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1.  The Association between Insomnia Symptoms and Mortality: A Prospective Study of US Men 
Circulation  2013;129(7):737-746.
Insomnia complaints are common in older adults and may be associated with mortality risk. However, evidence regarding this association is mixed. We thus prospectively examined whether men with insomnia symptoms had an increased risk of mortality during 6 years of follow-up.
Methods and Results
A prospective cohort study of 23,447 US men participating in the Health Professionals Follow-up Study and free of cancer, reported on insomnia symptoms in 2004 were followed through 2010. Deaths were identified from state vital statistic records, the National Death Index, family reports, and the postal system. We documented 2025 deaths during 6 years of follow-up (2004-2010). The multivariable-adjusted hazard ratios (HRs) of total mortality were 1.25 (95% confidence interval (CI):1.04-1.50) for difficulty initiating sleep, 1.09 (95%CI:0.97-1.24) for difficulty maintaining sleep, 1.04 (95%CI:0.88-1.22) for early-morning awakenings, and 1.24 (95%CI:1.05-1.46) for non-restorative sleep, comparing men with those symptoms most of the time to men without those symptoms, after adjusting for age, lifestyle factors and presence of common chronic conditions. Men with difficulty initiating sleep and non-restorative sleep most of the time had a 55% (HR:1.55; 95% CI:1.19-2.04; P-trend= 0.01) and 32% (HR:1.32; 95% CI:1.02-1.72; P-trend=0.002) increased risk of CVD mortality, respectively, relative to men without those symptoms.
Some insomnia symptoms, especially difficulty initiating asleep and non-restorative sleep, are associated with a modestly higher risk of mortality.
PMCID: PMC3987964  PMID: 24226807
Mortality; Sleep Disorders; Cardiovascular Outcomes; Insomnia
2.  The NFKB1 polymorphism (rs4648068) is associated with the cell proliferation and motility in gastric cancer 
BMC Gastroenterology  2015;15:21.
We have demonstrated previously that NFKB1 single nucleotide polymorphism (SNP) rs4648068 GG homozygote was associated with the increased risk of gastric cancer in Chinese Han population. In this study, we constructed the recombinant plasmid pGL3-AA, pGL3-GG, pGL3-AA-NFKB and pGL3-GG-NFKB to investigate the function of rs4648068 by cell biology experiments.
Quantitative real-time PCR was used to detect NFKB1 SNP rs4648068 genotype in the patients with gastric cancer. Anti-NF-κB1 p50 polyclonal antibodies were used for immunohistochemical analysis of the tissue specimens. The subsection of NFKB1 containing the promoter site and adjacent three consecutive exons were obtained by PCR technique and subcloned into the vector pGL3-Basic. Dual-Luciferase reporter assay was used to detect the transcriptional activity of the constructed promoter. Effects of transcription factor NFKB1 on C/EBPβ expression were determined by chromatin immunoprecipitation and Western analysis. Furthermore, proliferation and invasion ability of the transduced cell were also measured and compared.
Intensive staining for p50 expression was observed in the tissues of GG genotype patients, compared with those of GA group and AA genotype patients. The transcriptional activity of rs4648068 (A > G) by dual-Luciferase reporter assay suggested that the luciferase activity of homozygote group (pGL3-GG) was greater than that of the control (pGL3-AA), especially at the stimulation of LPS. We found that the luciferase activity was also influenced by pGL3-GG levels. The effects of NFKB1 rs4648068 were enhanced by rs4648065 on the transduced cells. The interaction between NFKB1 promoter nucleotide sequence and C/EBPβ was regulated by the functional SNP rs4648068 in SGC-7901 cells. Our data indicated that the transduction of pGL3 expression plasmid pGL3-GG-NFKB improved the proliferation and motility of gastric cancer cells. Correspondingly, the homozygote GG of SNP rs4648068 strengthened the transcriptional activity of NFKB1 and influenced the cell biological activity.
The transcriptional activity of NFKB1 was associated with SNP rs4648068, and this functional SNP site has the important effects on cell proliferation and motility.
PMCID: PMC4331381
NFKB1; Polymorphism; Gastric cancer; Susceptibility; Single nucleotide polymorphism
3.  Oxidized High-Density Lipoprotein Impairs Endothelial Progenitor Cells' Function by Activation of CD36-MAPK-TSP-1 Pathways 
Antioxidants & Redox Signaling  2015;22(4):308-324.
