👤 Hang Tong

Perfluorooctane sulfonate (PFOS), one persistent organic pollutant, has been widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on lipid metabolism, especially in aquatic organisms. The underlying mechanisms of hepatotoxicity induced by chronic PFOS exposure are still largely unknown. The present study defined the effects of chronic exposure to low level of PFOS on lipid metabolism using zebrafish as a model system. Our findings revealed a severe hepatic steatosis in the liver of males treated with 0.5μM PFOS as evidenced by hepatosomatic index, histological assessment and liver lipid profiles. Quantitative PCR assay further indicated that PFOS significantly increase the transcriptional expression of nuclear receptors (nr1h3, rara, rxrgb, nr1l2) and the genes associated with fatty acid oxidation (acox1, acadm, cpt1a). In addition, chronic PFOS exposure significantly decreased liver ATP content and serum level of VLDL/LDL lipoprotein in males. Taken together, these findings suggest that chronic PFOS exposure induces hepatic steatosis in zebrafish via disturbing lipid biosynthesis, fatty acid β-oxidation and excretion of VLDL/LDL lipoprotein, and also demonstrate the validity of using zebrafish as an alternative model for PFOS chronic toxicity screening. Show less

Cell-free RNA (cfRNA) transcripts known to be expressed by the fetal brain are detectable by quantitative reverse transcription PCR (RT-qPCR) in amniotic fluid and represent potential biomarkers of neurodevelopment. The aim of this study was to compare the cfRNA yields from amniotic fluid (AF) collected in a commercial RNA stabilization product with the traditional method of freezing alone. Thirteen women undergoing elective Cesarean birth at term without labor had whole AF collected at the time of uterine incision, prior to membrane rupture. Patient samples were split between Streck RNA blood collection tubes (BCT) and plain sterile polypropylene centrifuge tubes. Cell-free RNA from the AF supernatant was extracted according to a previously published protocol. RT qPCR was performed for the reference gene GAPDH, and three genes associated with neurodevelopment (NRXN3, NTRK3, and ZBTB18). The yield from samples collected in Streck RNA BCT and plain centrifuge tubes were compared with the paired t test. GAPDH, NRXN3 and ZBTB18 amplified successfully in all samples, but NTRK3 did not. The RNA yield was significantly lower in samples collected in the Streck RNA BCT compared with the traditional storage method of freezing alone for all three successfully amplified genes (p < 0.0001). Selected cfRNA neurodevelopment transcripts are consistently detectable in third trimester AF. There appears to be no benefit in collecting AF in Streck RNA BCT for quantitative studies of AF cell-free RNA. Show less

Diabetic patients have increased levels of advanced glycation end products (AGEs) and the role of AGEs in regulating cancer cell proliferation is unclear. Here, we found that treating colorectal and liver cancer cells with AGEs promoted cell proliferation. AGEs stimulated both the expression and activation of a key transcription factor called carbohydrate responsive element binding protein (ChREBP) which had been shown to promote glycolytic and anabolic activity as well as proliferation of colorectal and liver cancer cells. Using siRNAs or the antagonistic antibody for the receptor for advanced glycation end-products (RAGE) blocked AGEs-induced ChREBP expression or cell proliferation in cancer cells. Suppressing ChREBP expression severely impaired AGEs-induced cancer cell proliferation. Taken together, these results demonstrate that AGEs-RAGE signaling enhances cancer cell proliferation in which AGEs-mediated ChREBP induction plays an important role. These findings may provide new explanation for increased cancer progression in diabetic patients. Show less

Preeclampsia is a serious pregnancy complication. Soluble endoglin (sEng) is released from the placenta and contributes to the maternal endothelial dysfunction seen in preeclampsia. Recently oxysterols, which activate the Liver X Receptor (LXR), have been implicated in producing sEng, by upregulating matrix metalloproteinase-14 (MMP14; cleaves endoglin to produce sEng) and down-regulating tissue inhibitor of metalloproteinase-3 (TIMP-3; inhibitor of MMP14). The functional experiments in that study were performed on JAR cells (human choriocarcinoma cell line) and placental explants. We characterized LXR in severe preeclamptic placentas, and assessed whether oxysterols increase release of sEng from primary human umbilical vein endothelial cells (HUVECs), primary trophoblasts and placental explants. Given pravastatin is thought to block oxysterol production and inhibit the LXR, we examined whether pravastatin reduces sEng release. LXRα and β were localized to the syncytiotrophoblast and villous tips and were significantly up-regulated in preeclamptic placenta. Oxysterols upregulated sEng production in HUVECs and placental explants although the increases were far more modest than that recently reported. Oxysterols did not upregulate sEng in primary trophoblasts. Furthermore, mRNA expression of MMP14 and TIMP-3 were not altered by oxysterols in any tissue. Surprisingly, pravastatin did not decrease oxysterol-induced upregulation of sEng. LXR is up-regulated in preeclamptic placenta. Oxysterols upregulate sEng production from human tissues, but the increase is modest, suggesting this may not be the main mechanism for the very significant elevations in sEng seen in preeclampsia. Pravastatin does not decrease sEng production. Oxysterols modestly up-regulate sEng production which is not quenched by pravastatin. Show less

