in control group (p = 0.000). The expression of homozygous UGTlA8*1 was higher in control group (p = 0.000). Higher frequency of both heterozygous UGTlA8*1/*3 and UGTlA8*2/*3 were found in study group (p = 0.000; p = 0.000). The occurrence of colorectal cancer was mainly related to the presence of polymorphic UGTlA8*3 alleles (p = 0.000). Conclusion: Regulation of human UGT1A genes is tissue-specific. Individual variation in polymorphic expressions of UGTlA gene locus was noted in all types of colonic tissue tested, whereas hepatic tissue expression was uniform. The high incidence of UGTlA8 polymorphism exists in colorectal cancer patients. UGTlA8*1 allele is a protective factor and UGTlA8*3 allele is a risk factor. [ABSTRACT FROM AUTHOR]

Abstract: Background: Because there is evidence that certain neurotrophic factors are involved in depression and the mechanism of antidepressant treatment, it is hypothesized that neurotrophic factors may also play a functional role in the etiology of depression and treatment. Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor (TGF-β)-super-family. We performed a study to assess the impact of chronic unpredictable stress (CUS) and clomipramine treatment on GDNF expression in the rat hippocampus. Method: Using a rat model of CUS-induced depression, we administered clomipramine, one of the typical antidepressants, every day for 3weeks starting 2weeks after the beginning of the experiment. GDNF level in the hippocampus was detected by immunohistochemsitry, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR). Behavioral changes were measured by forced swimming test (FST) and open field test (OFT). Results: Animals exposed to CUS showed depression-like behavior and exhibited a significant decrease in GDNF expression in the hippocampus. Chronic clomipramine treatment reversed the behavioral deficits and the decrease in GDNF levels induced by CUS. Limitation: The relatively small number of the depression-model rats may cause some bias of behavioral tests. Conclusion: In our study, chronic clomipramine treatment restored GDNF expression in the hippocampus of CUS-induced depression rats, suggesting that GDNF is involved in the behavioral responses to antidepressants. The beneficial effects of clomipramine suggest that GDNF may be a viable target for new antidepressant drug development. [Copyright &y& Elsevier]

Abstract: Interleukin-17A (IL-17A)-induced migration and invasion of fibroblast-like synoviocytes (FLSs) is critical for the pathogenesis of rheumatoid arthritis (RA). More than 30% of RA patients are resistant to available therapies, despite the introduction of novel biologic agents. Therefore, it is necessary to develop new anti-arthritic agents. Recent studies have demonstrated that celastrol has anti-arthritic activity in an adjuvant-induced arthritis (AIA) model. However, the effect and molecular mechanisms of celastrol on the migration and invasion of RA-FLSs are not yet understood. Results showed that treatment of RA-FLSs with celastrol suppressed the IL-17A-induced migration and invasion abilities of the cells. In addition, celastrol inhibited IL-17A-induced matrix metalloproteinase (MMP)-9 mRNA and protein expression, and the proteolytic activity of MMP-9 in RA-FLSs. Furthermore, our results revealed that celastrol inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of nuclear factor-κB (NF-κB) in the MMP-9 promoter, and inhibited IκBα phosphorylation and nuclear translocation of NF-κB. In conclusion, celastrol can inhibit IL-17A-induced migration and invasion by suppressing NF-κB-mediated MMP-9 expression in RA-FLSs. These results provide a strong rationale for further testing and validation of celastrol as an adjunct with conventional drugs for the treatment of RA in humans. [Copyright &y& Elsevier]

