PRODH safeguards human naive pluripotency by limiting mitochondrial oxidative phosphorylation and reactive oxygen species production.

Naive human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naive hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naive hESCs, indicating that PRODH maintains naive pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naive pluripotency of hESCs.

ARID5B as a critical downstream target of the TAL1 complex that activates the oncogenic transcriptional program and promotes T-cell leukemogenesis.

The oncogenic transcription factor TAL1/SCL induces an aberrant transcriptional program in T-cell acute lymphoblastic leukemia (T-ALL) cells. However, the critical factors that are directly activated by TAL1 and contribute to T-ALL pathogenesis are largely unknown. Here, we identified AT-rich interactive domain 5B (ARID5B) as a collaborating oncogenic factor involved in the transcriptional program in T-ALL. ARID5B expression is down-regulated at the double-negative 2-4 stages in normal thymocytes, while it is induced by the TAL1 complex in human T-ALL cells. The enhancer located 135 kb upstream of the ARID5B gene locus is activated under a superenhancer in T-ALL cells but not in normal T cells. Notably, ARID5B-bound regions are associated predominantly with active transcription. ARID5B and TAL1 frequently co-occupy target genes and coordinately control their expression. ARID5B positively regulates the expression of TAL1 and its regulatory partners. ARID5B also activates the expression of the oncogene MYC Importantly, ARID5B is required for the survival and growth of T-ALL cells, and forced expression of ARID5B in immature thymocytes results in thymus retention, differentiation arrest, radioresistance, and tumor formation in zebrafish. Our results indicate that ARID5B reinforces the oncogenic transcriptional program by positively regulating the TAL1-induced regulatory circuit and MYC in T-ALL, thereby contributing to T-cell leukemogenesis.

Downregulation of KLF5 by EBER1 via the ERK signaling pathway in EBV-positive nasopharyngeal carcinoma cells: implications for latent EBV infection.

Nasopharyngeal carcinoma (NPC) carcinogenesis and malignant transformation are intimately associated with Epstein-Barr virus (EBV) infection. A zinc-fingered transcription factor known as Krüppel-like factor 5 (KLF5) has been shown to be aberrantly expressed in a number of cancer types. However, little is known about the regulatory pathways and roles of KLF5 in EBV-positive NPC. Our study found that KLF5 expression was significantly lower in EBV-positive NPC than in EBV-negative NPC. Further investigation revealed that EBER1, which is encoded by EBV, down-regulates KLF5 via the extracellular signal-regulated kinase (ERK) signalling pathway. This down-regulation of KLF5 by EBER1 contributes to maintaining latent EBV infection in NPC. Furthermore, we uncovered the biological roles of KLF5 in NPC cells. Specifically, KLF5 may influence the cell cycle, prevent apoptosis, and encourage cell migration and proliferation - all of which have a generally pro-cancer impact. In conclusion, these findings offer novel strategies for EBV-positive NPC patients' antitumour treatment.

Comprehensive Analysis Based on the TCGA Database Identified SCIN as a Key DNA Methylation-Driver Gene in Epstein-Barr Virus-Associated Gastric Cancer.