Aims: High-density lipoprotein (HDL) levels inversely correlate with cardiovascular events due to the protective effects on vascular wall and stem cells, which are susceptible to oxidative modifications and then lead to potential pro-atherosclerotic effects. We proposed that oxidized HDL (ox-HDL) might lead to endothelial progenitor cells (EPCs) dysfunction and investigated underlying mechanisms. Results: ox-HDL was shown to increase apoptosis and intracellular reactive oxygen species levels, but to reduce migration, angiogenesis, and cholesterol efflux of EPCs in a dose-dependent manner. p38 mitogen-activated protein kinase (MAPK) and NF-κB were activated after ox-HDL stimulation, which also upregulated thrombospondin-1 (TSP-1) expression without affecting vascular endothelial growth factor. Effects caused by ox-HDL could be significantly attenuated by pretreatment with short hairpin RNA-mediated CD36 knockdown or probucol. Data of in vivo experiments and the inverse correlation of ox-HDL and circulating EPC numbers among patients with coronary artery diseases (CAD) or CAD and type 2 diabetes also supported it. Meanwhile, HDL separated from such patients could significantly increase cultured EPC's caspase 3 activity, further supporting our proposal. Innovation: This is the most complete study to date of how ox-HDL would impair EPCs function, which was involved with activation of CD36-p38 MAPK-TSP-1 pathways and proved by not only the inverse relationship between ox-HDL and circulating EPCs in clinic but also pro-apoptotic effects of HDL separated from patients' serum. Conclusion: Activation of CD36-p38 MAPK-TSP-1 pathways contributes to the pathological effects of ox-HDL on EPCs' dysfunction, which might be one of the potential etiological factors responsible for the disturbed neovascularization in chronic ischemic disease. Antioxid. Redox Signal. 22, 308–324.
PMCID: PMC4298149  PMID: 25313537
4.  Effect of Menopausal Status on Carotid Intima-Media Thickness and Presence of Carotid Plaque in Chinese Women Generation Population 
Scientific Reports  2015;5:8076.
Menopause is an important physiological stage in women's life. The potential association of menopause with carotid intima-media thickness as well as with occurrence and stability of carotid plaque in Chinese female population is unclear. We conducted a population-based, cross-sectional study by recruiting 2,131 participants aged above 40 years from northeast of China. Carotid intima-media thickness (CIMT), presence of carotid plaque and its stability were evaluated by carotid duplex sonography. Among the participants, 1,133 (53.2%) were identified to be postmenopausal. After adjusting for potential confounding factors, presence of CIMT at 50th- 75th and ≥75th percentiles, carotid plaque and its unstable status were found to be significantly associated with the postmenopausal status (P < 0.001). When matched the participants by age, post-menopausal status was still associated with a higher risk of having unstable plaque. Moreover, our data show that postmenopausal status is a risk factor for intracranial arterial stenosis when compared with premenopausal status in the univariate analysis (OR = 1.314, P = 0.043), and such relationship is lost when the confounding factors are adjusted (OR = 0.828, P = 0.225). In conclusion, the vascular risk factors increase as the menopausal status changes. Compared with premenopausal status, postmenopausal status is associated with higher morbidity of CIMT, carotid plaque and its unstable status.
PMCID: PMC4308700  PMID: 25627797
5.  The Herbal Compound “Diwu Yanggan” Modulates Liver Regeneration by Affecting the Hepatic Stem Cell Microenvironment in 2-Acetylaminofluorene/Partial Hepatectomy Rats 
Ethnopharmacological Relevance. “Diwu Yanggan” (DWYG) has been reported to regulate liver regeneration, modulate the immune response, ameliorate liver injury, kill virus, ameliorate liver fibrosis, and suppress hepatic cancer. However, its mechanisms are still unknown. Objectives. To investigate the effects of DWYG on oval cell proliferation in 2-AAF/PH rats and determine its mechanism. Methods. Wistar rats were randomly distributed into normal group, sham group, vehicle group, and DWYG group. Hepatic pathological changes were examined by H&E staining. The oval cell markers CD34, AFP, CK-19 and hematopoietic cell markers CD45, Thy1.1, and hepatocyte marker ALB were examined with immunohistochemistry. The percentage of CD34/CD45 double-positive cells in bone marrow was detected by flow cytometry. Cytokine levels were measured with the Bio-plex suspension array system. Results. DWYG significantly increased the survival rates of 2-AAF/PH rats and promoted liver regeneration. Furthermore, DWYG increased the ratio of CD34/CD45 double-positive cells on days 10 and 14. In addition, DWYG gradually restored IL-1, GRO/KC, and VEGF levels to those of the normal group. Conclusions. DWYG increases 2-AAF/PH rat survival rates, suppresses hepatic precarcinoma changes, and restores hepatic tissue structure and function. DWYG may act by modulating the hepatic microenvironment to support liver regeneration.
