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Objective: Interstitial cystitis (IC)/bladder pain syndrome (BPS) is a chronic inflammatory disease that can cause bladder pain and accompanying symptoms, such as long-term urinary frequency and urgency. IC/BPS can be ulcerative or non-ulcerative. The aim of this study was to explore the core genes involved in the pathogenesis of ulcerative IC, and thus the potential biomarkers for clinical treatment. Materials and Methods: First, the gene expression dataset GSE11783 was downloaded using the Gene Expression Omnibus (GEO) database and analyzed using the limma package in R to identify differentially expressed genes (DEGs). Then, the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for Gene Ontology (GO) functional analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for pathway enrichment analysis. Finally, the protein-protein interaction (PPI) network was constructed, and key modules and hub genes were determined using the STRING and Cytoscape software. The resulting key modules were then analyzed for tissue-specific gene expression using BioGPS. Results: A total of 216 up-regulated DEGs and 267 down-regulated genes were identified, and three key modules and nine hub genes were obtained. Conclusion: The core genes (CXCL8, CXCL1, IL6) obtained in this study may be potential biomarkers of interstitial cystitis with guiding significance for clinical treatment.

Hyperglycemia has deleterious effects on pancreatic β-cells, causing dysfunction and insulin resistance that lead to diabetes mellitus (DM). The possible causes of injury can be caused by glucose- or fructose-induced oxidative and endoplasmic reticulum (ER) stress. Hawthorn (Crataegus pinnatifida) fruit has been widely used as a hypolipidemic agent in traditional herbal medicine. The study aimed to investigate whether high fructose-induced pancreatic β-cell dysfunction could be reversed through amelioration of ER stress by the treatment of polyphenol-enriched extract (PEHE) from hawthorn fruit. The extract was partitioned using ethyl acetate as a solvent from crude water extract (WE) of hawthorn fruits, followed by column fractionation. The results showed that the contents of total polyphenols, flavonoids and triterpenoids in PEHE could be enhanced by 2.2-, 7.7- and 1.1-fold, respectively, in comparison to the original obtained WE from hawthorn fruit. In ER stress studies, a sharp increase in the inhibitory activity on the gene expression levels of GRP79, ATF6, IRE1α and CHOP involved in ER stress was evident when dosages of PEHE at 50–100 μg/mL were used against high-fructose (150 mM)-treated cells. HPLC–MS/MS analysis showed that polyphenols and flavonoids collectively accounted for 87.03% of the total content of PEHE.

CerSs (ceramide synthases), a group of enzymes that catalyze the formation of ceramides from sphingoid base and acyl-CoA substrates. As far, six types of CerSs (CerS1-CerS6) have been found in mammals. Each of these enzymes have unique characteristics, but maybe more noteworthy is the ability of individual CerS isoform to produce a ceramide with a characteristic acyl chain distribution. As key regulators of sphingolipid metabolism, CerSs highlight their unique characteristics and have emerging roles in regulating programmed cell death, cancer and many other aspects of biology. However, the role of CerSs in lung cancer has not been fully elucidated. In this study, there was no significant change in the sequence or copy number of CerSs gene, which could explain the stability of malignant tumor development through COSMIC database. In addition, gene expression in lung cancer was examined using the OncomineTM database, and the prognostic value of each gene in non-small cell lung cancer (NSCLC) was analyzed by Kaplan-Meier analysis. The results showed that high mRNA expression levels of CerS2, CerS3, CerS4 and CerS5 in all NSCLC patients were associated with improved prognosis. Among them, CerS2 and CerS5 are also highly expressed in adenocarcinoma (Ade), but not in squamous cell carcinoma (SCC). In contrast, high or low expression of CerS1 and CerS6 no difference was observed in patients with NSCLC, Ade and SCC. Integrated the data of this study suggested that these CerSs may be a potential tumor markers or drug target of new research direction.

Background: miR-301b-3p is reported in various human cancers for its abnormal expression, while the role and molecular mechanisms in lung adenocarcinoma (LUAD) remain unclear, and this is the focus of the present study. Materials and Methods: TCGA database was consulted to know gene expression in LUAD tissue. CCK-8, colony formation assay and Transwell assay were applied to identify the role of target genes in regulating LUAD cell biological properties. Bioinformatics analysis plus dual-luciferase assay were performed to validate the potential connection between genes. Results: miR-301b-3p and DLC1 were the target genes of this study and respectively differentially up-regulated and down-regulated in LUAD. Functional experiments indicated that miR-301b-3p contributed to cancer cell proliferation, migration and invasion, while this effect was reversed with overexpressed DLC1 which was identified as a direct target of and regulated by miR-301b-3p. Conclusions: Collectively, miR-301b-3p was identified to actively function on LUAD malignant progression by suppressing DLC1 expression. This discovery provides a novel therapeutic strategy for LUAD patients, which helps improve the survival of patients.