Objective Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, may play a critical role in many chronic pain conditions. The possible involvement of BDNF in the altered gut sensation in patients with irritable bowel syndrome (IBS) was investigated in the present study. Methods Rectosigmoid biopsies were collected from 40 patients with IBS fulfilling the Rome II criteria and 21 healthy controls. Abdominal pain was quantified by a validated questionnaire. The presence of BDNF and nerve fibres in the mucosa was assessed by immunohistochemistry. The structure of mucosal nerve fibres was assessed by transmission electron microscopy. Mucosal BDNF release was measured by ELISA and correlated with abdominal pain scores. Animal studies using BDNF+/- mice were carried out to evaluate visceral sensitivity, mucosal nerve fibre density and ultrastructural changes. Alterations of visceral sensitivity and TrkB expression in dorsal root ganglia were examined in BDNF+/+ mice following different doses of BDNF administration. Results Biopsies from patients with IBS revealed a significant upregulation of BDNF (p¼0.003), as compared with controls. Total nerve fibres were also substantially increased in patients with IBS. Electron microscopy showed ultrastructural damage on the mucosal nerve fibres (eg, swollen mitochondria and nerve axons). Elevated BDNF release was significantly correlated with the abdominal pain scores. Meanwhile, abdominal withdrawal reflex scores to colorectal distension and mucosal protein gene product 9.5 immunoreactivity were significantly lowered in BDNF+/- than in BDNF+/+ mice. Electron microscopy showed degenerative changes on the mucosal nerve fibres in BDNF+/- mice. Exogenous BDNF induced an obvious dose-dependent increase in TrkB expression in dorsal root ganglia and dose-dependent decrease in threshold pressure in BDNF+/+ mice. Conclusions The increased expression of BDNF in colonic mucosa, together with the structural alterations of mucosal innervation, may contribute to the visceral hyperalgesia in IBS. [ABSTRACT FROM AUTHOR]

Abstract: In prior studies, Eph/ephrin system was demonstrated to be involved in inflammatory and neuropathic pain modulation. The present study was to investigate whether the spinal Eph/ephrin signaling was involved in modulation of spinal inflammatory cytokines in bone cancer pain (BCP) of rats. BCP was induced by intra-tibial inoculation of Walker 256 mammary gland carcinoma cells. The expressions of EphB1/ephrinB1 in spinal cord (SC) and dorsal root ganglia (DRG) were determined. At 16 days post inoculation, the pain relieving effect and the mRNA levels of inflammatory cytokines were detected after intrathecal administration of EphB1-Fc (blocker of EphB1 receptor, 10μg). The results showed that the EphB1/ephrinB1 expression was significantly increased in SC, but ephrinB1 was decreased in DRG after Walker 256 inoculation. The mechanical allodynia induced by bone cancer was significantly alleviated by intrathecal administration of EphB1-Fc. Furthermore, the RT-PCR analysis showed that the mRNA levels of IL-1β, IL-6 and TNF-α were significantly increased at 16 days post Walker 256 inoculation and were significantly suppressed by intrathecal administration of EphB1-Fc in SC. We concluded that Eph/ephrin might be involved in the maintenance of mechanical allodynia, via modulating the expression of spinal inflammatory cytokines, in the present rat model of BCP. This study suggested that Eph/ephrin signaling would be a potential target for the treatment of BCP. [Copyright &y& Elsevier]

Summary: Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na+,K+-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation. [ABSTRACT FROM AUTHOR]

Abstract: Escherichia coli secreted protein A (EspA) is a component of the type 3 secretion system (T3SS). The high level of expression when self-stimulated suggests that EspA may be used as a fusion partner. In the present study, EspA was used as a “fusion partner” to construct a fusion expression system, pEspA, in order to improve the expression and solubility of proteins from prokaryotes and eukaryotes. Target proteins were linked to the C-terminus of EspA by a linker containing a YAPQDP sequence, multiple cloning sites and an enterkinase cleavage site. Six proteins, IL-24, Stx2A1, Stx2B, S1, IntiminC300 and GFP, were expressed as EspA-fusion proteins using this vector. The expression level of each protein was enhanced by EspA and the majority of them (Stx2B, IntiminC300, GFP, Stx2A1, IL-24) were expressed in soluble form. EspA-fusion proteins can be purified by affinity chromatography (Sepharose chelated with EspA-specific monoclonal antibody) and by Ni2+ affinity chromatography for they contain a 6× His tag at their C-terminus. In addition, IL-24 remains soluble and demonstrates certain anti-tumor activity after the removal of EspA by enterkinase. The EspA fusion expression system was efficient in enhancing expression levels and the solubility of target proteins. [Copyright &y& Elsevier]