PMCID: PMC4299675  PMID: 25628749
6.  Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury 
Biochimica et biophysica acta  2013;1842(1):10.1016/j.bbadis.2013.10.006.
Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R.
First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3−/−) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending coronary artery (LAD) for 21 days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for up to 3 days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production.
TLR3−/− mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3−/− mice. TLR3 deficiency increases Bcl2 levels and attenuates I/R-increased Fas, FasL, FADD, Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial NF-κB binding activity, TNF-α and IL-1β production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium.
TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.
PMCID: PMC3879925  PMID: 24140513
TLRs; myocardial I/R; apoptosis; NF-κB; inflammatory cytokines
7.  Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes 
World Journal of Gastroenterology : WJG  2014;20(48):18284-18295.
AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.
METHODS: The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA.
RESULTS: The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 108 ± 2.747 × 107 vs 5.368 × 107 ± 1.016 × 107, P < 0.05; 1.049 × 108 ± 2.747 × 107 vs 5.243 × 107 ± 1.194 × 107, P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells.
CONCLUSION: Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.
PMCID: PMC4277965  PMID: 25561795
Hepacivirus; Replicon; Internal ribosome entry site; Tricistronic expression
8.  Clinical trial with traditional Chinese medicine intervention ''tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment'' for chronic hepatitis B-associated liver failure 
World Journal of Gastroenterology : WJG  2014;20(48):18458-18465.
AIM: To study the clinical efficacy of traditional Chinese medicine (TCM) intervention “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) for treating liver failure due to chronic hepatitis B.
METHODS: We designed the study as a randomized controlled clinical trial. Registration number of Chinese Clinical Trial Registry is ChiCTR-TRC-12002961. A total of 144 patients with liver failure due to infection with chronic hepatitis B virus were enrolled in this randomized controlled clinical study. Participants were randomly assigned to the following three groups: (1) a modern medicine control group (MMC group, 36 patients); (2) a “tonifying qi and detoxification” (“TQD”) group (72 patients); and (3) a “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) group (36 patients). Patients in the MMC group received general internal medicine treatment; patients in the “TQD” group were given a TCM formula “tonifying qi and detoxification” and general internal medicine treatment; patients in the “TTK” group were given a TCM formula of “TTK” and general internal medicine treatment. All participants were treated for 8 wk and then followed at 48 wk following their final treatment. The primary efficacy end point was the patient fatality rate in each group. Measurements of various virological and biochemical indicators served as secondary endpoints. The one-way analysis of variance and the t-test were used to compare patient outcomes in the different treatment groups.
RESULTS: At the 48-wk post-treatment time point, the patient fatality rates in the MMC, “TQD”, and “TTK” groups were 51.61%, 35.38%, and 16.67%, respectively, and the differences between groups were statistically significant (P < 0.05). However, there were no significant differences in the levels of hepatitis B virus DNA or prothrombin activity among the three groups (P > 0.05). Patients in the “TTK” group had significantly higher levels of serum total bilirubin compared to MMC subjects (339.40 μmol/L ± 270.09 μmol/L vs 176.13 μmol/L ± 185.70 μmol/L, P = 0.014). Serum albumin levels were significantly increased in both the “TQD” group and “TTK” group as compared with the MMC group (31.30 g/L ± 4.77 g/L, 30.72 g/L ± 2.89 g/L vs 28.57 g/L ± 4.56 g/L, P < 0.05). There were no significant differences in levels of alanine transaminase among the three groups (P > 0.05). Safety data showed that there was one case of stomachache in the “TQD” group and one case of gastrointestinal side effect in the “TTK” group.
CONCLUSION: Treatment with “TTK” improved the survival rates of patients with liver failure due to chronic hepatitis B. Additionally, liver tissue was regenerated and liver function was restored.
PMCID: PMC4277987  PMID: 25561817
Clinical study; “Tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”); Liver regeneration; Treatment with integrated traditional and Western medicine; Chronic hepatitis B-associated liver failure
9.  Surgical treatment for ruptured dural arteriovenous fistula with large intracranial hematoma 
The rupture of dural arteriovenous fistula (DAVF) is a serious complication endangering the lives of patients. It is difficult to treat such ruptured DAVF with large intracranial hematoma since lacking of early diagnostic methods. Meanwhile, there was no consensus of how to surgically treat these patients in early stage. In this study, we tried to use 4D-CTA to diagnose DAVF and guide surgical treatment. Based on the result of 4D-CTA, we attempted to eliminate DAVF at the same time we removed hematoma. The result was encouraging. 7 patients with ruptured DAVF presented as large spontaneous intracranial hemorrhage were included in this research between May, 2010 and August, 2012 in our hospital. 4D-CTA was performed in all cases. All results of 4D-CTA inspections were studied by both neurosurgeon and neuroradiologist. The therapeutic options were evaluated based on the clinical and angiographic results. All fistulas of seven patients were eliminated at the same time the hematoma being evacuated. 4D-CTA was sufficient for detecting and recognizing basic vessel angioarchitecture of DAVF to guide surgical treatment. Main arterial supplies, fistula location and CVDs found during surgery are consistent with the results 4D-CTA. All seven cases achieved completely fistula occlusion in operation without new neurological complication. We favor one stage surgical treatment for ruptured DAVF with large intracranial hemorrhage. 4D-CTA plays an important role in preoperative emergent inspection for its safety, rapidity and accuracy. However, it still needs further and larger investigations to optimize such treatment methods and to find out other potential risks.