E Du,1,* Chao Lu,1,* Fei Sheng,1 Changying Li,1 Hong Li,2 Na Ding,1 Yue Chen,1 Ting Zhang,1 Kuo Yang,1 Yong Xu1 1Central Laboratory, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, Tianjin 300211, People’s Republic of China; 2The Institute of Molecular Cardiology, Medical school, University of Louisville, Louisville, KY, USA *These authors contributed equally to this work Background: Dysfunction of primary cilia (PC), which could influence cell cycle and modulate cilia-related signaling transduction, has been reported in several cancers. However, there is no evidence of their function in bladder cancer (BLCA). This study was performed to investigate the presence of PC in BLCA and to explore the potential molecular mechanisms underlying the PC in BLCA.Patients and methods: The presence of PC was assessed in BLCA and adjacent non-cancerous tissues. The gene expression dataset GSE52519 was employed to obtain differentially expressed genes (DEGs) associated with PC. The mRNA expression of the DEGs were confirmed by Gene Expression Profiling Interactive Analysis. The DEGs properties and pathways were analyzed by Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Genomatix software was used to predict putative transcription factor binding sites (TFBS) in the promoter region of DEGs, and the transcription factors were achieved according to the shared TFBS, which were supported by the ChIP-Sequence data.Results: PC were found to be reduced in BLCA tissue samples in this study. Seven DEGs were observed to be associated with PC, and gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that these DEGs exhibited the properties and functions of PC, and that the Hedgehog signaling pathway probably participated in the pathogenesis and progression of BLCA. The mRNA expression of the seven DEGs in 404 BLCA and 28 normal tissue samples were analyzed, and five DEGs including CENPF, STIL, AURKA, STK39 and OSR1 were identified. Five TFBS including CREB, E2FF, EBOX, ETSF and HOXF in the promoter region of five DEGs were calculated and the transcription factors were obtained according to the shared TFBS.Conclusion: PC were found to be reduced in BLCA, and the potential molecular mechanisms of PC in BLCA helped to provide novel diagnosis and therapeutic targets for BLCA. Keywords: bladder cancer, primary cilia, differentially expressed genes, microarray analysis, transcription factors

With its unique cellular plasticity, the small intestinal mucosa exhibits efficient adaptability upon feeding. However, little is known about the effect of high-fat diet (HFD) feeding on this adaption and its underlying mechanism. Herein, we demonstrated that the cell proliferation ability, mitochondrial morphology, and global transcriptomic profile of the small intestine exhibited a prominent discrepancy between the fasted and refed state in mice, which were markedly attenuated by long-term HFD feeding. The retinol (Vitamin A, VA) metabolism pathway was dramatically affected by HFD feeding in the small intestine. Both VA and its active metabolite retinoic acid (RA), with the administration of lipid micelles, promoted the expression of genes involved in lipid absorption and suppressed the expression of genes involved in the cell proliferation of intestinal organoids. Via chip-qPCR and RT-qPCR, genes involved in lipid metabolism and cell proliferation were target genes of RARα/RXRα in small intestinal organoids treated with RA and lipid micelles. The role of VA in the in vivo attenuation of intestinal adaptability, in response to HFD, was evaluated. Mice were fed a normal chow diet, HFD, or HFD diet supplemented with additional 1.5-fold VA for 12 weeks. VA supplementation in HFD accelerated the attenuation of intestinal adaptability upon feeding induced by HFD, promoted lipid absorption gene expression, and increased body weight and serum cholesterol levels. In conclusion, the discrepancy of the small intestine between the fasted and refed state was dramatically attenuated by HFD feeding, in which VA and RA might play important roles.

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

One of the common challenges in using glucocorticoid in the long term is the development of femoral head necrosis. To address this challenge, the use of glucocorticoid suppressors like plant polysaccharides has been considered. In this study, Moringa oleifera polysaccharide was isolated through hot water–ethanol precipitation method and purified by DEAE-Sepharose fast flow column. Then, they were characterized by FT-IR, NMR, methylation, and chromatography assays. The polysaccharide biocompatibility was investigated by MTT assay and its effect on osteoblasts was evaluated by controlling gene expression. Also, the effect of polysaccharide on dexamethasone-induced femoral head necrosis in rats was assessed by hydroxyproline, hexosamine and morphometric parameters. The results show that 2 Da molecular weight polysaccharide is mainly composed of Rha, Ara, Fru, Xyl, Man and Gal in the molar ratio of 1.7:2.1:3.4:5.9:5.8:1.3. Meanwhile, MTT results on osteoblasts cells showed polysaccharide biocompatibility, while significantly reducing the negative effects of glucocorticoid. Likewise, polysaccharide significantly reduced the levels of apoptosis and intracellular ROS of glucocorticoid-induced femoral necrosis. Moreover, the results of gene expression indicated a decrease in the expression of TNF-α and IL-6 genes using polysaccharide, which is very effective in preventing apoptotic activity. Also, Polysaccharide increased bone density, bone volume per tissue volume, trabecular thickness, and the hexosamine to hydroxyproline ratio in the rat serum in the presence of glucocorticoids, which are very effective in the process of femoral head necrosis. Furthermore, polysaccharide significantly increases the OCN, RUNX2 and COL-1 genes expression in cartilage tissue, which is in line with the result of morphometric parameters. Overall, this study suggests that the use of polysaccharide could result in the treatment of femoral head necrosis.