An important feature of EBV-associated gastric cancer (EBVaGC) is extensive methylation of viral and host genomes. This study aims to analyze DNA methylation-driven genes (DMDG) in EBVaGC through bioinformatics methods, providing an important bioinformatics basis for the differential diagnosis and treatment of potential methylation biomarkers in EBVaGC. We downloaded the mRNA expression profiles and methylation datasets of EBVaGC and EBV-negative gastric cancer (EBVnGC) through the TCGA database to screen methylated-differentially expressed genes (MDEGs). DNA methylation-driver genes were identified based on MethylMix algorithm and key genes were further identified by LASSO regression and Random Forest algorithm. Then, we performed gene enrichment analysis for key genes and validated them by GEO database. Gene expression differences in EBVaGC and EBVnGC cell lines was determined by quantitative real-time PCR (qRT-PCR) and western blotting and in GT38 cell and SNU719 cell which all treated by 5-Aza-CdR. Finally, the effect of key gene on the migration and proliferation capacity of EBVaGC cells was determined by Transwells assay and Cell counting Kit-8 (CCK-8) assay. We obtained a total of 687 hypermethylation-low expression genes (Hyper-LGs) and further obtained 53 DNA methylation-driver genes based on the MethylMix algorithm. A total of six key genes (SCIN, ETNK2, PCDH20, PPP1R3C, MATN2, and HOXA5) were identified by LASSO regression and Random Forest algorithm. Among them, SCIN expression was significantly lower in EBVaGC cell lines than in EBVnGC cell lines, and its expression was significantly recovered in EBVaGC cell lines treated with 5-Aza-CdR. Overexpression of SCIN can promote the proliferation and migration capacity of EBVaGC cells. Our study will provide some bioinformatics basis for the study of EBVaGC-related methylation. SCIN may be used as potential methylation biomarkers for the diagnosis and treatment of EBVaGC.

Betaine Alleviates High-Fat Diet Induced Excessive Lipid Deposition in Gibel Carp Hepatopancreas and L8824 Cells by Enhancing VLDL Secretion through HNF4α/MTTP Pathway.

Betaine, a methyl donor, plays a crucial role in lipid metabolism. Previous studies have shown that appropriate betaine supplementation in a high-fat diet reduces triglycerides (TG) of serum and hepatopancreas in fish. However, the underlying mechanism remains unclear. This study examined whether betaine can enhance the secretion of very low-density lipoprotein (VLDL) and sought to identify the specific mechanisms through which this enhancement occurs. A lipid accumulation model was established in gibel carp and L8824 cells using a high-fat diet and oleic acid, respectively. Different doses of betaine (1, 4, and 16 g/kg in the diet; 400 µmol in cell culture) were administered, and measurements were taken for lipid deposition, gene expression of HNF4α, MTTP, and ApoB, as well as the regulation of Mttp and Apob promoters by HNF4α. The results showed that betaine supplementation mitigated lipid droplet accumulation, TG levels, and VLDL production induced by the high-fat diet in gibel carp hepatopancreas and L8824 cells. Moreover, betaine not only increased VLDL content in the cell culture supernatant but also reversed the inhibitory effects of the high-fat diet on protein expression of MTTP, ApoB, and HNF4α in both gibel carp hepatopancreas and L8824 cells. Additionally, HNF4α exhibits transactivating activity on the promoter of Mttp in gibel carp. These findings suggest that betaine supplementation exerts its effects through the HNF4α/MTTP/ApoB pathway, promoting the assembly and secretion of VLDL and effectively reducing lipid accumulation in the hepatopancreas of farmed gibel carp fed a high-fat diet.

Oncogenic viruses and host lipid metabolism: a new perspective.

As noncellular organisms, viruses do not have their own metabolism and rely on the metabolism of host cells to provide energy and metabolic substances for their life cycles. Increasing evidence suggests that host cells infected with oncogenic viruses have dramatically altered metabolic requirements and that oncogenic viruses produce substances used for viral replication and virion production by altering host cell metabolism. We focused on the processes by which oncogenic viruses manipulate host lipid metabolism and the lipid metabolism disorders that occur in oncogenic virus-associated diseases. A deeper understanding of viral infections that cause changes in host lipid metabolism could help with the development of new antiviral agents as well as potential new therapeutic targets.

Use of Zebrafish Embryos To Reproduce Human Norovirus and To Evaluate Human Norovirus Infectivity Decay after UV Treatment.

