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1.  Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis 
The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia against diseases such as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC50 value less than 1 µg/mL. Therefore, the present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structure of 1 was assigned by means of 1D and 2D NMR data and high-resolution mass spectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulation assay with IC50 values of 1.7 and 11 nM, respectively, with a therapeutic index up to 71,000. The antiallergic activities of A. malaccensis seeds and aquimavitalin (1) have never been revealed before. The results indicated that A. malaccensis seeds and the pure compound have the potential for use in the treatment of allergy.
PMCID: PMC4813253  PMID: 27007372
Aquilaria malaccensis seeds; antiallergic; degranulation; phorbol ester; bioactivity-guided fractionation
2.  Assessment of ALK gene fusions in lung cancer using the differential expression and exon integrity methods 
Oncology Letters  2016;11(3):1651-1656.
Anaplastic lymphoma kinase (ALK) gene fusion is a driving mutation underlying the development of non-small cell lung cancer (NSCLC). Accurate detection of ALK fusion is critical for the use of ALK inhibitors in the treatment of NSCLC. Commonly utilized methods for ALK detection include fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). However, these methods are time-consuming and costly. In the present study, a method for assessing ALK gene fusion based on the differential expression levels of the ALK kinase and non-kinase domains was developed and evaluated, with the aim of providing a convenient and reliable method for the detection of ALK fusion. In addition, another method was established to determine the integrity of exons 19–20 and 20–21 of ALK, two genomic loci that are typically broken in ALK fusions. These novel methods were applied to detect ALK fusion in 100 NSCLC patients, and were compared with IHC and FISH methods. The novel methods developed in the present study successfully detected ALK fusions in 10 samples. The concordances between the novel methods and IHC and FISH were 100%. Furthermore, the differential expression method was able to detect ALK fusions in cell-free urine samples, which was advantageous over FISH and IHC. The novel methods developed in the present study are cost-effective and easy to perform, and may provide simple and convenient techniques for the clinical assessment of ALK fusions, facilitating the use of targeted therapy for NSCLC.
PMCID: PMC4774594  PMID: 26998057
anaplastic lymphoma kinase; lung cancer; targeted therapy; crizotinib
3.  Exercise-Related Changes of Networks in Aging and Mild Cognitive Impairment Brain 
Aging and mild cognitive impairment (MCI) are accompanied by decline of cognitive functions. Meanwhile, the most common form of dementia is Alzheimer’s disease (AD), which is characterized by loss of memory and other intellectual abilities serious to make difficulties for patients in their daily life. MCI is a transition period between normal aging and dementia, which has been used for early detection of emerging dementia. It converts to dementia with an annual rate of 5–15% as compared to normal aging with 1% rate. Small decreases in the conversion rate of MCI to AD might significantly reduce the prevalence of dementia. Thus, it is important to intervene at the preclinical stage. Since there are still no effective drugs to treat AD, non-drug intervention is crucial for the prevention and treatment of cognitive decline in aging and MCI populations. Previous studies have found some cognitive brain networks disrupted in aging and MCI population, and physical exercise (PE) could effectively remediate the function of these brain networks. Understanding the exercise-related mechanisms is crucial to design efficient and effective PE programs for treatment/intervention of cognitive decline. In this review, we provide an overview of the neuroimaging studies on physical training in normal aging and MCI to identify the potential mechanisms underlying current physical training procedures. Studies of functional magnetic resonance imaging, electroencephalography, magnetoencephalography and positron emission tomography on brain networks were all included. Based on our review, the default mode network, fronto-parietal network and fronto-executive network are probably the three most valuable targets for efficiency evaluation of interventions.
PMCID: PMC4779936  PMID: 27014055
exercise; aging; mild cognitive impairment; functional magnetic resonance imaging; electroencephalogram; magnetoencephalography; positron emission tomography
4.  Whole Exome Sequencing Identifies Frequent Somatic Mutations in Cell-Cell Adhesion Genes in Chinese Patients with Lung Squamous Cell Carcinoma 
Scientific Reports  2015;5:14237.