The use of steroid drugs such as dexamethasone in long-term treatment poses a challenge called femoral head necrosis. To reduce the destructive effects of dexamethasone, the use of herbal suppressants such as Curculigo orchioides polysaccharides (COPs) is recommended despite the ambiguities in their chemical composition and the effects of each component on the anti-necrosis activity of the femoral head. In this study, after separation of COPs through hot water–ethanol precipitation method and purification through DEAE-Sepharose fast flow column, their properties were explained by FTIR, NMR, methylation and chromatography. Moreover, the COPs biocompatibility and its inhibitory effects against dexamethasone-induced cytotoxicity were evaluated by MTT assay and gene expression on human primary osteoblasts cells. The results exhibit that the 4756 Da molecular weight COPs is generally composed of Rha, Ara, Fru, Xyl, Man, Glc, Gal, Glu A and Gal A. Also, MTT results recognized high biocompatibility of COPs and suppressive effect on dexamethasone. Also, COPs significantly reduced dexamethasone-induced intracellular levels of apoptosis and ROS. Furthermore, the use of COPs has significantly increased ALP activity, collagen content and mineralization, which are very effective in differentiating HPOS cells to repair bone tissue. Meanwhile, the gene expression outcomes indicate an increase in Bcl-2 expression gene and decrease in BAX, Caspase-9 and Caspase-3 gene expression in the presence of COPs, which are important in controlling apoptotic activity and bone regeneration. Overall, this study revealed that COPs can lead to the treatment of femoral head necrosis.

Background: Idiopathic achalasia is a primary disorder of esophageal motility characterized by the absence of lower esophageal sphincter (LES). However, the pathogenesis of idiopathic achalasia remains unclear. To further understand the pathogenesis of idiopathic achalasia, we conducted lncRNA and mRNA microarray analyses.Methods: LES specimens used for microarray were collected from five idiopathic achalasia and four control patients undergoing total gastrectomy for gastric cancer. Potential target genes with significantly changed lncRNA and mRNA were predicted using cis/trans-regulatory algorithms, followed by Gene Ontology (GO) analysis and KEGG pathway enrichment analysis to further understand the biophysical effect.Results: In this study, 3136 significantly upregulated and 3997 significantly downregulated mRNAs were identified, along with 6892 significantly dysregulated lncRNAs (4900 increased and 1992 decreased lncRNAs). Biophysical function analysis of these RNAs revealed that the cell adhesion molecule (CAM) pathway was a common pathway. The predicted lncRNA targets of NRXN1 (Down FC: 9.07), NTNG2 (UP FC: 2.75), CADM1 (Down FC: 2.26), NLGN1 (Down FC: 4.60), NEGR1 (Down FC: 2.335), CD22 (Down FC: 5.62), HLA-DQB1 (Down FC: 5.06), and HLA-DOA (Down FC: 2.31) were inputted in this pathway, which were mainly located in the synapse part of the neural system and immune system. Conclusions: Our study demonstrates the lncRNAs and corresponding mRNAs that play critical roles in idiopathic achalasia. Our findings provide new insights into the pathogenesis of this disorder.

Background This study aimed to explore potential novel genes correlated with osteoarthritis (OA). Methods The gene expression profile of GSE48422 was downloaded from the Gene Expression Omnibus (GEO) database. This dataset included five arthritic cartilage samples and five non-arthritic cartilage samples from five female OA patients. Differentially methylated genes (DMGs) between the two kinds of samples were identified, followed by their functional analysis and protein–protein interaction (PPI) analysis. Furthermore, the Comparative Toxicogenomics Database (CTD) was used to further identify OA-related genes among these DMGs. Results In total, 965 hypermethylated genes and 112 hypomethylated genes were identified in the arthritic cartilage samples. The hypermethylated genes (e.g., ADCY4 and ADCY6 ) were significantly related to the calcium signaling pathway and gonadotropin-releasing hormone signaling pathway, while the hypomethylated genes were implicated in the mammalian target of rapamycin signaling pathway. In the PPI network, several genes had a higher degree, such as ADCY4 , ADCY6 and GPR17 , and they interacted with each other. Additionally, 565 DMGs were predicted to be associated with OA, and five of them (e.g., COMP and EDIL3 ) were previously identified as OA markers. Conclusions The methylation of genes ADCY4 , ADCY6 and GPR17 , as well as the gonadotropin-releasing hormone signaling pathway, was newly found to be potentially associated with OA. They may be novel OA markers.

Intramuscular fat (IMF) is an important economic trait for pork quality and a complex quantitative trait regulated by multiple genes. The objective of this work was to investigate the novel transcriptional effects of a multigene pathway on IMF deposition in the longissimus dorsi (LD) muscles of pigs. Potential signaling pathways were screened by mining data from three gene expression profiles in the Gene Expression Omnibus (GEO) database. We designed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) arrays for the candidate signaling pathways to verify the results in the LD muscles of two pig breeds with different IMF contents (Large White and Min). Western blot analysis was used to detect the expression levels of several candidate proteins. Our results showed that the AMPK signaling pathway was screened via bioinformatics analysis. Ten key hub genes of this signaling pathway (AMPK, ADIPOR1, ADIPOR2, LKB1, CAMKK&beta, CPT1A, CPT1B, PGC-1&alpha, CD36, and ACC1) were differentially expressed between the Large White and Min pigs. Western blot analysis further confirmed that LKB1/CaMKK2-AMPK-ACC1-CPT1A axis dominates the activity of AMPK signaling pathway. Statistical analyses revealed that AMPK signaling pathway activity clearly varied among the two pig breeds. Based on these results, we concluded that the activation of the AMPK signaling pathway plays a positive role in reducing IMF deposition in pigs.