This study reports an essential improvement of the method for replication of human norovirus (HNoV) with the use of zebrafish (Danio rerio) embryos. With three HNoV genotypes and P-types GII.2[P16], GII.4[P16], and GII.17[P31], we demonstrated that this tool had higher efficiency and robustness than the zebrafish larvae as reported previously. When zebrafish larvae were injected with virus (1.6 ± 0.3 log genome copies/10 larvae), a significant increase of virus genome copies was detected at 2 days postinfection (dpi; 4.4 ± 0.8 log genome copies/10 larvae, P < 0.05) and the viral loads started to decrease gradually from 3 dpi. In comparison, when the viruses were injected into the zebrafish embryos, significant virus replication was noticed from 1 dpi and lasted to 6 dpi (P < 0.05). The virus levels detected at 3 dpi had the highest mean value and the smallest variation (7.7 ± 0.2 log genome copies/10 larvae). The high levels of virus replication enabled continuous passaging for all three strains up to four passages. The zebrafish embryo-generated HNoVs showed clear patterns of binding to human histo-blood group antigens (HBGAs) in human saliva by a simple saliva-binding reverse transcription-quantitative PCR (RT-qPCR). Last, in a disinfection study, it was shown that a dose of 6 mJ/cm2 UV254 was able induce a >2-log reduction in HNoV infectivity for all three HNoV strains tested, suggesting that HNoVs were more UV susceptible than multiple enteric viruses and commonly used HNoV surrogates as tested before. IMPORTANCE HNoVs are a leading cause of gastroenteritis outbreaks worldwide. The zebrafish embryo tool as developed in this study serves as an efficient way to generate viruses with high titers and clean background and a straightforward platform to evaluate HNoV inactivation efficacies. It is expected that this tool will not only benefit epidemiological research on HNoV but also be used to generate HNoV inactivation parameters which are highly needed by the water treatment and food industries.

Effect of miRNA-146a-mediated TLR4 Signal Pathway on the Pain of Lumbar Disc Herniation.

The current experiment was carried out to explore the effect of the miR-146a-mediated TLR4 signaling pathway on the lumbar disc herniation pains. For this aim, a total of 32 rats were divided randomly into 4 groups - the blank group (Group C), Model group (M), miR-146a overexpression group (agomiR-146a group) and negative control group (NC group), with 8 rats in each group. Rats in Group M were prepared for the construction of lumbar disc herniation models, while those in the agomiR-146a group or NC group, in addition to the model construction, would receive the intrathecal injection of agomiR-146a or agomiRNA-146a NC. Thereafter, a series of tests were performed for rats, including the mechanical pain test and heat pain test to measure the pain threshold, RT-PCR to detect the expression of miR-146a, and the transcription of TLR4, IRAK1, TRAF6, IL-6 and TNF-α, Western blot to determine the expression of IRAK1 and TRAF6 and ELISA to determine the expression of IL-6 and TNF-α. Results showed that as compared to the blank group, rats in Group M were more sensitive to the pains, presenting with declines in the thresholds in the pain, and upregulation in the TRL4 signaling pathway (TLR4, IRAK1 and TRAF6) and pro-inflammatory factors, including IL-6 and TNF-α. In comparison with Group M, intrathecal injection of agomiR-146a relieved the pains, with significant upregulation of miR-146a and downregulation of TLR4, IRAK1, TRAF6, IL-6 and TNF-α. Then upregulation of miR-146a could reduce the activity of the TLR4 signaling pathway and the release of pro-inflammatory factors, which may be a potential strategy for the treatment of lumbar disc herniation.

The globally re-emerging norovirus GII.2 manifests higher heat resistance than norovirus GII.4 and Tulane virus.

AIMS: To compare the heat stability of two globally prevalent human norovirus (HuNoV) strains (GII.2[P16] and GII.4[P16]) and a commonly used HuNoV surrogate, Tulane virus (TV). METHODS AND RESULTS: With the use of a newly developed zebrafish larvae platform, we measured the change of infectivity of HuNoV GII.2[P16] and GII.4[P16] toward mild heat treatment at 55°C for 5 min. TV was tested with the same experimental design. As a result, the virus infectivity measurement clearly indicated the higher heat resistance of HuNoV GII.2[P16] (no reduction) than GII.4[P16] (>0.8-log TCID50  ml-1 reduction) and TV (2.5-log TCID50  ml-1 reduction). Further exploration revealed higher virus structural stability of HuNoV GII.2 than GII.4 strains by the use of different clinical samples with different evaluation methods. CONCLUSION: The inactivation data generated from the surrogate virus TV cannot be used directly to describe the inactivation of HuNoV. The phylogenetic classification of HuNoVs may correlate with the virus stability and/or circulation dynamics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is expected to serve as an important reference when revisiting the numerous previous data evaluating HuNoV inactivation conditions in foods with the use of TV as the cultivable surrogate or with genuine HuNoV but using molecular methods. The higher resistance of NoV GII.2 strains than GII.4 strains toward inactivation treatment supplies a possible explanation for the global re-emerging of NoV GII.2 epidemic in recent years.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