Lung squamous cell carcinoma (SQCC) accounts for about 30% of all lung cancer cases. Understanding of mutational landscape for this subtype of lung cancer in Chinese patients is currently limited. We performed whole exome sequencing in samples from 100 patients with lung SQCCs to search for somatic mutations and the subsequent target capture sequencing in another 98 samples for validation. We identified 20 significantly mutated genes, including TP53, CDH10, NFE2L2 and PTEN. Pathways with frequently mutated genes included those of cell-cell adhesion/Wnt/Hippo in 76%, oxidative stress response in 21%, and phosphatidylinositol-3-OH kinase in 36% of the tested tumor samples. Mutations of Chromatin regulatory factor genes were identified at a lower frequency. In functional assays, we observed that knockdown of CDH10 promoted cell proliferation, soft-agar colony formation, cell migration and cell invasion, and overexpression of CDH10 inhibited cell proliferation. This mutational landscape of lung SQCC in Chinese patients improves our current understanding of lung carcinogenesis, early diagnosis and personalized therapy.
PMCID: PMC4621504  PMID: 26503331
5.  Euphol from Euphorbia tirucalli Negatively Modulates TGF-β Responsiveness via TGF-β Receptor Segregation inside Membrane Rafts 
PLoS ONE  2015;10(10):e0140249.
Transforming growth factor-β (TGF-β) responsiveness in cultured cells can be modulated by TGF-β partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. Lipid rafts are plasma membrane microdomains with an important role in cell survival signaling, and cholesterol is necessary for the lipid rafts’ structure and function. Euphol is a euphane-type triterpene alcohol that is structurally similar to cholesterol and has a wide range of pharmacological properties, including anti-inflammatory and anti-cancer effects. In the present study, euphol suppressed TGF-β signaling by inducing TGF-β receptor movement into lipid-raft microdomains and degrading TGF-β receptors.
PMCID: PMC4598150  PMID: 26448474
6.  Rickettsia massiliae and Rickettsia conorii Israeli Spotted Fever Strain Differentially Regulate Endothelial Cell Responses 
PLoS ONE  2015;10(9):e0138830.
Rickettsiae primarily target microvascular endothelial cells. However, it remains elusive how endothelial cell responses to rickettsiae play a role in the pathogenesis of rickettsial diseases. In the present study, we employed two rickettsial species with high sequence homology but differing virulence to investigate the pathological endothelial cell responses. Rickettsia massiliae is a newly documented human pathogen that causes a mild spotted fever rickettsiosis. The “Israeli spotted fever” strain of R. conorii (ISF) causes severe disease with a mortality rate up to 30% in hospitalized patients. At 48 hours post infection (HPI), R. conorii (ISF) induced a significant elevation of IL-8 and IL-6 while R. massiliae induced a statistically significant elevated amount of MCP-1 at both transcriptional and protein synthesis levels. Strikingly, R. conorii (ISF), but not R. massiliae, caused a significant level of cell death or injury in HMEC-1 cells at 72 HPI, demonstrated by live-dead cell staining, annexin V staining and lactate dehydrogenase release. Monolayers of endothelial cells infected with R. conorii (ISF) showed a statistically significant decrease in electrical resistance across the monolayer compared to both R. massiliae-infected and uninfected cells at 72 HPI, suggesting increased endothelial permeability. Interestingly, pharmacological inhibitors of caspase-1 significantly reduced the release of lactate dehydrogenase by R. conorii (ISF)-infected HMEC-1 cells, which suggests the role of caspase-1 in mediating the death of endothelial cells. Taken together, our data illustrated that a distinct proinflammatory cytokine profile and endothelial dysfunction, as evidenced by endothelial cell death/injury and increased permeability, are associated with the severity of rickettsial diseases.
PMCID: PMC4578955  PMID: 26394396
7.  Urban-Rural Differences in Cardiovascular Disease Risk Factors: A Cross-Sectional Study of Schoolchildren in Wuhan, China 
PLoS ONE  2015;10(9):e0137615.
China’s rapid population growth and urban migration has developed healthcare inequity across the urban-rural divide. Past studies comparing cardiovascular disease (CVD) risk factor prevalence amongst urban-rural Chinese children are sparse and conflicting. We examined the association between urban-rural residence and risk of offspring CVD in Chinese children.
A cross-sectional study was conducted in Wuhan, China, during May and June 2010. CVD risk factors include; waist circumference (WC), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), triglycerides (TG), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, body mass index (BMI), cardiorespiratory fitness (CRF), metabolic syndrome (MetS), and metabolic risk score (MRS). Analysis of covariance and multivariable logistic regression were used to estimate associations between urban-rural residence and offspring CVD risks.