The urothelium is an epithelial barrier lining the bladder that protects against infection, fluid exchange and damage from toxins. The nuclear receptor Pparg promotes urothelial differentiation in vitro, and Pparg mutations are associated with bladder cancer. However, the function of Pparg in the healthy urothelium is unknown. Here we show that Pparg is critical in urothelial cells for mitochondrial biogenesis, cellular differentiation and regulation of inflammation in response to urinary tract infection (UTI). Superficial cells, which are critical for maintaining the urothelial barrier, fail to mature in Pparg mutants and basal cells undergo squamous-like differentiation. Pparg mutants display persistent inflammation after UTI, and Nf-KB, which is transiently activated in response to infection in the wild type urothelium, persists for months. Our observations suggest that in addition to its known roles in adipogegnesis and macrophage differentiation, that Pparg-dependent transcription plays a role in the urothelium controlling mitochondrial function development and regeneration., The nuclear receptor Pparg regulates urothelial differentiation in vitro but its role in healthy urothelium is unclear. Here, the authors show that PPAR gamma mediates urothelial development during both homeostasis (via mitochondrial function) and following infection, via an inflammatory response.

Insulin-like growth factor binding protein 6 (IGFBP6) is a key factor in regulating the effects of insulin-like growth factor 2 (IGF2) on the animal growth and development, but the mechanism is far from known. In this study, the 5'-terminal sequence of the IGFBP6 gene (from −920 to −1 bp) which may regulate gene expression was sequenced in the Bama mini-pigs, the Tibetan mini-pigs, the Landrace pigs, the Large White pigs and the Northeast wild boars to screen for the single nucleotide polymorphisms (SNPs) and analyze the relations with the body size traits of swine through a chi-square test analysis. The genotype frequencies of the SNPs in the 5'-terminal sequence have shown that c.-726C > T, c.-722T > C, c.-535C > A, c.-488T > C, c.-403A > G, and c.-378T > C may be related to the dwarf traits of the Bama mini-pig and the Tibetan mini-pigs (P

SET7, serving as the only histone methyltransferase that monomethylates ‘Lys-4’ of histone H3, has been proved to function as a key regulator in diverse biological processes, such as cell proliferation, transcriptional network regulation in embryonic stem cell, cell cycle control, protein stability, heart morphogenesis and development. What′s more, SET7 is involved inthe pathogenesis of alopecia aerate, breast cancer, tumor and cancer progression, atherosclerosis in human carotid plaques, chronic renal diseases, diabetes, obesity, ovarian cancer, prostate cancer, hepatocellular carcinoma, and pulmonary fibrosis. Therefore, there is urgent need to develop novel SET7 inhibitors. In this paper, based on DC-S239 which has been previously reported in our group, we employed scaffold hopping- and 2D fingerprint-based similarity searches and identified DC-S285 as the new hit compound targeting SET7 (IC50 = 9.3 μM). Both radioactive tracing and NMR experiments validated the interactions between DC-S285 and SET7 followed by the second-round similarity search leading to the identification ofDC-S303 with the IC50 value of 1.1 μM. In cellular level, DC-S285 retarded tumor cell proliferation and showed selectivity against MCF7 (IC50 = 21.4 μM), Jurkat (IC50 = 2.2 μM), THP1 (IC50 = 3.5 μM), U937 (IC50 = 3.9 μM) cell lines. Docking calculations suggested that DC-S303 share similar binding mode with the parent compoundDC-S239. What′s more, it presented good selectivity against other epigenetic targets, including SETD1B, SETD8, G9a, SMYD2 and EZH2. DC-S303 can serve as a drug-like scaffold which may need further optimization for drug development, and can be used as chemical probe to help the community to better understand the SET7 biology.

Background: The Wnt signaling pathway controls the development of thymic epithelial cells by regulating the expression of FoxN1. Thymoma is a type of malignant tumor arising from the thymic epithelial cells. To determine whether Wnt4 and FoxN1 are involved in the pathogenesis of thymoma, this study determined the mRNA and protein levels of Wnt4 and Foxn1 in thymoma, and analyzed the effect of thymoma cell apoptosis and tumor growth in nude mice after Wnt4 and FoxN1 downregulation. Methods: Wnt4 and FoxN1 mRNA and protein levels in thymoma tissues were analyzed by RT-qPCR and immunohistochemistry, respectively. Thymoma cells were cultured and transfected with siRNA targeting the Wnt4, JNK , and FoxN1 genes. Apoptosis of thymoma cells were analyzed after Wnt4 and FoxN1 downregulation. In addition, thymoma cells were inoculated into nude mice and tumor growth was analyzed. Results: The rates of expression of Wnt4 and FoxN1 protein were 64.3% and 58.9%, while the levels of mRNA expression were 2.56±0.04 and 1.83±0.11, respectively. With increasing malignancy of thymoma, the rates of positivity for Wnt4 and FoxN1 mRNA and protein expression gradually increased. Upon interfering with Wnt4, JNK , and FoxN1 gene expression by using siRNA technology, the inhibition rates were 56.7%, 72.6%, and 63.2%, respectively. The expression of FoxN1 mRNA and protein was decreased after Wnt4 and JNK downregulation. After downregulation of Wnt4 and FoxN1 gene expression, the apoptosis rate of thymoma cells increased and the tumor volume decreased in nude mice. Conclusions: High expression of Wnt4 and FoxN1 may play an important role in the generation and development of thymoma. The FoxN1 gene produced a marked downstream effect through the regulation of Wnt4 . Determining the positivity for both Wnt4 and FoxN1 can help us to evaluate the level of malignancy of thymoma.

