Generation of JAG1 gene c.1615C > T heterozygous mutation human embryonic stem cell line (SDQLCHe001-A) using cytosine base editor.

Pathogenic variants in Jagged-1 (JAG1), which encodes the ligand of the Notch receptor, had been demonstrated to cause Alagille syndrome. However, there is no evidence to support any genotype-phenotype correlations. Here, we generated a gene-edited human embryonic stem cell (hESC) line (H9) carrying the c.1615C > T mutation in JAG1 that was identified in a patient with Alagille syndrome (ALGS). This modified cell line was accomplished by using cytosine base editor (CBE), and may serve as a valuable model for JAG1 mutaion related disease, and facilitate to gain more insight into the biological function of JAG1.

Curcumin inhibits the formation of atherosclerosis in ApoE-/- mice by suppressing cytomegalovirus activity in endothelial cells.

BACKGROUND: Curcumin (Cur) is a hydrophobic polyphenol compound derived from the rhizome of the herb Curcuma longa. Cur has a wide spectrum of biological and pharmacological activities. It has been shown that human cytomegalovirus (HCMV) infection was an important risk factor for atherosclerosis (AS) and Cur exhibited an outstanding anti-HCMV effect. However, anti-AS effects of Cur remain unclear when HCMV infected endothelial cells. AIMS: This study will investigate the anti-AS activities and mechanism of Cur,when HCMV infected in vivo and in vitro. MATERIALS AND METHODS: Cur (0.5, 1, and 2 µM) was used to explore the anti-AS activities and mechanism after HCMV infected endothelial cells in vitro. ApoE-/- mice were fed a high fat and cholesterol diet (HD) and given 4000,000 copies/mouse MCMV infection by intraperitoneal and treated with ganciclovir (5 mg/kg/d), Cur (25, 15 mg/kg/d) for 10 weeks in vivo. KEY FINDINGS: As our results showed that Cur inhibited CMV replication and proliferation, reduced the intracellular ROS overproduction, decreased the release of inflammatory cytokines, down-regulated the level of HMGB1-TLRS-NF-κB signaling pathway-related proteins in vitro experiments. Cur reduced the serum levels of LDL-C, TC and TG, significantly decreased the formation of atherosclerotic plaque in the aorta, reduced the lipid deposition in liver and inflammatory damage in heart, lung and kidney in vivo experiments. SIGNIFICANCE: This study showed that Cur prevent AS progression by inhibiting CMV activity and CMV-induced HMGB1-TLRS-NF-κB signaling pathway.

Determinants of gefitinib pharmacokinetics in healthy Chinese male subjects: A pharmacogenomic study of cytochrome p450 enzymes and transporters.

WHAT IS KNOWN AND OBJECTIVE: Gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, exhibited a wide interindividual variability in pharmacokinetics. In the present study, we aimed to evaluate the impact of single-nucleotide polymorphisms in the metabolizing enzymes and transporters on gefitinib disposition in healthy Chinese subjects. METHODS: Fourteen single-nucleotide polymorphisms, including polymorphisms of ATP-binding cassette (ABC) transporters and cytochrome P450 enzymes, were genotyped by Sanger sequencing, and the concentration of gefitinib was measured by ultrafast liquid chromatography-tandem mass spectrometry. The association between the pharmacokinetic parameters (peak plasma concentration [Cmax ], time to reach Cmax , plasma half-life, area under the concentration-time curve from 0 to 168 hours [AUC(0-168h) ], AUC(0-∞) and plasma clearance [CL/F]) and genotypes was evaluated using unpaired t test or Mann-Whitney U test. A stepwise multiple linear regression analysis was applied to assess the relationships between multiple factors and gefitinib pharmacokinetics. Thirty-nine healthy Chinese male subjects were enrolled in the pharmacokinetic study. RESULTS AND DISCUSSION: Subjects carrying an ABCG2 A allele (c.421CA + c.421AA genotypes) exhibited 33 and 37% increases in the mean gefitinib AUC(0-168h) and AUC(0-∞) values (P < .05), respectively, compared to that of subjects carrying wild-type ABCG2 (c.421CC). Additionally, the mean CL/F of the c.421A allele carriers was 32% less than that of the c.421CC carriers (P < .05). No associations were found between polymorphisms in other metabolic enzymes or ABC transporters and gefitinib pharmacokinetics. WHAT IS NEW AND CONCLUSION: Our results suggested that a single-nucleotide polymorphism in ABCG2 (c.421C>A) significantly affected the pharmacokinetics of gefitinib. Further studies are required to evaluate the effects of single-nucleotide polymorphism on the pharmacokinetics, pharmacodynamics and toxicity of gefitinib.