Sox2 is a key factor for maintaining embryonic stem cell (ESS) pluripotency, but little is known about its posttranslational regulation. Here we present evidence that the precise level of Sox2 proteins in ESCs is regulated by a balanced methylation and phosphorylation switch. Set7 monomethylates Sox2 at K119, which inhibits Sox2 transcriptional activity and induces Sox2 ubiquitination and degradation. The E3 ligase WWP2 specifically interacts with K119-methylated Sox2 through its HECT domain to promote Sox2 ubiquitination. In contrast, AKT1 phosphorylates Sox2 at T118 and stabilizes Sox2 by antagonizing K119me by Set7 and vice versa. In mouse ESCs, AKT1 activity toward Sox2 is greater than that of Set7, leading to Sox2 stabilization and ESC maintenance. In early development, increased Set7 expression correlates with Sox2 downregulation and appropriate differentiation. Our study highlights the importance of a Sox2 methylation-phosphorylation switch in determining ESC fate. Show less

The glucose-responsive transcription factor carbohydrate responsive element binding protein (ChREBP) plays an important role in regulating glucose metabolism in support of anabolic synthesis in both hepatocytes and cancer cells. In order to further investigate the molecular mechanism by which ChREBP regulates transcription, we used a proteomic approach to identify proteins interacting with ChREBP. We found several potential ChREBP-interacting partners, one of which, flightless I homolog (FLII) was verified to interact and co-localize with ChREBP in HCT116 colorectal cancer and HepG2 hepatocellular carcinoma cells. FLII is a member of the gelsolin superfamily of actin-remodeling proteins and can function as a transcriptional co-regulator. The C-terminal 227 amino acid region of ChREBP containing the DNA-binding domain interacted with FLII. Both the N-terminal leucine-rich repeat (LRR) domain and C-terminal gelsolin homolog domain (GLD) of FLII interacted and co-localized with ChREBP. ChREBP and FLII localized in both the cytoplasm and nucleus of cancer cells. Glucose increased expression and nuclear localization of ChREBP, and had minimal effect on the level and distribution of FLII. FLII knockdown using siRNAs increased mRNA and protein levels of ChREBP-activated genes and decreased transcription of ChREBP-repressed genes in cancer cells. Conversely, FLII overexpression negatively regulated ChREBP-mediated transcription in cancer cells. Our findings suggest that FLII is a component of the ChREBP transcriptional complex and negatively regulates ChREBP function in cancer cells. Show less

Hypertrophic cardiomyopathy (HC) is a hereditary heterogeneous cardiovascular disorder. Existing data have been of predominantly Caucasian samples, and a large study is needed in Chinese population. The present study was intended to explore the genetic basis and clinical characteristics correlated with different genotypes in a large cohort of Chinese patients. Direct gene sequencing of β-myosin heavy chain (MYH7), myosin binding protein-C (MYBPC3), and cardiac troponin T (TNNT2) was performed in 136 unrelated Chinese HC patients. Clinical evaluations were conducted. In total, 32 mutations were identified in 36 patients (27%), including 10 novel ones. Distribution of mutations was 56% (MYBPC3), 31% (MYH7), and 13% (TNNT2), respectively. Double mutations were identified in 3% patients. The occurrence of HC-associated sarcomeric mutations was associated with an earlier age of onset, increased left ventricular hypertrophy, a higher incidence of syncope, previous family history, and sudden cardiac death. No statistical difference was identified in patients carrying MYBPC3 and MYH7 mutations with regard to clinical characteristics and outcomes. Patients with double mutations were associated with malignant progression in the study. In conclusion, MYBPC3 is the most predominant gene in HC. Multiple mutations are common in MYH7, MYBPC3, and TNNT2. The present study suggests a large diversity of HC and a prognostic role of genotype. Show less