During adipocyte differentiation, significant epigenomic changes occur in association with the implementation of the adipogenic program. We have previously shown that histone acetylation increases during differentiation in a manner dependent on acetyl coenzyme A (acetyl-CoA) production by the enzyme ATP-citrate lyase (ACL). Whether ACL regulates nuclear targets in addition to histones during differentiation is not clear. In this study, we report that DNA methyltransferase 1 (DNMT1) levels in adipocytes are controlled in part by ACL and that silencing of DNMT1 can accelerate adipocyte differentiation. DNMT1 gene expression is induced early in 3T3-L1 adipocyte differentiation during mitotic clonal expansion and is critical for maintenance of DNA and histone H3K9 methylation patterns during this period. In the absence of DNMT1, adipocyte-specific gene expression and lipid accumulation occur precociously. Later in differentiation, DNMT1 levels decline in an ACL-dependent manner. ACL-mediated suppression of DNMT1 occurs at least in part by promoting expression of microRNA 148a (miR-148a), which represses DNMT1. Ectopic expression of miR-148a accelerates differentiation under standard conditions and can partially rescue a hypermethylation-mediated differentiation block. The data suggest a role for DNMT1 in modulating the timing of differentiation and describe a novel ACL-miR-148a-dependent mechanism for regulating DNMT1 during adipogenesis.

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research., Author summary Classical swine fever (CSF), caused by classical swine fever virus (CSFV), and is a highly contagious, often fatal porcine disease that causes significant economic losses. Due to the economic importance of this virus to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread, and it is only a matter of time before the virus is reintroduced and the next round of disease outbreaks occurs. These findings highlight the necessity and urgency for developing effective approaches to eradicate the challenging CSFV. In this study, we successfully produced anti-CSFV TG pigs by combining RNAi technology and CRISPR/Cas9 technologies, and viral challenge results confirmed that these TG pigs could effectively limit the replication of CSFV in vivo and in vitro. Additionally, we confirmed that the disease resistance traits in the TG founders were stably transmitted to their F1-generation offspring. Altogether, our work reported the combinational application of CRISPR/Cas9 and RNA interference (RNAi) technology in the generation of anti-CSFV TG pigs, it provided an alternative strategy to change the virus. The results of this study suggested that these TG pigs offered potential benefits over commercial vaccination and reduced CSFV-related economic losses.

Breast cancer is currently the most common malignancy affecting women worldwide. It had been shown that the allopregnanolone biosynthesis was associated with tumorigenesis and PK11195, the Translocator Protein 18 KDa (TSPO) antagonist, had the effects of the allopregnanolone biosynthesis. However, little is known about the association between the effects of PK11195 on the breast cancer and the allopregnanolone biosynthesis. To evaluate this, the breast cancer cell lines MCF-7 and T47D were cultured. Cell viability and proliferation were determined by CCK-8 assay. The IC50 of PK11195 on the MCF-7 and T47D were 5.4 nM and 6 nM. The cell viability and proliferation of AC-5216 (TSPO selective ligand, 3 and 6 nM) was blocked by PK11195 (5.4 nM and 6 nM). Moreover, we evaluated the role of allopregnanolone biosynthesis in the effects of TSPO on breast cancer. Enzyme-Linked ImmunoSorbent Assay (ELISA) was used in the measurement of the allopregnanolone level. We found that the allopregnanolone level was increased by AC-5216 (3 and 6 nM) and the increase was reversed by PK11195 (5.4 nM and 6 nM, resepectively) in MCF-7 and T47D. The TSPO mRNA level was determined by real time polymerase chain reaction (PCR). The TSPO mRNA level were increased by AC-5216 (6 nM), which the increases were reversed by PK11195 (5.4 nM and 6 nM, resepectively) in MCF-7 and T47D. Collectedly, it firstly indicated that the effects of PK11195 on MCF-7 and T47D were associated with the decrease of allopregnanolone biosynthesis, which was mediated by TSPO.

Ulcerative colitis (UC) is a common inflammatory bowel disease (IBD) producingintestinal inflammation and tissue damage. The precise aetiology of UC remainsunknown. In this study, we applied a rank-based expression profile comparativealgorithm, gene set enrichment analysis (GSEA), to evaluate the expression profilesof UC patients and small interfering RNA (siRNA)-perturbed cells to predict proteinsthat might be essential in UC from publicly available expression profiles. We usedquantitative PCR (qPCR) to characterize the expression levels of those genespredicted to be the most important for UC in dextran sodium sulphate (DSS)-inducedcolitic mice. We found that bromo-adjacent homology domain (BAHD1), a novelheterochromatinization factor in vertebrates, was the most downregulated gene. Wefurther validated a potential role of BAHD1 as a regulatory factor for inflammationthrough the TNF signalling pathway in vitro. Our findings indicate thatcomputational approaches leveraging public gene expression data can be used to inferpotential genes or proteins for diseases and BAHD1 might act as an indispensablefactor in regulating the cellular inflammatory response in UC.