PMCID: PMC4307474  PMID: 25664027
DAVF; 4D-CTA; intracranial hemorrhage; surgical treatment
10.  Interplay between Bladder Microbiota and Urinary Antimicrobial Peptides: Mechanisms for Human Urinary Tract Infection Risk and Symptom Severity 
PLoS ONE  2014;9(12):e114185.
Resident bacterial communities (microbiota) and host antimicrobial peptides (AMPs) are both essential components of normal host innate immune responses that limit infection and pathogen induced inflammation. However, their interdependence has not been investigated in the context of urinary tract infection (UTI) susceptibility. Here, we explored the interrelationship between the urinary microbiota and host AMP responses as mechanisms for UTI risk. Using prospectively collected day of surgery (DOS) urine specimens from female pelvic floor surgery participants, we report that the relative abundance and/or frequency of specific urinary microbiota distinguished between participants who did or did not develop a post-operative UTI. Furthermore, UTI risk significantly correlated with both specific urinary microbiota and β-defensin AMP levels. Finally, urinary AMP hydrophobicity and protease activity were greater in participants who developed UTI, and correlated positively with both UTI risk and pelvic floor symptoms. These data demonstrate an interdependency between the urinary microbiota, AMP responses and symptoms, and identify a potential mechanism for UTI risk. Assessment of bacterial microbiota and host innate immune AMP responses in parallel may identify increased risk of UTI in certain populations.
PMCID: PMC4259481  PMID: 25486068
11.  Immunomodulatory Effects of Polysaccharide from Marine Fungus Phoma herbarum YS4108 on T Cells and Dendritic Cells 
Mediators of Inflammation  2014;2014:738631.
YCP, as a kind of natural polysaccharides from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune response, but little is known about the molecular mechanisms. In the present study, we mainly focused on the effects and mechanisms of YCP on the specific immunity mediated by dendritic cells (DCs) and T cells. T cell /DC activation-related factors including interferon- (IFN-) γ, interleukin-12 (IL-12), and IL-4 were examined with ELISA. Receptor knock-out mice and fluorescence-activated cell sorting are used to analyze the YCP-binding receptor of T cells and DCs. RT-PCR is utilized to measure MAGE-A3 for analyzing the tumor-specific killing effect. In our study, we demonstrated YCP can provide the second signal for T cell activation, proliferation, and IFN-γ production through binding to toll-like receptor- (TLR-) 2 and TLR-4. YCP could effectively promote IL-12 secretion and expression of markers (CD80, CD86, and MHC II) via TLR-4 on DCs. Antigen-specific immunity against mouse melanoma cells was strengthened through the activation of T cells and the enhancement of capacity of DCs by YCP. The data supported that YCP can exhibit specific immunomodulatory capacity mediated by T cells and DCs.
PMCID: PMC4267005  PMID: 25525304
12.  Design and Evaluation of a PEGylated Lipopeptide Equipped with Drug-Interactive Motifs as an Improved Drug Carrier 
The AAPS Journal  2013;16(1):114-124.
Micelles are attractive delivery systems for hydrophobic drugs due to their small size and the ease of application. However, the limited drug loading capacity and the intrinsic poor stability of drug-loaded formulations represent two major issues for some micellar systems. In this study, we designed and synthesized a micelle-forming PEG-lipopeptide conjugate with two Fmoc groups located at the interfacial region, and two oleoyl chains as the hydrophobic core. The significance of Fmoc groups as a broadly applicable drug-interactive motif that enhances the carrier–drug interaction was examined using eight model drugs of diverse structures. Compared with an analogue without carrying a Fmoc motif, PEG5000-(Fmoc-OA)2 demonstrated a lower value of critical micelle concentration and three-fold increases of loading capacity for paclitaxel (PTX). These micelles showed tubular structures and small particle sizes (∼70 nm), which can be lyophilized and readily reconstituted with water without significant changes in particle sizes. Fluorescence quenching study illustrated the Fmoc/PTX π–π stacking contributes to the carrier/PTX interaction, and drug-release study demonstrated a much slower kinetics than Taxol, a clinically used PTX formulation. PTX/PEG5000-(Fmoc-OA)2 mixed micelles exhibited higher levels of cytotoxicity than Taxol in several cancer cell lines and more potent inhibitory effects on tumor growth than Taxol in a syngeneic murine breast cancer model (4T1.2). We have further shown that seven other drugs can be effectively formulated in PEG5000-(Fmoc-OA)2 micelles. Our study suggests that micelle-forming PEG-lipopeptide surfactants with interfacial Fmoc motifs may represent a promising formulation platform for a broad range of drugs with diverse structures.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-013-9536-9) contains supplementary material, which is available to authorized users.