Perfluorooctane sulfonate promotes doxycycline-induced liver tumor progression in male Krasv12 transgenic zebrafish.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that has been widely detected in the environment and has caused growing international concern. The liver is the main target organ of PFOS exposure. Animal experiments have shown that PFOS exposure can increase the risk of liver tumorigenesis. However, whether PFOS can accelerate liver tumor progression is still unclear. In this study, transgenic zebrafish Tg(fabp10:rtTA2s-M2; TRE2:EGFP-KRASG12V), a hepatocellular carcinoma (HCC) model that can cause liver tumorigenesis by doxycycline (DOX) induction, was used to investigate the effect of PFOS exposure in HCC progression. The male krasV12 transgenic zebrafish were exposed to 20 mg/L DOX, 500 µg/L PFOS or combined 20 mg/L DOX and 500 µg/L PFOS for 10 d. The results showed that co-treated with PFOS and DOX caused oncogenic Kras-induced liver enlargement, increased the percentages of zebrafish with HCC, and aggravated metabolic reprogramming of liver. To the best of our knowledge, this study for the first proved that PFOS could promote liver tumor progression. Decreased vitamin D level and increased fatty acid intake caused by PFOS might be responsible for the tumor-promoting effects. The results suggest that attention should be paid to the tumor-promoting effects of PFOS when assessing its environmental health risks, and these findings provide new insights into the toxicity of PFOS.

The toxic effect of triclosan and methyl-triclosan on biological pathways revealed by metabolomics and gene expression in zebrafish embryos.

The omnipresence of antimicrobial triclosan (TCS) and by-products in aquatic environments is a threat to aquatic organisms. Traditionally, the adverse effects of TCS and its by-products have been evaluated by examining the phenotypic output relevant to predicting acute toxicity rather than studying the perturbation of biological pathways. Identifying alterations in the key pathways and molecular mechanisms caused by toxic chemicals helps researchers assess the ecological risks of TCS and its by-products to aquatic environments. In this study, we used metabolomics and reverse transcription qPCR to investigate the adverse effects of a wide range of concentrations of triclosan and its derivative methyl-triclosan (MTCS), ranging from relatively low environmentally relevant levels (ng/L) to high-dose concentrations (sublethal concentration), on zebrafish (Danio rerio) embryos. The metabolism and transcriptome analysis revealed changes in the metabolite and transcripts expression of zebrafish embryos after 96 h exposure at 30 µg/L and 300 µg/L of TCS, 400 µg/L of MTCS and the TCS/MTCS mixture (30 µg/L TCS + 3 µg/L MTCS and 300 µg/L TCS + 30 µg/L MTCS). Significant dysregulations in the expression of the urea transporter (UT), glucose-6-phosphate dehydrogenase (G6PD), alanine transaminase (ALT), glutamate dehydrogenase (GDH), phosphoglucomutase (PGM), and fatty acid synthase (FASN), together with changes in alanine, urea, glucose, 6-phosphogluconalactone, and palmitic acid were observed in the TCS, MTCS, and TCS/MTCS treatments. Particularly, the MTCS treatment group showed fold changes in the mRNA expression of nitrogen metabolism, energy metabolism, and fatty acid synthesis, indicating a disruption of the zebrafish embryos' biological pathways. The changes in the metabolites and gene expressions induced by the TCS, MTCS and the TCS/MTCS mixture treatment demonstrate the pathway changes in starch and sucrose metabolism, nitrogen metabolism, fatty acid synthesis, and phenylalanine, tyrosine and tryptophan biosynthesis. Therefore, our study provides better insights into the risks of the parental compound (TCS) and its by-product (MTCS), as well as the perturbation in biological pathways induced by these two compounds in aquatic environments.