The de novo synthesis of the nonessential amino acid serine is often upregulated in cancer. In this study, we demonstrate that the serine catabolic enzyme, mitochondrial serine hydroxymethyltransferase (SHMT2), is induced when MYC-transformed cells are subjected to hypoxia. In mitochondria, SHMT2 can initiate the degradation of serine to CO2 and NH4+, resulting in net production of NADPH from NADP+. Knockdown of SHMT2 in MYC-dependent cells reduced cellular NADPH:NADP+ ratio, increased cellular reactive oxygen species, and triggered hypoxia-induced cell death. In vivo, SHMT2 suppression led to impaired tumor growth. In MYC-amplified neuroblastoma patient samples, there was a significant correlation between SHMT2 and hypoxia-inducible factor-1 α (HIF1α), and SHMT2 expression correlated with unfavorable patient prognosis. Together, these data demonstrate that mitochondrial serine catabolism supports tumor growth by maintaining mitochondrial redox balance and cell survival. Significance: In this study, we demonstrate that the mitochondrial enzyme SHMT2 is induced upon hypoxic stress and is critical for maintaining NADPH production and redox balance to support tumor cell survival and growth. Cancer Discov; 4(12); 1406–17. ©2014 AACR. See related commentary by Martínez-Reyes and Chandel, p. 1371 This article is highlighted in the In This Issue feature, p. 1355

Lipoxin A 4 inhibits TNF-α-induced production of interleukins and proliferation of rat mesangial cells. Background Studies have shown that lipoxin A 4 (LXA 4 ) and its analogues inhibited proliferation of glomerular mesangial cells induced by leukotriene D 4 (LTD 4 ) or platelet-derived growth factor (PDGF), reduced the production of proinflammatory cytokines such as interleukin (IL)-1β and IL-6 in renal tissue of ischemic injury. In the present studies, we examine whether LXA 4 have inhibitory effects on tumor necrosis factor-α (TNF-α)-induced productions of IL-1β and IL-6 and proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal pathway of LXA 4 . Methods Cultured glomerular mesangial cells were treated with TNF-α (10 ng/mL), with or without preincubation with LXA 4 at the different concentrations. Cell proliferation was assessed by [ 3 H]-thymidine incorporation. Proteins of IL-1β and IL-6 in supernatant were analyzed by enzyme-linked immunosorbent assay (ELISA). Expressions of mRNA of IL-1β and IL-6 were determined by real-time polymerase chain reaction (PCR) and cyclin E by reverse transcription (RT)-PCR. Proteins of cyclin E, threonine phosphorylated Akt 1 at 308 site (Thr 308 ) and p27 kip1 were analyzed by Western blotting studies. Activities of signal transducers and activators of transcription-3 (STAT 3 ), nuclear factor-κB (NF-κB) were determined by electrophroretic mobility shift assay (EMSA). Expression of Src homology (SH) 2-containing protein-tyrosine phosphatase (SHP-2) was assessed by immunoprecipitation and immunoblotting. Results TNF-α-stimulated proliferation, release of proteins and expressions of mRNA of IL-1β and IL-6 in mesangial cells were inhibited by LXA 4 in a dose-dependent manner. The marked increments in mRNA expression and protein synthesis of cyclin E induced by TNF-α in parallel with proliferation of mesangial cells were down-regulated by LXA 4 . LXA 4 antagonized the phosphorylation of SHP-2 and activity of NF-κB induced by TNF-α. Pretreatment of the cells with NF-κB inhibitor pyrrolidine dithio-carbamate (PDTC) blocked the productions of IL-1β, IL-6, and activation of NF-κB induced by TNF-α. Stimulation of mesangial cells with TNF-α resulted in enhanced DNA-binding activity of STAT 3 . This increment was inhibited by LXA 4 in a dose-dependent manner. Threonine phosphorylated Akt 1 protein at 308 site stimulated by TNF-α was reduced by LXA 4. TNF-α-induced decrement in expression of p27 kip1 protein was ameliorated by LXA 4 in a dose-dependent manner. Conclusion TNF-α-induced proliferation and increment of cyclin E of rat mesangial cells can be inhibited by LXA 4 , and these inhibitory effects might be through the mechanisms of STAT 3 and Akt 1 /p27 kip1 pathway-dependent signal transduction. LXA 4 also antagonized TNF-α-stimulated IL-1β and IL-6 synthesis, and these antagonisms were related to SHP-2 and NF-κB pathway-dependent signal transduction.

As an oncoprotein, MDM4 plays a key part in P53 tumor suppressor pathway through negatively regulating P53 function. It has been reported that an rs4245739 A>C polymorphism locating in the MDM4 3'-untranslated region creates a miR-191 target site and results in decreased MDM4 expression. Therefore, we investigated the association between this polymorphism and esophageal squamous cell carcinoma (ESCC) risk as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1128 ESCC cases and 1150 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The impact of the polymorphism on MDM4 expression was examined with esophagus tissues. Our results demonstrated that the MDM4 rs4245739 AC and CC genotypes were significantly associated with decreased ESCC risk compared with the AA genotype in both case-control sets (Jinan set: OR = 0.54, 95% CI = 0.35-0.82, P = 0.004; Huaian set: OR = 0.68, 95% CI = 0.45-0.99, P = 0.049). Stratified analyses revealed that a multiplicative interaction between rs4245739 and smoking or drinking was evident (Gene-smoking: P(interactioin) = 0.022; gene-drinking: P(interactioin) = 0.032). After detecting In vivo MDM4 mRNA expression, we found that the rs4245739 AC and CC genotype carriers had significantly decreased MDM4 expression in normal esophagus tissues compared with AA genotype carriers, indicating a consistent genotype-phenotype correlation. Our results elucidate that the MDM4 rs4245739 polymorphism contributes to susceptibility of ESCC and support the hypothesis that genetic variants, interrupting miRNA-mediated gene regulation, may modify cancer risk.