PMCID: PMC3889538  PMID: 24281690
drug-interactive motif; micelle; paclitaxel; slow release
13.  KCTD10 Is Involved in the Cardiovascular System and Notch Signaling during Early Embryonic Development 
PLoS ONE  2014;9(11):e112275.
As a member of the polymerase delta-interacting protein 1 (PDIP1) gene family, potassium channel tetramerisation domain-containing 10 (KCTD10) interacts with proliferating cell nuclear antigen (PCNA) and polymerase δ, participates in DNA repair, DNA replication and cell-cycle control. In order to further investigate the physiological functions of KCTD10, we generated the KCTD10 knockout mice. The heterozygous KCTD10+/− mice were viable and fertile, while the homozygous KCTD10−/− mice showed delayed growth from E9.0, and died at approximately E10.5, which displayed severe defects in angiogenesis and heart development. Further study showed that VEGF induced the expression of KCTD10 in a time- and dose-dependent manner. Quantitative real-time PCR and western blotting results revealed that several key members in Notch signaling were up-regulated either in KCTD10-deficient embryos or in KCTD10-silenced HUVECs. Meanwhile, the endogenous immunoprecipitation (IP) analysis showed that KCTD10 interacted with Cullin3 and Notch1 simultaneously, by which mediating Notch1 proteolytic degradation. Our studies suggest that KCTD10 plays crucial roles in embryonic angiogenesis and heart development in mammalians by negatively regulating the Notch signaling pathway.
PMCID: PMC4234411  PMID: 25401743
14.  A Role for the Rap GTPase YlRsr1 in Cellular Morphogenesis and the Involvement of YlRsr1 and the Ras GTPase YlRas2 in Bud Site Selection in the Dimorphic Yeast Yarrowia lipolytica 
Eukaryotic Cell  2014;13(5):580-590.
Yarrowia lipolytica is a dimorphic yeast species that can grow in the ovoid yeast form or in the elongated pseudohyphal or hyphal form depending on the growth conditions. Here, we show that the Rap GTPase Rsr1 of Y. lipolytica (YlRsr1) plays an important role in cellular morphogenesis in this microorganism. Cells deleted for YlRSR1 exhibited impaired polarized growth during yeast-form growth. Pseudohyphal and hyphal development were also abnormal. YlRsr1 is also important for cell growth, since the deletion of YlRSR1 in cells lacking the Ras GTPase YlRas2 caused lethality. Y. lipolytica cells bud in a bipolar pattern in which the cells produce the new buds at the two poles. YlRsr1 plays a prominent role in this bud site selection process. YlRsr1's function in bud site selection absolutely requires the cycling of YlRsr1 between the GTP- and GDP-bound states but its function in cellular morphogenesis does not, suggesting that the two processes are differentially regulated. Interestingly, the Ras GTPase YlRas2 is also involved in the control of bud site selection, as Ylras2Δ cells were severely impaired in bipolar bud site selection. The GTP/GDP cycling and the plasma membrane localization of YlRas2 are important for YlRas2's function in bud site selection. However, they are not essential for this process, suggesting that the mechanism by which YlRas2 acts is different from that of YlRsr1. Our results suggest that YlRsr1 is regulated by the GTPase-activating protein (GAP) YlBud2 and partially by YlCdc25, the potential guanine nucleotide exchange factor (GEF) for YlRas2.