Promotion effect of liver tumor progression in male kras transgenic zebrafish induced by tris (1, 3-dichloro-2-propyl) phosphate.

A previous study reported that exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) could promote the progression of hepatocellular carcinoma (HCC) in female HCC model zebrafish. Due to the existence of gender disparity in the development of HCC between females and males, whether the promotion effect of TDCIPP still exists in male HCC model zebrafish remains unclear. In this study, Tg(fabp10:rtTA2s-M2; TRE2:EGFP-krasG12V), referred as kras transgenic zebrafish which was shown to be an inducible liver tumor model, was applied as experimental model to assess the promotion potential of TDCIPP for HCC in males. In brief, kras males were exposed to 20 mg/L doxycycline (DOX), 0.3 mg/L TDCIPP and a binary mixture of 20 mg/L DOX with 0.3 mg/L TDCIPP, and after exposure liver size, histopathology and transcriptional profiles of liver from these treatments were examined. With the involvement of TDCIPP, the liver size was significantly increased and the lesion of hepatocyte became more aggressive. Furthermore, expressions of genes involved in DNA replication and inflammatory response were simultaneously up-regulated in the treatment of TDCIPP compared with the solvent control and in the treatment of the binary mixture of the two chemicals compared to the single DOX treatment. Overall, our results suggested that TDCIPP had promotion effect on the progression of liver tumor in kras males.

Molecular Cloning, Characterization, and Expression Regulation of Acyl-CoA Synthetase 6 Gene and Promoter in Common Carp Cyprinus carpio.

Omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), particularly docosahexaenoic acids (22:6n-3, DHA), have positive effects on multiple biologic and pathologic processes. Fish are the major dietary source of n-3 LC-PUFA for humans. Growing evidence supports acyl-coenzyme A (acyl-CoA) synthetase 6 (acsl6) being involved in cellular DHA uptake and lipogenesis in mammals, while its molecular function and regulatory mechanism remain unknown in fish. The present study focused on investigating the molecular characterization and transcription regulation of the acsl6 gene in the freshwater teleost common carp (Cyprinus carpio). First, the full length of acsl6 cDNA contained a coding region of 2148 bp for 715 amino acids, which possessed all characteristic features of the acyl-CoA synthetase (ACSL) family. Its mRNA expression was the highest in the brain, followed by in the heart, liver, kidney, muscle, and eyes, but little expression was detected in the ovary and gills. Additionally, a candidate acsl6 promoter region of 2058 bp was cloned, and the sequence from -758 bp to -198 bp was determined as core a promoter by equal progressive deletion and electrophoretic mobility shift assay. The binding sites for important transcription factors (TFs), including stimulatory protein 1 (SP1), CCAAT enhancer-binding protein (C/EBPα), sterol-regulatory element binding protein 1c (SREBP1c), peroxisome proliferator activated receptor α (PPARα), and PPARγ were identified in the core promoter by site-directed mutation and functional assays. Furthermore, the intraperitoneal injection of PPARγ agonists (balaglitazone) increased the expression of acsl6 mRNA, coupling with an increased proportion of DHA in the muscle, while opposite results were obtained in the injection of the SREBP1c antagonist (betulin). However, the expression of acsl6 and DHA content in muscle were largely unchanged by PPARα agonist (fenofibrate) treatment. These results indicated that acsl6 may play an important role for the muscular DHA uptake and deposition in common carp, and PPARγ and SREBP-1c are the potential TFs involved in the transcriptional regulation of acsl6 gene. To our knowledge, this is the first report of the characterization of acsl6 gene and its promoter in teleosts.

Transgenic expression of tgfb1a induces hepatic inflammation, fibrosis and metastasis in zebrafish.