In this study, we aimed to investigate changes in the expression of human Clock (hClock), a gene at the core of the circadian gene family, in colorectal carcinomas (CRCs) and to discuss the possible effects. Previous studies have revealed that the disruption of circadian rhythms is one of the endogenous factors that contribute to the initiation and development of CRCs. However, the underlying molecular changes to the circadian genes associated with CRCs have not been explored. Immunofluorescence and quantitative polymerase chain reaction (qPCR) analysis of the hCLOCK protein and gene expression were performed in 30 cases of CRC. The hCLOCK protein was expressed in all specimens obtained from 30 CRC patients. Higher levels of hCLOCK expression were observed in human CRC tissues compared with the paired non-cancerous tissues. hCLOCK expression was significantly higher in poorly differentiated, or late-stage, Dukes' grade tumors and in 64.3% of tumor cases with lymph node metastasis. The hClock gene was expressed in all specimens. A significantly higher expression of hClock was found in human CRC cases compared with paired non-cancerous tissues. There was a strong positive linear correlation between hClock gene expression and protein expression in human CRCs. A strong positive linear correlation was also found between hClock gene expression and ARNT, HIF-1α and VEGF expression in human CRCs. There was no significant correlation between hClock and Bak, Bax, Bid, tumor necrosis factor receptor I (TNFR I) and TNFR II. The circadian gene hClock was stably expressed in human colorectal mucosa and was important in regulating the expression of downstream clock-controlled genes. hCLOCK may interact with HIF-1α/ARNT and activate VEGF to stimulate tumor angiogenesis and metastasis.

Somatic mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) contribute to the pathogenesis of cancer via production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). Elevated D-2HG can block differentiation of malignant cells by functioning as a competitive inhibitor of alpha-ketoglutarate (alpha-KG) dependent enzymes, including Jumonji family histone lysine demethylases. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although cancer-associated IDH1/2 mutations exclusively produce D-2HG, biochemical studies have demonstrated that L-2HG functions as a more potent inhibitor of alpha-KG-dependent enzymes. Here we report that under conditions of oxygen limitation, mammalian cells selectively produce L-2HG via enzymatic reduction of alpha-KG. Hypoxia-induced L-2HG is not mediated by IDH1 or IDH2, but instead results from promiscuous substrate usage by lactate dehydrogenase A (LDHA). During hypoxia, the resulting increase in L-2HG is necessary and sufficient for the induction of increased methylation of histone repressive marks, including histone 3 lysine 9 (H3K9me3). Thus, L-2HG appears to function as a metabolic signaling intermediate, translating information about oxygen availability into epigenetic modifications that can influence gene expression and cellular differentiation. Citation Format: Craig B. Thompson, Andrew M. Intlekofer, Raymond G. Dematteo, Sriram Venneti, Lydia W. S. Finley, Chao Lu, Ariën S. Rustenburg, Patrick B. Grinaway, John D. Chodera, Justin R. Cross, Alexander R. Judkins. Stereochemistry matters: L-2HG as a tumor response to hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY43-01. doi:10.1158/1538-7445.AM2015-SY43-01

To investigate the effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs).Bone marrow was obtained from a healthy young man and hBMSCs were isolated and cultured. Two siRNAs (Par-4-siRNA-1 and -2) targeting Par-4 gene were chemically synthesized. Eukaryocytic expression vectors containing these Par-4 siRNA sequences were established and transfected into the hBMSCs. The hBMSCs were divided into 4 groups: non-transfected hBMSCs (normal control group), blank Pae-4 plasmid transfected hBMSCs (Par4 control group), Par4-siRNA-1 transfected hBMSCs, and Par-4-siRNA-2 transfected hBMSCs. The expression of Par-4 mRNA was detected by real-time PCR. Another hBMSCs were inoculated in DMEM and divided into 4 groups: non-transfected normal hBMSCs, glutamate (an apoptosis inducer) + non-transfected hBMSC group, glutamine + Par-4-siRNA-1 hBMSC group, and glutamate + Par4-SiRNS-2 hBMSC group. Flow cytometry was used to detect the apoptotic rate. The relative activity of caspase-3 was determined by colorimetric assay. Western blotting was used to detect the Smac protein expression.The relative mRNA expression levels of Par-4 gene of the Par-4-siRNA-1 hBMSCs, Par-4 SiENA-2 hBMSCs, and Par-4 control hBMSCs were 0.12 +/- 0.03, 0.33 +/- 0.09, and 0.97 +/- 0.02 respectively, decreased by 88%, 67%, and 3% respectively compared with that of the normal control. The percentages of apoptotic cells of the glutamate + Par-4-siRNA-1 hBMSCs was (37.2 +/- 6.3)%, significantly lower than that of the glutamate + non-transfected hBMSC group [(58.9 +/- 8. 9)%, F = 58.26, P0.01). The Smac protein expression level of the glutamate + non-transfected hBMSC group was significantly higher than that of the normal control group (P0.01); however, the Smac protein expression level of the Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected hBMSC group (P0.01). The caspase-3 activity of the glutamate + Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected BMSC group (P0.01).Par-4-siRNA-1 inhibits markedly the apoptosis of the hBMSCs induced by glutamate. Par-4 gene silencing induced by siRNA inhibits the apoptosis of hBMSCs. The mechanism of the inhibition may be closely related to suppression of the up-regulation of Smac gene expression and caspase-3 activity.

To study thyroid hormone receptor (TR), we developed an in vivo expression system in yeast by using a copper-responsive yeast metallothionein promoter and ubiquitin-fusion protein technology. The cDNA encoding full-length rat liver TR beta was expressed under the control of copper. The [125I]T3 binding activities to yeast extracts were significantly correlated with the added copper sulfate into the medium. Partially purified TR from the transformed yeast had a high hormone binding affinity (Kd = 0.34) for T3 and could bind thyroid hormone response element in gel retardation analysis.