PMCID: PMC4060475  PMID: 24610659
15.  Association of CDKN2BAS Polymorphism rs4977574 with Coronary Heart Disease: A Case-Control Study and a Meta-Analysis 
The goal of our study was to explore the significant association between a non-protein coding single nucleotide polymorphism (SNP) rs4977574 of CDKN2BAS gene and coronary heart disease (CHD). A total of 590 CHD cases and 482 non-CHD controls were involved in the present association study. A strong association of rs4977574 with CHD was observed in females (genotype: p = 0.002; allele: p = 0.002, odd ratio (OR) = 1.57, 95% confidential interval (CI) = 1.18–2.08). Moreover, rs4977574 was more likely to be a risk variant of CHD under the recessive model in females (χ2 = 10.29, p = 0.003, OR = 2.14, 95% CI = 1.31–2.77). A breakdown analysis by age had shown that there was an 87% increased risk of CHD for females younger than 65 years (genotype: χ2 = 14.64, degrees of freedom (df) = 2, p = 0.0002; allele: χ2 = 11.31, df = 1, p = 0.0008, OR = 1.87, 95% CI = 1.30–2.70). Similar observation was also found in males younger than 65 years (genotype: χ2 = 8.63, df = 2, p = 0.04; allele: χ2 = 7.55, df = 1, p = 0.006, OR = 1.45, 95% CI = 1.11–1.90). p values were adjusted by age, sex, smoking, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). Meta-analysis of 23 studies among 36,452 cases and 39,781 controls showed a strong association between rs4977574 and the risk of CHD (p < 0.0001, OR = 1.27, 95% CI = 1.22–1.31).
PMCID: PMC4227174  PMID: 25268619
coronary heart disease; single nucleotide polymorphism (SNP); CDKN2BAS; meta–analysis
16.  Bone Marrow Transplantation Concurrently Reconstitutes Donor Liver and Immune System across Host Species Barrier in Mice 
PLoS ONE  2014;9(9):e106791.
Liver immunopathologic mechanisms during hepatotropic infection, malignant transformation, and autoimmunity are still unclear. Establishing a chimeric mouse with a reconstituted liver and immune system derived from a single donor across species is critical to study regional donor immune responses in recipient liver. Using a strain of mice deficient in tyrosine catabolic enzyme fumarylacetoacetate hydrolase (fah-/-) and bone marrow transplantation (BMT), we reconstituted the donor's hepatocytes and immune cells across host species barrier. Syngeneic, allogeneic or even xenogeneic rat BMT rescued most recipient fah-/- mice against liver failure by donor BM-derived FAH+ hepatocytes. Importantly, immune system developed normally in chimeras, and the immune cells together with organ architecture were intact and functional. Thus, donor BM can across host species barrier and concurrently reconstitutes MHC-identical response between immune cells and hepatocytes, giving rise to a new simple and convenient small animal model to study donor's liver immune response in mice.
PMCID: PMC4156390  PMID: 25191899
17.  Relationship between C - Reactive Protein and Stroke: A Large Prospective Community Based Study 
PLoS ONE  2014;9(9):e107017.
Previous studies have suggested that C-reactive protein (CRP) was associated with risk of stroke. There were few studies in Asian population, or on stroke subtypes other than ischemic stroke. We thus investigated the relationship between CRP and the risks of all stroke and its subtypes in a Chinese adult population.
In the current study, we included 90,517 Chinese adults free of stroke and myocardial infarction at baseline (June 2006 to October 2007) in analyses. Strokes were classified as ischemic stroke (IS), intracranial heamorrhage (ICH) and subarachnoid heamorrhage (SAH). High-sensitivity CRP (hs-CRP) were categorized into three groups: <1 mg/L, 1 to 3 mg/L, and >3 mg/L. Cox proportional hazards regression was used to calculate the association between hs-CRP concentrations and all stroke, as well as its subtypes.
During a median follow-up time of 49 months, we documented 1,472 incident stroke cases. Of which 1,049 (71.3%) were IS, 383 (26.0%) were ICH, and 40 (2.7%) were SAH. After multivariate adjustment, hs-CRP concentrations ≥1 mg/L were associated with increased risks of all stroke (hs-CRP 1–3 mg/L: hazard ratio (HR) 1.17, 95% confidential interval (CI) 1.03–1.33; hs-CRP>3 mg/L: HR 1.25, 95% CI 1.07–1.46) and IS (hs-CRP 1–3 mg/L: HR 1.17, 95% CI 1.01–1.36; hs-CRP>3 mg/L: HR 1.33, 95% CI 1.11–1.60), but not with ICH and SAH. Subgroup analyses showed that higher hs-CRP concentration was more prone to be a risk factor for all stroke and IS in non-fatal stroke, male and hypertensive participants.
We found that higher hs-CRP concentrations were associated with a higher risk of IS, particularly for non-fatal stroke, male and hypertensive subjects. In contrast, we did not observe significant associations between hs-CRP and ICH/SAH.
PMCID: PMC4156395  PMID: 25191699
18.  Short-term glucosamine infusion increases islet blood flow in anesthetized rats 
Islets  2013;5(5):201-206.