A total of 579 Chinese children (338 boys and 241 girls) aged 9.6 (0.7) years participated in this study. Rural boys had significantly lower CRF and higher FBG, TG, and MRS, while urban boys had significantly higher LDL and DBP. Rural girls had significantly higher BMI, FBG, and TG, as well as lower CRF. Rural children were at increased risks for decreased CRF, elevated MRS, and TG, (OR:2.04, 95%CI:1.29–3.25), (OR:2.33, 95%CI:1.50–3.62), and (OR:2.40, 95%CI:1.62–3.57), respectively. Rural girls and mothers were at increased risks for overweight(OR:7.19, 95%CI:1.64–31.6)/obesity (OR:1.683, 95%CI:1.01–2.82). However, rural boys and fathers were less likely to have overweight(OR:0.62, 95%CI:0.34–1.12)/obesity (OR:0.68, 95%CI:0.48–0.97).
Rural residence was significantly associated with increased CVD risks amongst Chinese children. It is important to provide interventions aiming at China’s urban-rural healthcare inequity and community-based approaches that reduce familial CVD risk.
PMCID: PMC4564102  PMID: 26352586
8.  Coumarin derivatives from Ainsliaea fragrans and their anticoagulant activity 
Scientific Reports  2015;5:13544.
Coumarin derivatives are an important class of C6–C3 plant metabolites that show a variety of bioactivities. Currently, most clinical anticoagulant agents are coumarins, such as warfarin, dicoumarol and acenocoumarol, and patients taking these drugs must be monitored for adverse reactions. In a search for safe and effective anticoagulant compounds from Chinese herbal medicine, a screening procedure on the whole plant of Ainsliaea fragrans was performed. The phytochemical investigation of this plant afforded five new coumarin derivatives, including a pair of natural 4-hydroxycoumarin enantiomers (1), a pair of coumarin enantiomers with a rare polycyclic pyrano[3-2c] carbon skeleton (2) and a 7-hydroxycoumarin derivative (3), together with 5 known biogenetically related compounds (4–8). Enantioseparation of 1 and 2 produced optically pure compounds 1a, 1b, 2a and 2b. The absolute configurations of the new compounds were confirmed by single-crystal X-ray diffraction analysis. In addition, we evaluated the anticoagulant activity of all isolates via activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) assays in vitro and in vivo. Of note, compound 3 displayed potent anticoagulant activity and no significant hepatic or renal toxicity, which could make it a promising agent for further preclinical evaluation for preventing abnormal blood clotting.
PMCID: PMC4551958  PMID: 26315062
9.  Clinical Characteristics for the Relationship between Type-2 Diabetes Mellitus and Cognitive Impairment: A Cross-Sectional Study 
Aging and Disease  2015;6(4):236-244.
We explored the potential differences in cognitive status, lipid and glucose metabolism, ApoEε4 alleles and imaging between diabetic and non-diabetic subjects. 83 subjects with normal cognitive function and 114 mild cognitive impaired patients were divided into four groups by history of diabetes. General demographics was collected from all participants followed by MRI scan, biochemical examinations and a series of neuropsychological tests. Student’s t test, multiple regressions and one-way ANOVA were applied to investigate the differences between groups. Comparing diabetic patients with non-diabetic subjects in the mild cognitive impaired group, we found several decreased items in recall of three words in MMSE (p=0.020), AVLT and SCWT (p<0.050). The multiple linear regression revealed that two-hour glucose level (B= −0.255, p<0.001) and fasting C-peptide (B= −0.466, p=0.001) had negative effects on the score of MMSE. In addition, diabetic patients treated with insulin and other diabetes medication performed better in part of the AVLT (p<0.050) compared to patients with insulin treatment or oral antidiabetic medication only. Patients with metformin medication had a better memory outcome compared to patients with sulphonylurea medication in the AVLT long delay free recall (p =0.010). These findings show that patients of mild cognitive impairment with diabetes mellitus have a worse outcome in attention, information processing speed and memory compared to non-diabetic patients. Higher two-hour glucose level and C-peptide level may be risk factors for severe cognitive impairment in type-2 diabetes mellitus patients. The results of this study also suggest that medication may have effects on cognitive function.
PMCID: PMC4509472
Type-2 diabetes mellitus; mild cognitive impairment; C-peptide; blood glucose
10.  Cancer risk among patients with type 2 diabetes mellitus: a population-based prospective study in China 
Scientific Reports  2015;5:11503.