A potent, selective glucagon receptor antagonist 9m, N-[(4-{(1S)-1-[3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-β-alanine, was discovered by optimization of a previously identified lead. Compound 9m is a reversible and competitive antagonist with high binding affinity (IC(50) of 6.6 nM) and functional cAMP activity (IC(50) of 15.7 nM). It is selective for glucagon receptor relative to other family B GPCRs, showing IC(50) values of 1020 nM for GIPR, 9200 nM for PAC1, and >10000 nM for GLP-1R, VPAC1, and VPAC2. Compound 9m blunted glucagon-induced glucose elevation in hGCGR mice and rhesus monkeys. It also lowered ambient glucose levels in both acute and chronic mouse models: in hGCGR ob/ob mice it reduced glucose (AUC 0-6 h) by 32% and 39% at 3 and 10 mpk single doses, respectively. In hGCGR mice on a high fat diet, compound 9m at 3, and 10 mpk po in feed lowered blood glucose levels by 89% and 94% at day 10, respectively, relative to the difference between the vehicle control and lean hGCGR mice. On the basis of its favorable biological and DMPK properties, compound 9m (MK-0893) was selected for further preclinical and clinical evaluations. Show less

γ-Secretase inhibitors (GSIs) are being investigated for their potential to modify the progression of Alzheimer disease based on their ability to regulate amyloid-β (Aβ) accumulation. BMS-708163 (avagacestat) is an oral GSI designed for selective inhibition of Aβ synthesis currently in development for the treatment of mild to moderate and predementia AD. In addition to the desired effect on Aβ synthesis, GSIs affect Notch processing, which is thought to mediate some toxic adverse effects reported with this drug class. Avagacestat produced up to 190-fold greater selectivity for Aβ synthesis than Notch processing in preclinical studies and may therefore produce less toxic adverse events than other less selective compounds. Presented here are the results of the first in-human study for this new GSI compound. The goal of this study was to assess the tolerability profile, pharmacokinetic properties, and effects on pharmacodynamic markers (Aβ, trefoil factor family 3 protein, dual specificity phosphatase 6, and hairy and enhancer of split-1) of single, oral doses of avagacestat in healthy, young, male volunteers. This was a multicenter, randomized, double-blind, placebo-controlled, single-ascending dose study in 8 healthy young men (age, 18-45 years) per dosing panel. Each study participant was randomized to receive a single dose of placebo (n = 2) or avagacestat (n = 6 for each dose) as an oral solution in 1 of 9 sequential dose panels (0.3, 1.5, 5, 15, 50, 100, 200, 400, and 800 mg). For determination of avagacestat, blood samples were obtained before dosing and for up to 144 hours after dosing. For participants in the 800-mg avagacestat dose panel, additional samples were obtained at 216, 312, and 648 hours. For 40-amino acid isoform of Aβ (Aβ(1-40)) assessment, plasma samples were collected before avagacestat administration and up to 72 hours after dosing. Avagacestat concentrations peaked quickly after oral administration and then had a biphasic decrease in concentrations with a prolonged terminal phase. Exposures were proportional with doses up to 200 mg. Avagacestat was well tolerated at single oral doses up to 800 mg, with a biphasic effect on plasma Aβ(1-40). Adverse events were predominately mild to moderate in severity with no evidence of dose dependence up to 200 mg. Results from this single-ascending dose study suggest that avagacestat was tolerated at a single-dose range of 0.3 to 800 mg and suitable for further clinical development. Show less

Infantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs. HemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR. HEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes. HES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells. Show less

Tumor cells are metabolically reprogrammed to fuel cell proliferation. Most transformed cells take up high levels of glucose and produce ATP through aerobic glycolysis. In cells exhibiting aerobic glycolysis, a significant fraction of glucose carbon is also directed into de novo lipogenesis and nucleotide biosynthesis. The glucose-responsive transcription factor carbohydrate responsive element binding protein (ChREBP) was previously shown to be important for redirecting glucose metabolism in support of lipogenesis in nonproliferating hepatocytes. However, whether it plays a more generalized role in reprogramming metabolism during cell proliferation has not been examined. Here, we demonstrated that the expression of ChREBP can be induced in response to mitogenic stimulation and that the induction of ChREBP is required for efficient cell proliferation. Suppression of ChREBP resulted in diminished aerobic glycolysis, de novo lipogenesis, and nucleotide biosynthesis, but stimulated mitochondrial respiration, suggesting a metabolic switch from aerobic glycolysis to oxidative phosphorylation. Cells in which ChREBP was suppressed by RNAi exhibited p53 activation and cell cycle arrest. In vivo, suppression of ChREBP led to a p53-dependent reduction in tumor growth. These results demonstrate that ChREBP plays a key role both in redirecting glucose metabolism to anabolic pathways and suppressing p53 activity. Show less