Impaired glucose tolerance and type 2 diabetes in rodents are associated with increased islet blood flow. If this is important for modulation of the endocrine function is at present unknown. We evaluated if glucosamine infusion, which induces peripheral insulin resistance and glucose intolerance, could be used to acutely increase islet blood flow. We infused anaesthetized Sprague-Dawley rats for 2 h with glucosamine (6 mg/kg body weight), in some cases followed by glucose administration. The former induced a 2-fold increase in serum insulin concentrations while plasma glucose remained unchanged. In vitro an augmented insulin response to hyperglycemia and decreased insulin content in batch type islet incubations with glucosamine for 24 h were seen. After 2 h glucosamine exposure in vitro, insulin release was decreased. In vivo glucosamine infusion increased islet blood flow, without affecting other regional blood flow values. Glucose increased islet blood flow to the same extent in control and glucosamine-infused rats. When exposed to 10 mmol/L glucosamine arterioles of isolated perfused islets showed a 10% dilation of their vascular smooth muscle. Thus, application of this model leads to acute hyperinsulinemia in vivo but a decreased insulin release in vitro, which suggests that effects not located to β cells are responsible for the effects seen in vivo. An increased islet blood flow in previously healthy animals was also seen after glucose administration, which can be used to further dissect the importance of blood flow changes in islet function.
PMCID: PMC4010572  PMID: 24275157
pancreatic islets; insulin resistance; islet blood flow; glucosamine; glucose tolerance
19.  TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro 
Cellular and Molecular Immunology  2014;11(5):477-494.
Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4+ T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2−/− and TLR4−/− mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2−/− and TLR4−/− mice. In addition, CD4+ T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4+ T cells from TLR2−/− and TLR4−/− mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2−/− and TLR4−/− mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2−/− and TLR4−/− mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response.
PMCID: PMC4197203  PMID: 24769793
BCSP31; Brucella abortus; cytokine; macrophage; Th1 immune response; TLR
20.  Conferring Virulence: Structure and Function of the chimeric A2B5 Typhoid Toxin 
Nature  2013;499(7458):350-354.
Salmonella Typhi differs from most other salmonellae in that it causes a life-threatening systemic infection known as typhoid fever1. The molecular bases for its unique clinical presentation are unknown2. Here we found that in an animal model, the systemic administration of typhoid toxin, a unique virulence factor of S. Typhi, reproduces many of the acute symptoms of typhoid fever. We identified specific carbohydrate moieties on specific surface glycoproteins that serve as receptors for typhoid toxin, which explains its broad cell target specificity. We present the atomic structure of typhoid toxin, which shows an unprecedented A2B5 organization with two covalently-linked A subunits non-covalently-associated to a pentameric B subunit. The structure provides insight into the toxin’s receptor-binding specificity and delivery mechanisms and reveals how the activities of two powerful toxins have been coopted into a single, unique toxin that can induce many of the symptoms characteristic of typhoid fever. These findings may lead to the development of potentially life-saving therapeutics against typhoid fever.
PMCID: PMC4144355  PMID: 23842500
Salmonella typhi; typhoid fever; bacterial toxins; Podocalyxin; CD45; bacterial pathogenesis; glycobiology; AB5 toxins
21.  Design and Characterization of PEG-Derivatized Vitamin E as a Nanomicellar Formulation for Delivery of Paclitaxel 
Molecular pharmaceutics  2013;10(8):2880-2890.
Various PEG-Vitamin E conjugates including D-alpha-tocopheryl polyethylene glycol succinate 1000 (TPGS) have been extensively studied as a nonionic surfactant in various drug delivery systems. However, limited information is available about the structure-activity relationship of PEG-Vitamin E conjugates as a micellar formulation for paclitaxel (PTX). In this study, four PEG-Vitamin E conjugates were developed that vary in the molecular weight of PEG (PEG2K vs PEG5K) and the molar ratio of PEG/Vitamin E (1/1 vs 1/2) in the conjugates. These conjugates were systematically characterized with respect to CMC, PTX loading efficiency, stability, and their efficiency in delivery of PTX to tumor cells in vitro and in vivo. Our data show that PEG5K-conjugates have lower CMC values and are more effective in PTX loading with respect to both loading capacity and stability. The conjugates with two Vitamin E molecules also worked better than the conjugates with one molecule of Vitamin E, particularly for PEG2K-system. Furthermore, all of the PEG-Vitamin E conjugates can inhibit P-gp function with their activity being comparable to that of TPGS. More importantly, PTX-loaded PEG5K-VE2 resulted in significantly improved tumor growth inhibitory effect in comparison to PTX formulated in PEG2K-VE or PEG2K-VE2, as well as Cremophor EL (Taxol) in a syngeneic mouse model of breast cancer (4T1.2). Our study suggests that PEG5K-Vitmin E2 may hold promise as an improved micellar formulation for in vivo delivery of anticancer agents such as PTX.