Summary Purpose: Basal cell carcinoma (BCC) is the most common human neoplasm. Much interest lies in determining the genetic basis of BCC to explain the unique locally invasive phenotype and infrequent metastatic behavior of these skin tumors. Objective: We sought to examine a gene expression profile for BCC to elucidate new molecules responsible for its unique growth characteristics. Methods: We analyzed gene expression patterns of 50 BCC tumors using spotted cDNA microarrays of 1718 characterized human genes related to cancer and immunity. This is the largest and most comprehensive gene expression study ever performed for BCC. Nodular and sclerosing histological subtypes of BCC were examined and compared to normal control skin. After statistical filtering, 374 significantly dysregulated genes were sorted by hierarchical clustering to determine trends of gene expression and similarities between patient gene expression profiles. Results: A total of 165 upregulated genes and 115 downregulated genes were identified. These covered a range of categories, including extracellular matrix, cell junctions, motility, metastasis, oncogenes, tumor suppressors, DNA repair, cell cycle, immune regulation and angiogenesis. Clusters of genes were either commonly dysregulated across the 50 patient sample, or selectively affected in subsets of tumors. Histological subtypes were not distinguishable by hierarchical clustering. Many of the genes elucidated, including collagen type IV subunits and other novel candidates, possess functions related to extracellular matrix remodeling and metastasis. Conclusion: These results suggest a gene profile which may explain the invasive growth yet rarely metastatic behavior of BCC. The genes identified may also be potential targets for therapeutics aimed at further controlling invasiveness and local destruction of BCC.

Background Normalization is an important step for microarray data analysis to minimize biological and technical variations. Choosing a suitable approach can be critical. The default method in GeneChip expression microarray uses a constant factor, the scaling factor (SF), for every gene on an array. The SF is obtained from a trimmed average signal of the array after excluding the 2% of the probe sets with the highest and the lowest values. Results Among the 76 U34A GeneChip experiments, the total signals on each array showed 25.8% variations in terms of the coefficient of variation, although all microarrays were hybridized with the same amount of biotin-labeled cRNA. The 2% of the probe sets with the highest signals that were normally excluded from SF calculation accounted for 34% to 54% of the total signals (40.7% ± 4.4%, mean ± sd). In comparison with normalization factors obtained from the median signal or from the mean of the log transformed signal, SF showed the greatest variation. The normalization factors obtained from log transformed signals showed least variation. Conclusions Eliminating 40% of the signal data during SF calculation failed to show any benefit. Normalization factors obtained with log transformed signals performed the best. Thus, it is suggested to use the mean of the logarithm transformed data for normalization, rather than the arithmetic mean of signals in GeneChip gene expression microarrays.

Plaque rupture and subsequent embolism as well as thrombosis are major causes of acute myocardial infarction and stroke secondary to atherosclerosis. Pai-1, t-PA, TF and ET-1 are thrombosis- and thrombolysis-related factors which play important roles in thrombosis formation and plaque rupture. Since acute myocardial infarction and stroke are more likely to occur between 6 a.m. and 12 p.m. than at another time of the day, we studied the relationship between circadian rhythm and Pai-1, t-PA, TF and ET-1 in normal and atherosclerotic mice. Atherosclerosis was developed in apoE -/- mice fed a normal diet or a high cholesterol diet. The expression of Pai-1, t-PA, TF and ET-1 in the hearts of control C57BL/6J mice and atherosclerotic mice was measured by real-time RT-PCR at different Zeitgeber times (ZT) including ZT0, ZT4, ZT8, ZT10, ZT12, ZT14, ZT16 and ZT20. The expression of Pai-1, t-PA, TF and ET-1 peaked between ZT14 and ZT16 and bottomed at ZT10 in C57BL/6J mice. Their expression in apoE -/- mice fed a normal diet lost circadian rhythm. Their expression in apoE -/- mice fed a high cholesterol diet peaked at ZT4, indicating a reverse circadian rhythm. Our result indicates that circadian changes in the expression of Pai-1, t-PA, TF and ET-1 may be involved in the onset of myocardial infarction and stroke.

Tryptase is involved in proteinase-activated receptor-2 (PAR-2) mediated up-regulation of IL-8 expression. The present report showed the effects of tryptase on gene expression and activation, including up-regulation IL-8 expression. The expression of mRNA for NF-kappaB first increased at 1 h after tryptase-treatment (1 ng/ml) and reached the plateau after 4 h. The NF-kappaB mRNA increased by 3-fold (n = 3, P0.05), AP-1 by 2-fold (n = 3, P0.05), and PKB by 10-fold (n = 3, P0.05). However, tryptase-treatment did not affect the expression of JNK and p38 MAPK when compared with control cells at mRNA level. Furthermore, in addition to increasing phosphorylation of p38 MAPK, tryptase-treatment also increased phosphorylation of PKB by 2-fold at 15 min following the treatment. The up-regulation and phosphorylation of PKB by tryptase could be abolished by either phosphoinositol-3-kinase (PI3K) inhibitor (LY294002) at 10 microM or antisense PKB cDNA transfection. The up-regulation of NF-kappaB expression could be inhibited by LY294002 and antisense PKB cDNA. These results indicate that tryptase can activate PI3K-PKB pathway and enhance IL-8 expression.