Cytomegalovirus Infection Exacerbates Experimental Colitis by Promoting IL-23 Production.

Many studies have demonstrated an association between cytomegalovirus (CMV) infection and inflammatory bowel disease (IBD). Moreover, CMV infection is more common in patients with severe or steroid-refractory IBD. However, it is not clarified whether CMV worsens IBD or if it is merely a surrogate marker for IBD. Here, we used the dextran sodium sulfate (DSS)-induced colitis model to investigate if CMV infection exacerbates colitis. The mice were injected intraperitoneally with 10 MOI of murine CMV (MCMV) and thereafter, chronic colitis was induced by one cycle of DSS exposure. Anti-IL-23R mAb at 20 µg/mice and pyrrolidine dithiocarbamate (PDTC), an effective NF-κB inhibitor, at 50 mg/kg were administrated to the mice. The MCMV-infected mice had a shorter colon length and a higher histopathology score than the mock inoculum-treated mice, while anti-IL-23R mAb administration ameliorated the pathological changes. Expression of IL-23, phospho-NF-κB p65, and phospho-IκBα was upregulated in colon tissues of the MCMV-infected mice compared to mock inoculum-treated mice, while treatment with PDTC attenuated colonic IL-23 production. These data demonstrated that CMV infection could accelerate IBD development. This effect may be due to its activation on NF-κB signaling pathway and subsequently IL-23 production.

Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation.

Clusterin (CLU) is a stress-responding protein associated with cytoprotection in a broad range of pathological processes. However, clusterin's function in diabetes-induced endothelial dysfunction has not been defined. Herein, using two diabetes models, we investigated the role of clusterin in endothelial dysfunction triggered by diabetes and the molecular mechanisms involved. The results revealed that clusterin overexpression inhibited ICAM-1/VCAM-1 expression in aortas and improved endothelium-dependent vasodilatation in db/db diabetic mice and streptozotocin (STZ)-induced diabetes models. Consistently, in vitro, adenoviral clusterin overexpression reduced the expression of a range of pro-inflammatory cytokines and suppressed monocyte adhesion to endothelial cells subjected to high glucose and high palmitate. Further study indicated that clusterin overexpression mitigated mitochondrial excessive fission and reduced mitochondrial ROS production. Conversely, silencing clusterin aggravated mitochondrial fission and endothelial inflammatory activation in high glucose-exposed endothelial cells. Accumulating evidence indicates that impaired mitochondrial dynamics plays a considerable role in promoting endothelial dysfunction in diabetic subjects. Therefore, treatments targeting mitochondrial undue fission may be promising measures to prevent vascular complications of diabetes. Furthermore, AMP-activated protein kinase (AMPK) activation contributed to the modulation of mitochondrial dynamics executed by clusterin. Mechanistically, clusterin promoted the phosphorylation of AMPKα and its downstream target acetyl-CoA carboxylase (ACC), while the inhibition of AMPKα negated the improvement in mitochondrial dynamics provided by clusterin overexpression. Over all, these findings suggest that clusterin exerts beneficial effects in endothelial cells under diabetic conditions via inhibiting mitochondrial fragmentation mediated by AMPK.

Aberrant DNA methylation of PAX1, SOX1 and ZNF582 genes as potential biomarkers for esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly invasive malignant tumor and the majority of patients have advanced stage of ESCC at diagnosis with poor outcome. Identification of sensitive and specific biomarkers for early screening of ESCC is critical for improving patient overall survival. METHODS: Pyrosequencing was used to determine the magnitude of DNA methylation on the selected regions PAX1 (paired box gene1), SOX1(sex determining region Y-box-1), and ZNF582 (zinc finger protein 582) in ESCC. RESULTS: The methylation levels ofPAX1, SOX1, and ZNF582 genes were significantly higher in the cancerous tissues compared to those in the non-cancerous (all P < 0.0001). The accuracy, sensitivity and specificity of PAX1 methylation for the detection of cancer were respectively 0.754, 96.0% and 51.4%; for SOX1 which were 0.781, 89.2% and 59.5%; for ZNF582 which were 0.898, 93.2% and 75.7%. Most importantly, both the sensitivity and specificity of ZNF582 methylation testing achieved 100% in female ESCC patients. Hypermethylation of PAX1/SOX1/ZNF582 exhibited as an independent risk factor for ESCC development. In addition, ZNF582 methylation level in tumor tissue from the female patients was higher than that from male patients, and it was higher in the moderate and poor differentiated tumors compared to that in well-differentiated tumors. SOX1 methylation level was significantly higher in tumors located in the upper region than those located in the middle or lower regions. CONCLUSION: The methylation levels ofPAX1, SOX1 and ZNF582 genes were all higher in cancer tissues than in paired-adjacent and normal tissues, suggesting that detection of PAX1/SOX1/ZNF582 methylation status may serve as a promising biomarker for ESCC screening and diagnosis.