Abstract: Cumulative evidences suggest that electroacupuncture (EA) can modulate immune function, but the mechanism needs further study. In the present study, the contribution of EA on toll-like receptors 2 and 4 (TLR2/TLR4) and pro-inflammatory cytokine expression after surgical trauma stress were investigated. The mRNA level of both TLR2/4 and pro-inflammatory cytokine was measured by quantitative real-time PCR. ELISA and Western blot assay were chosen for TLR2/TLR4 protein expression and pro-inflammatory cytokine production, respectively. The results showed that surgical trauma stress increased TLR2 mRNA and TLR2/4 proteins in the spleen and augmented pro-inflammatory cytokines (e.g. IL-1β) mRNA and protein expression in the spleen and plasma. These effects could be deteriorated by adrenalectomy (ADX). EA at “Zusanli” acupoint significantly inhibited surgical trauma-induced TLR2 mRNA and TLR2/4 protein expression in spleen and pro-inflammatory cytokine expression in the spleen and plasma. ADX, however, could not block the effect of EA. These results suggested that surgical trauma stress primes the innate immune system for enhanced TLR2 expression and pro-inflammatory cytokine production. EA inhibits TLR2/4 and pro-inflammatory cytokines to produce an anti-inflammatory effect in a surgical trauma stress model, without adrenal gland involvement. [Copyright &y& Elsevier]

The HECT-domain protein family is one of the most important classes of E3 ligases. While the roles of this family in human diseases have been intensively studied, the information for plant HECTs is limited. In the present study, we performed the identification of HECT genes in Brassica rapa and Brassica oleracea, followed by analysis of phylogeny, gene structure, additional domains, putative cis-regulatory elements, chromosomal location, synteny, and expression. Ten and 13 HECT genes were respectively identified in B. rapa and B. oleracea and then resolved into seven groups along with their Arabidopsis orthologs by phylogenetic analysis. This classification is well supported by analyses of gene structure, motif composition within the HECT domain and additional protein domains. Ka/Ks ratio analysis showed that these HECT genes primarily underwent purifying selection with varied selection pressures resulting in different rates of evolution. RNA-Seq data analysis showed that the overwhelming majority of them were constitutively expressed in all tested tissues. qRT-PCR based expression analysis of the 10 B. rapa HECT genes under salt and drought stress conditions showed that all of them were responsive to the two stress treatments, which was consistent with their promoter sequence analysis revealing the presence of an important number of phytohormone-responsive and stress-related cis-regulatory elements. Our study provides useful information and lays the foundation for further functional determination of each HECT gene across the Brassica species. [ABSTRACT FROM AUTHOR]

Several studies have brought about increasing evidence to support the hypothesis that miRNAs play a pivotal role in multiple processes of carcinogenesis, including cell growth, apoptosis, differentiation, and metastasis. In this study, we investigated the potential role of miR-31 in colorectal cancer (CRC) aggressiveness and its underlying mechanisms. We found that miR-31 increased in CRC cells originated from metastatic foci and human primary CRC tissues with lymph node metastases. Furthermore, the high-level expression of miR-31 was significantly associated with a more aggressive and poor prognostic phenotype of patients with CRC (p < 0.05). The stable over-expression of miR-31 in CRC cells was sufficient to promote cell proliferation, invasion, and migration in vitro. It facilitated tumor growth and metastasis in vivo too. Further studies showed that miR-31 can directly bind to the 3’untranslated region (3’UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2. Ectopic expression of SATB2 by transiently transfected with pCAG-SATB2 vector encoding the entire SATB2 coding sequence could reverse the effects of miR-31 on CRC tumorigenesis and progression. In addition, ectopic over-expression of miR-31 in CRC cells induced epithelial-mesenchymal transition (EMT). Our results illustrated that the up-regulation of miR-31 played an important role in CRC cell proliferation, invasion, and metastasis in vitro and in vivo through direct repressing SATB2, suggesting a potential application of miR-31 in prognosis prediction and therapeutic application in CRC. [ABSTRACT FROM AUTHOR]