To screen the MYBPC3 gene mutations in Han Chinese patients with hypertrophic cardiomyopathy (HCM). Sixty-six patients with HCM were enrolled for the study. The exons in the functional regions of MYBPC3 were amplified with PCR and the products were sequenced. Four novel mutations and four common polymorphisms were identified in this patient cohort. A Lys301fs mutation in exon10 was evidenced in a H30, and when he was 47 years old, he had the chest tightness, shortness of breath with septal hypertrophy of 18.7mm; a Asp463stop mutation in exon17 was detected in a H48, he was 24 years old 24-year-old when a medical examination showed ventricular septal hypertrophy of 15.4 mm; both Gly523Arg mutation in exon18 and Tyr847His mutation in exon26 were found in a H53 with onset age 36 years old, feeling chest tightness after excise and his ventricular septal hypertrophy was 27 mm that time. MYBPC3 mutations occurred in 4.5% patients in this cohort. These mutations were not found in 100 non-HCM control patients. MYBPC3 mutation is presented in a small portion of Han Chinese patients with HCM. Show less

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. Show less

To investigate the gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs), Y5 receptor antisense, mismatched ODNs or vehicle was intracerebroventricularly injected and cDNA microarrays were undertaken. Central administration of NPY-Y5 receptor antisense ODNs decreased food intake, body weight and serum insulin compared with both vehicle and mismatched ODNs. The average area of adipocytes both at retroperitoneal and epididymal adipose tissue were fall in antisense group while only the weight of the retroperitoneal fat pats was reduced in antisense group. cDNA microarrays containing 18,000 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 404, 81, and 34 genes were differently expressed over twofold, threefold, and fivefold, respectively. The analysis of gene expression profiles indicated that 332 genes were up-regulated and 187 genes were down-regulated in response to Y5 receptor antisense ODNs treatment. Different clusters of genes associated with apoptosis, signal transduction, energy metabolism, lipid metabolism, etc., such as FXR1, PHLDA1, MAEA, PIK3R1, ICAM2, PITPN, CALM2, CAMK2D, PKIA, DRD2, SLC25A14, CKB, AADAC, LIPA, ACOX3, FADS1, were concerned. Analysis of differentially expressed genes will help to understand the effects of Y5 receptor antisense ODNs therapy. Show less

The aim of this study was to screen the disease-causing gene mutations and investigate the genotype-phenotype correlation in 10 Chinese pedigrees with familial hypertrophic cardiomyopathy (HCM). There are 91 family members from these 10 pedigrees and 5 members were normal mutated carriers, 23 members were HCM patients (14 male) aged from 1.5 to 73 years old. The functional regions of myosin heavy chain gene (MYH7), cardiac myosin-binding protein C (MYBPC3) and cardiac troponin T gene (TNNT2) were screened with PCR and direct sequencing technique. Clinical information from all patients was also evaluated in regard to the genotype. Mutations were found in 5 out of 10 pedigrees. Mutations in MYH7 (Arg663His, Glu924Lys and Ile736Thr) were found in 3 pedigrees and 3 patients from these pedigrees suffered sudden death at age 20-48 years old during sport. Mutations in MYBPC3 were found in 2 pedigrees, 1 with complex mutation (Arg502Trp and splicing mutation IVS27 + 12C > T) and 1 with novel frame shift mutation (Gly347fs) and the latter pedigree has sudden death history. No mutation was identified in TNNT2. Although the Han Chinese is a relatively homogeneous ethnic group, different HCM gene mutations were responsible for familiar HCM suggesting the heterogeneity nature of the disease-causing genes and HCM MYH7 mutations are associated with a higher risk of sudden death in this cohort. Furthermore, identical mutation might result in different phenotypes suggesting that multiple factors might be involved in the pathogenesis of familiar HCM. Show less