Blockade of angiogenin by thalidomide inhibits the tumorigenesis of murine hemangioendothelioma.

Thalidomide, a well-known immunomodulatory compound, has an anti-angiogenic activity, which may be utilized for the treatment of angiogenesis-related diseases such as hemangioendothelioma. The aim of the present study was to investigate both the antitumor role of thalidomide on hemangioendothelioma and the underlying mechanism. By using the xenograft mouse model, we found that thalidomide can inhibit the progression of hemangioendothelioma in vivo. Moreover, thalidomide shows no effect on the proliferation of hemangioendothelioma endothelial cell (EOMA), but significantly impairs the pro-angiogenic capacity of the EOMA cells in vitro. By qRT-PCR screening, we observed that the expression of angiogenin was downregulated by thalidomide treatment. We next performed tissue array analysis and found a positive correlation between angiogenin expression level and hemangioendothelioma occurrence in patients. Moreover, we confirmed that the antitumoral role of thalidomide is dependent on angiogenin expression both in vivo and in vitro. Taken together, we concluded that thalidomide can inhibit the progression of hemangioendothelioma by downregulating the expression of pro-angiogenic factor angiogenin and therefore can be used as a potent therapeutic to treat hemangioendothelioma.

Occurrence of acute pulmonary embolism induced by recombinant erythropoietin during treatment of pure red cell aplasia associated with thymoma: A case report.

RATIONALE: Thymoma is a type of rare tumor in the thymus gland, and among patients with thymoma, less than 10% will develop pure red cell aplasia (PRCA), whereas less than 5% of patients with PRCA have a thymoma. The optimal approach for PRCA in thymoma is immunosuppressive therapy, such as steroids, cyclosporine, and human antithymocyte globulin. PATIENT CONCERNS: A sixty-one-year-old male was diagnosed with thymoma with PRCA after he complained fatigue, tinnitus, and weakness for 1 month, he received therapy with recombinant erythropoietin (rhEPO) for 1 month after the tumor was totally resected and readmitted with pulmonary embolism and received anticoagulation therapy with enoxaparin for 3 months. DIAGNOSES: Thymoma, pure red cell aplasia, pulmonary embolism. INTERVENTION: He received cyclosporine A, prednisone and rhEPO treatment. Two months after the thymectomy and postoperative radiation, he was readmitted with pulmonary embolism. OUTCOMES: Thymoma and pulmonary embolism become complete response (CR), PRCA become partial response (PR). LESSONS: Clinicians should be alert to the possibility of the increased risk of thrombosis induced by rhEPO when it used to treat PRCA associated with thymoma. If other medication is effective for managing PRCA, rhEPO should be avoided.

Polymorphisms in IGF2/H19 gene locus are associated with platinum-based chemotherapeutic response in Chinese patients with epithelial ovarian cancer.