TGFB signaling pathway plays a key role on liver disease progression. In our previous study, we have demonstrated the oncogenic ability of Tgfb signaling pathway as a chronic induction of tgfb1a specifically in hepatocytes led to both hepatocellular carcinoma (HCC) and cholangiocarcinoma in zebrafish. Here we would like to examine the potential mechanisms of Tgfb1a induced tumorigenesis. As majority of HCC developed from the background of liver inflammation and fibrosis, by immune-fluorescent staining on markers of liver inflammation, we indeed observed a progressively increased liver inflammation during tumorigenesis. Examination of liver fibrosis also revealed marked increase of liver fibrosis during early liver tumorigenesis and it was dramatically dropped in late liver tumorigenesis. Hence, induction of tgfb1a drives HCC through association of liver inflammation and fibrosis. Furthermore, we found high expression of EMT markers in late liver tumorigenesis, indicating a tumor metastasis potential. These observations are generally consistent with the molecular mechanisms of hepatocarcinogenesis in human.

Transcriptomic analyses of oncogenic hepatocytes reveal common and different molecular pathways of hepatocarcinogenesis in different developmental stages and genders in krasG12V transgenic zebrafish.

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is mainly due to genetic changes in hepatocytes. However, molecular expression in hepatocytes during hepatocarcinogenesis has not been characterized. In this study, using an inducible kras transgenic zebrafish models for HCC, transcriptomic profiles of oncogenic hepatocytes from larvae, male and female adult fish following a brief induction of oncogenic kras were investigated. We found that oncogenic hepatocytes from all the three sources possess most of the cancer hallmarks at molecular level, including Sustaining proliferative signaling, Evading growth suppressors, Resisting cell death, Avoiding immune destruction, Inflammation, Reprogramming of energy metabolism, Angiogenesis, and Activating invasion and metastasis, suggesting the malignant transformation at molecular level could occur at the early stage of hepatocarcinogensis and can be captured in hepatocytes. However, each group of oncogenic hepatocytes also had their own characteristics. Larval oncogenic hepatocytes have cancer stem cell features. Female oncogenic hepatocytes showed resemblance to a mild human HCC subtype while male oncogenic hepatocytes resembled a severe HCC subtype, consistent with the observed sex disparity of HCC in both zebrafish and human. Finally, the two adult groups were more similar to each other than to the larval group, indicating an overwhelming effect of development over the gender.

Anti-inflammatory and anti-osteoarthritis effects of Cm-02 and Ck-02.

Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive deterioration of articular cartilage. There have been reports that small molecule inhibitors have anti-osteoarthritis effects; however, the effects of 3-(4-chloro-2-fluorophenyl)-6-(2,4-difluorophenyl)-2H-benzo[e] [1,3]oxazine-2,4(3H)-dione (Cm-02) and 6-(2,4-difluorophenyl)-3-(3,4-difluorophenyl)-2H-benzo[e] [1,3]oxazine-2,4(3H)-dione (Ck-02), small molecule inhibitors which share many structural similarities with quercetin (a potent anti-inflammatory flavonoid), remain unclear. In this study, TNF-α-stimulated porcine and human chondrocyte models were used to investigate the inhibitory effects of Cm-02 and Ck-02 on the molecular mechanisms underlying the anti-OA effects. TNF-α was used to stimulate porcine and human chondrocytes to mimic immunomodulatory potency in-vitro. Anti-osteoarthritic effects were characterized in terms of protein and mRNA levels associated with the pathogenesis of OA. We also examined (1) the inducible nitric oxide synthase (iNOS)-nitric oxide (NO) system in cultured chondrocytes, (2) matrix metalloproteinases (MMPs) in cultured chondrocytes, and (3) aggrecan degradation in cartilage explants. Finally, we tested the activation of nuclear factor-kappaB (NF-κB), interferon regulatory factor-1 (IRF-1), and activate the protein-1 (AP-1), and we tested the signal transduction and activation of transcription-3 (STAT-3). Our results indicate that, in chondrocytes, Cm-02 and Ck-02 inhibit TNF-α induced NO production, iNOS, MMP, the expression of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and the enzyme activity of MMP-13. Furthermore, both Cm-02 and Ck-02 were found to stimulate TNF-α, which has been shown to suppress the activation of several transcription factors, including NF-κB, STAT-3, and IRF-1 in porcine and human chondrocytes. Cm-02 and Ck-02 were also found to help prevent the release of proteoglycans from cartilage explants. Our findings demonstrate that both Cm-02 and Ck-02 have potent anti-inflammatory activities and the ability to protect cartilage in an OA cell model. These findings indicate that Cm-02 and Ck-02 have the potential to be further developed for the therapeutic treatment of OA.