PMCID: PMC3778165  PMID: 23768151
Nanomicelles; Paclitaxel; TPGS; Controlled and sustained drug delivery; Cancer; Nanotechnology
22.  Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation 
Cell Research  2014;24(8):912-924.
Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.
PMCID: PMC4123296  PMID: 24980960
autophagy; Beclin1; Dapper1; neurodegeneration; Vps34
23.  Epidemiologic studies of particulate matter and lung cancer 
Chinese Journal of Cancer  2014;33(8):376-380.
Particulate matter (PM) plays an important role in air pollution, especially in China. European and American researchers conducted several cohort-based studies to examine the potential relationship between PM and lung cancer and found a positive association between PM and lung cancer mortality. In contrast, the results regarding PM and lung cancer risk remain inconsistent. Most of the previous studies had limitations such as misclassification of PM exposure and residual confounders, diminishing the impact of their findings. In addition, prospective studies on this topic are very limited in Chinese populations. This is an important problem because China has one of the highest concentrations of PM in the world and has had an increased mortality risk due to lung cancer. In this context, more prospective studies in Chinese populations are warranted to investigate the relationship between PM and lung cancer.
PMCID: PMC4135366  PMID: 25011458
Particulate matter; lung cancer; epidemiologic study
24.  Establishing a rapid animal model of osteoporosis with ovariectomy plus low calcium diet in rats 
The objective of this study was to rapidly develop osteoporotic model animals by combining ovariectomy with a low calcium diet in rats. Thirty, eight-week-old, female, Sprague-Dawley rats were either sham-operated (Sham) or ovariectomized (Ovx) and divided into three groups: Sham, Ovx, and Ovx + low calcium diet. Rats in the Sham and Ovx groups were fed a standard diet containing 1.1% w/w calcium while rats in the Ovx + low calcium diet group were fed a diet containing 0.1% w/w calcium. Serum osteocalcin and bone mineral density (BMD) of the lumbar vertebrae were measured 4 and 8 weeks after surgery. The rats were euthanized 12 weeks after surgery, and the BMD of the right femur and histomorphometry of the femoral neck were assessed at that time. The Ovx + low-calcium diet group had a significantly lower mean BMD of the lumbar vertebra and higher mean serum osteocalcin concentration than the Sham and Ovx groups. Twelve weeks after surgery, rats in the Ovx + low calcium diet group had a significantly lower BMD, smaller Tb.Th and Tb.N, and larger Tb.Sp of the right femoral neck than did rats in the Sham and Ovx groups. These data indicate that a low calcium diet can significantly accelerate bone loss in ovariectomized rats. Combining ovariectomy and a low calcium diet can save considerable time in the creation of osteoporotic model animals.
PMCID: PMC4152076  PMID: 25197386
Animal model; osteoporosis; ovariectomy; low calcium diet
25.  Elevated Plasma Total Cholesterol Level Is Associated with the Risk of Asymptomatic Intracranial Arterial Stenosis 
PLoS ONE  2014;9(7):e101232.
Intracranial arterial stenosis (ICAS) is one of the most common causes of stroke, and dyslipidemia was one of the most common risk factors related to ICAS. However, the correlation between the plasma total cholesterol level (PTC) and ICAS, especially asymptomatic ICAS (AICAS) is not clear.
Materials and Methods
5,300 participants were enrolled in this study. The diagnosis of AICAS was made by transcranial Doppler ultrasonography. The participants were then divided into 5 essentially equal-sized groups based on their PTC levels. The multivariate logistic regression was used to analyze the correlation between the PTC level and the prevalence of AICAS.
13.0% of the participants were diagnosed with AICAS. The prevalence of AICAS gradually increased with the increasing PTC level. After adjusted by the possible confounding factors, the Odds Ratios (OR) of the AICAS prevalence between the 1st quintile group and the other 4 groups were 1.13, 1.23, 1.63 and 1.75 with 95% confident intervals (CI) of 0.84–1.52, 0.91–1.66, 1.20–2.22 and 1.23–2.47, respectively. The further subgroup analysis revealed that the PTC level was stronger for males (OR 1.42 95%CI 1.23–1.64), regarding the prevalence of AICAS.
In this large community-based study, the prevalence of AICAS is 13.0%, subjects with higher PTC levels showed a mild increase in the prevalence of AICAS. The PTC level is an independent risk factor of AICAS. Males seem to be significantly more vulnerable to the risk of AICAS.
PMCID: PMC4081648  PMID: 24992466

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