AIM: The present study aimed to assess the association between IGF2/H19 genetic variants and susceptibility to platinum-based chemotherapy in epithelial ovarian cancer (EOC). METHODS: A total of 43 platinum-resistant (PR) and 138 platinum-sensitive (PS) EOC patients were recruited in our study. 21 polymorphisms in IGF2/H19 locus were genotyped by Sequenom MassARRAY assay. RESULTS: The frequencies of GG genotype in both rs3842761(C/G) and rs4244809(A/G) were significantly lower in PR group compared with those in PS group (9.76 vs 23.36%, p = 0.049; 9.76 vs 26.09%, p = 0.045; respectively). Compared with the AA genotype, rs4244809 GG genotype was associated with significantly reduced risk of platinum resistance (adjusted OR: 0.30; 95% CI: 0.10-0.91; p = 0.033). Further stratified analyses revealed that the SNPs of rs3842761 and rs4244809 were greatly related to PR risk in FIGO stage III-IV (rs3842761GG/CC+CG: adjusted OR: 0.15; 95% CI: 0.02-1.21; rs4244809 GG/AA+AG: adjusted OR: 0.24; 95% CI: 0.07-0.84; respectively) and serous adenocarcinoma subgroups (rs3842761 GG/CC+CG: adjusted OR: 0.21; 95% CI: 0.05-0.94; rs4244809 GG/AA+AG: adjusted OR: 0.19; 95% CI: 0.04-0.5; respectively), while rs7924316 polymorphism was associated with reduced risk of PR in serous adenocarcinoma subgroup as analyzed by a recessive model (rs7924316 GG/TT+TG: adjusted OR: 0.22; 95% CI: 0.05-0.98). In addition, both TCT haplotypes of rs3741206/rs3842761/rs7924316 and TC haplotype of rs3741206/rs3842761 were associated with elevated risk of PR (for the TCT haplotype of rs3741206/rs3842761/rs7924316: p = 0.049; OR: 1.69; 95% CI: 1.00-2.87; for the TC haplotype of rs3741206/rs3842761: p = 0.044; OR: 1.71; 95% CI: 1.01-2.88). CONCLUSION: These results suggest that polymorphisms in IGF2/H19 gene locus are associated with PR risk in EOC.

Potential Prognostic and Diagnostic Values of CDC6, CDC45, ORC6 and SNHG7 in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is a common human malignancy. The aims of this study are to investigate the gene expression profile of CRC and to explore potential strategy for CRC diagnosis, therapy and prognosis. METHODS: We use affy and Limma package of Bioconductor R to do differential expression genes (DEGs) and differential expression lncRNAs (DELs) analysis from the gene datasets (GSE8671, GSE21510, GSE32323, GSE39582 and TCGA) respectively. Then, DEGs were analyzed by GO and KEGG pathway and Kaplan-Meier survival curve and Cox regression analyses were used to find aberrantly expressed genes associated with survival outcome of CRC patients. Real-time PCR assay was used to verify the aberrantly expressed genes expression in CRC samples. RESULTS: 306 up-regulation and 213 down-regulation common DEGs were found. A total of 485 DELs were identified, of which 241 up-regulated and 244 down-regulated. Then, GO and KEGG pathway analyses showed that DEGs were involved in cell cycle, mineral absorption, DNA replication, and Nitrogen metabolism. Among them, Kaplan-Meier survival curve and Cox regression analyses revealed that CDC6, CDC45, ORC6 and SNHG7 levels were significantly associated with survival outcome of CRC patients. Finally, real-time PCR assay was used to verify that the CDC6, CDC45, ORC6 and SNHG7 expression were up-regulated in 198 CRC samples compared with the expression levels in individual-matched adjacent mucosa samples. CONCLUSION: CDC6, CDC45, ORC6 and SNHG7 are implicated in CRC initiation and progression and could be explored as potential diagnosis, therapy and prognosis targets for CRC.

VDR Gene variation and insulin resistance related diseases.

BACKGROUND: Vitamin D status may influence the risk of Insulin resistance related diseases such as Type 2 diabetes (T2DM), metabolic syndrome (MetS), and polycystic ovarian syndrome (PCOS). Several studies have assessed vitamin D receptor (VDR) gene polymorphism in relationship with these diseases; however, results remain inconsistent. Our study was conducted to elucidate whether VDR Gene polymorphisms could predict insulin resistance on a large scale. METHODS: A meta-analysis using MEDLINE and EMBASE, was performed up to December 16th, 2016. Studies reporting association of vitamin D gene polymorphism with incident T2DM, MetS and PCOS outcomes were included and sub-group analysis by pigment of skin and latitude were performed. RESULTS: A total of 28 articles based on four gene variation, and comprising 9232 participants with 5193 Insulin resistance related diseases patients were included. No significant associations of the VDR ApaI, BsmI, FokI and TaqI variant with Insulin resistance related diseases were found. However, sub-group analysis analysis showed that PCOS in TaqI (OR = 1.47, 95% CI = 1.03-2.09, P = 0.03) for T allele and MetS for G allele (OR = 1.41, 95% CI = 1.07-1.85, P = 0.01) in BsmI was significant association with VDR gene polymorphism. Simultaneously, sub-group analysis showed VDR ApaI rs7975232(G > T)variant was associated with insulin resistance related diseases in Asians (GG/GT + TT) (OR, 1.62; 95% CI, 1.03-2.53; P = 0.04) and population who lived in middle latitude district (30-60°) (GG/GT + TT) (OR, 1.22; 95% CI, 1.04-1.43; P = 0.02), VDR BsmI rs1544410 (A > G)and VDR Taq1rs731236 (T/C) variant were associated with insulin resistance related diseases in Caucasian (dark-pigmented). CONCLUSION: The results suggested that the association between insulin resistance related diseases and VDR ApaI, BsmI, FokI variant was more obvious in dark-pigmented Caucasians and Asians but not in Caucasian with white skin.