Effects of sex hormones on liver tumor progression and regression in Myc/xmrk double oncogene transgenic zebrafish.

Hepatocellular carcinoma (HCC) shows clear sex disparity with men being more prone to developing HCC and having higher mortality than women. Previous studies have indicated that sex hormones play important roles in HCC initiation and development, but the effects of sex hormones on HCC in clinical trials remain inconsistent. Using zebrafish liver tumor model co-induced by oncogenes Myc and xmrk, we observed similar sex disparity between male and female zebrafish in liver tumor progression and regression; i.e. male Myc/xmrk transgenic zebrafish developed HCC significantly faster and regressed HCC significantly slower than female Myc/xmrk transgenic zebrtafish. To investigate the effects of sex hormones on liver tumor progression and regression, Myc/xmrk fish were treated with either androgen or estrogen, we observed that androgen promoted HCC progression and retarded HCC regression in females, while estrogen attenuated HCC progression and accelerated HCC regression in males. Furthermore, androgen promoted cell proliferation while estrogen inhibited it. Overall, the present study suggested that sex hormones affected liver tumor progression and regression in the Myc/xmrk transgenic zebrafish.

Correlation between high expression levels of jumonji domain-containing 4 and short survival in cases of colon adenocarcinoma.

In this study, we investigated the expression of jumonji domain-containing 4 (JMJD4) in colon adenocarcinoma (CA) look for evidences for future studies on clinical diagnostic and prognostic value. The immunohistochemical (IHC) reactivity of JMJD4 was assessed in human tissue microarrays using monoclonal antibodies. An initial investigation revealed that the expression of JMJD4 protein was significantly higher in tumor tissue of the colon and liver than in normal tissue. Upon further investigation, we observed significant positivity of JMJD4 between 59 paired samples from CA tissue and adjacent normal tissue. JMJD4 protein expression in CA differed significantly according to the histological grade and M-class (distant metastasis). We also determined that the mRNA or protein expression of JMJD4 was significantly associated with poor survival in patients with CA. Finally, univariate and multivariate analysis revealed that JMJD4 expression could be a prognostic indicator for patients with CA and may provide a new target for the development of novel therapies for the treatment of CA.

Investigation of the molecular mechanisms of hepatic injury upon naphthalene exposure in zebrafish (Danio rerio).

Naphthalene has been used worldwide as a commercial insecticide for decades, which when detected in the environment can have various negative effects on non-target organism, such as hepatotoxicity. However, the molecular mechanisms of how naphthalene acts to affect the liver in zebrafish (Danio rerio) remains unknown. In this study, we evaluated the potential toxic effects of naphthalene on livers in female adult zebrafish over a 21-day subacute exposure. Global hepatic gene expression was examined by microarrays and the results indicated the regulated genes were associated significantly with vital hepatic injury pathways and GO categories upon naphthalene exposure, such as disruptions in lipid metabolism, inflammatory response, and the carcinogenic processes. According to our observations of liver histology, nuclear enlargement as a potential indicator of cancers and hepatic lipometabolic disorder precisely were supported. The 96 h acute naphthalene tests on Tg(lysC:DsRed) and LiPan lines larvae revealed recruitment of neutrophils by the liver, as well as decreased liver size, which further confirmed hepatic inflammation response to naphthalene exposure. Thus, these findings advance the field of ecotoxicology by unveiling a new role of naphthalene as a leading cause of liver damage and provide potential biomarker-genes for environmental naphthalene monitoring.