TNF-α and LT-α polymorphisms and the risk of leukemia: a meta-analysis.

AIM: The aim of this study is to investigate whether TNF-α or LT-α polymorphisms are associated with the risk of leukemia. METHODS: A meta-analysis was performed to examine the association between the TNF-α -308 G>A and LT-α +252 A>G polymorphisms and the incidence of leukemia. We also performed subgroup analyses based on the classification of leukemias. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the association. RESULTS: A total of 19 publications comprising 1,509 cases and 4,075 controls were selected in the study. An association between the risk of leukemia and the LT-α +252 AA genotype was found (GG + AG vs. AA, OR = 0.485, 95% CI 0.368-0.639, p = 0.000). After multivariable analysis TNF-α polymorphism showed no consistent association with leukemia. CONCLUSIONS: This meta-analysis suggests that the LT-α +252 AA polymorphism is associated with the risk of leukemia.

Analysis of ABCG2 methylation in stool samples of Chinese healthy males by pyrosequencing.

ABCG2, an efflux pump protein-BCRP coding gene, is involved in the acquisition of chemotherapeutic drug resistance. In recent years, the epigenetic regulation of ABCG2, such as DNA methylation, has become a research hotspot and been attracting widespread attention. Methylation Special PCR (MSP) has been the mainly used method for gene methylation detection for a long time. With the development of pyrosequencing (PSQ) instrument and the convenience, simpleness, and economical benefit it brings, it will become the mainstream method for gene methylation detection in the near future. This study aims to establish a pyrosequencing method for detecting the methylation sites on ABCG2 gene promoter up-stream region, the promoter region and the first exon region, and to detect the methylation level of each site in stool samples, respectively. Thus, it cannot only lay the methodological foundation for the study of BCRP-mediated multi-drug resistance mechanisms in tumor cells, but also can give knowledge of ABCG2 methylation distribution in the intestine of Chinese healthy males by detecting the ABCG2 methylation levels in stool samples as the exfoliated intestinal epithelial cells constantly shed into the stool.

Marked Alteration of Rosuvastatin Pharmacokinetics in Healthy Chinese with ABCG2 34G>A and 421C>A Homozygote or Compound Heterozygote.

Rosuvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor used to lower blood low-density lipoprotein cholesterol, is a substrate of the membrane ABCG2 exporter. ABCG2 variants have been shown to alter rosuvastatin disposition. The objective of this study is to determine the impact of ABCG2 34/421 compound haplotypes on rosuvastatin pharmacokinetics in healthy Chinese volunteer subjects. Eight hundred healthy Chinese males were genotyped by polymerase chain reaction-pyrosequencing for ABCG2 34G>A, ABCG2 421C>A, SLCO1B1 521T>C, and CYP2C9*3 variants. Sixty-two male subjects with wild-type SLCO1B1 c.521TT and CYP2C9*3 were recruited for this pharmacokinetic study of rosuvastatin. A single oral dose of 10 mg rosuvastatin was administrated to each subject, and blood samples were collected before and at various time points after drug administration. Plasma concentration of rosuvastatin was determined by high-performance liquid chromatography-tandem mass spectrometry, and pharmacokinetic analysis was carried out using the WinNonlin program. In Chinese males, high allele frequency of ABCG2 c.34G>A (0.275) and c.421C>A (0.282) was observed, resulting in a considerable portion (23.3%) of subjects being ABCG2 34/421 compound heterozygotes. Compared with subjects with ABCG2 wild-type (c.34GG/421CC), plasma rosuvastatin Cmax and area under the curve, AUC0-∞, were significantly higher, while the apparent oral clearance, CL/F, was significantly lower in subjects with c.34AA, c.421AA, and c.34GA/421CA genotypes. Both t1/2 and Tmax were similar among subjects with different genotypes. A high frequency of ABCG2 c.34G>A and c.421C>A variants was present in Chinese males, and the disposition of rosuvastatin was significantly affected by both variants. These data suggest that it is advisable to genotype these variants when prescribing rosuvastatin to Chinese subjects, leading to a precise dose for each individual.