Evidence indicates an increased cancer risk among type 2 diabetes mellitus (T2DM) patients, yet studies in mainland China are scarce. Based on Diabetes Surveillance System linking to Cancer Surveillance System of Zhejiang Province in China, we explored the cancer risk among T2DM patients. Totally, 327,268 T2DM patients were identified and followed from January 1, 2007 to December 31, 2013. Standardized incidence ratios (SIRs) with 95% confidence intervals (CIs) were reported. Overall cancer risk was found significantly increased with an SIR of 1.15 (95% CI 1.12–1.19) and 1.25 (95% CI 1.21–1.30) in males and females, respectively. Regarding specific cancer sites, risks of liver, colon, rectum, pancreas, and kidney were significantly increased with SIRs of 1.26 (95% CI 1.16–1.36), 1.47 (95% CI 1.29–1.67), 1.25 (95% CI 1.09–1.43), 2.81 (95% CI 2.50–3.16) and 1.61 (95% CI 1.28–2.03) in males, 1.53 (95% CI 1.35–1.73), 1.33 (95% CI 1.15–1.54), 1.29 (95% CI 1.10–1.51), 3.62 (95% CI 3.20–4.09) and 1.71 (95% CI 1.28–2.29) in females, respectively. A significant increased SIR was noted for prostate (1.80, 95% CI 1.58–2.06). Significant increased SIRs for lung (1.32, 95% CI 1.20–1.44) and stomach (1.16, 95% CI 1.03–1.30) were observed in females. We suggested an increased cancer risk among T2DM patients.
PMCID: PMC4469976  PMID: 26082067
11.  Loss of IRF8 Inhibits the Growth of Diffuse Large B-cell Lymphoma 
Journal of Cancer  2015;6(10):953-961.
IRF8 is a transcription factor with a critical role in B lymphocyte development and functions. Its role in human diffuse large B-cell lymphoma (DLBCL), however, remained elusive. In this study, using shRNA-mediated knockdown of IRF8 expression, we found that the loss of IRF8 significantly reduced the proliferation of DLBCL cells (P<0.05). Mechanistically, decreasing the levels of IRF8 led to a suppression of the phosphorylation of p38 and ERK, molecules critical for B cell proliferation. Furthermore, using a xenograft lymphoma mouse model, we found that the loss of IRF8 significantly inhibited the growth of lymphomas in vivo (P<0.05). Immunohistochemical analysis of human DLBCL tissues revealed that the levels of IRF8 were significantly greater in non-germinal center B-cell-like (non-GCB) subtype than that in GCB subtype (P<0.05). Analysis of public available data also suggested that the expression levels of IRF8 mRNA in human DLBCL tissues were inversely correlated with patients' overall survival time. Taken together, this study suggested that IRF8 may play an oncogenic role in human DLBCL by promoting cell proliferation.
PMCID: PMC4543755  PMID: 26316891
lymphoma; IRF8; DLBCL
12.  Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3−/− Macrophages 
PLoS ONE  2014;9(11):e111539.
In this study, we have utilized wild-type (WT), ASC−/−, and NLRP3−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.
PMCID: PMC4221042  PMID: 25372293
13.  Ursolic Acid Inhibits Superoxide Production in Activated Neutrophils and Attenuates Trauma-Hemorrhage Shock-Induced Organ Injury in Rats 
PLoS ONE  2014;9(10):e111365.
Neutrophil activation is associated with the development of organ injury after trauma–hemorrhagic shock. In the present study, ursolic acid inhibited the superoxide anion generation and elastase release in human neutrophils. Administration of ursolic acid attenuated trauma–hemorrhagic shock-induced hepatic and lung injuries in rats. In addition, administration of ursolic acid attenuated the hepatic malondialdehyde levels and reduced the plasma aspartate aminotransferase and alanine aminotransferase levels after trauma–hemorrhagic shock. In conclusion, ursolic acid, a bioactive natural compound, inhibits superoxide anion generation and elastase release in human neutrophils and ameliorates trauma–hemorrhagic shock-induced organ injury in rats.
PMCID: PMC4216084  PMID: 25360589
14.  Serum-Free Medium Optimization Based on Trial Design and Support Vector Regression 
BioMed Research International  2014;2014:269305.
The Plackett-Burman design and support vector machine (SVM) were reported to be used on many fields such as some feature selections, protein structure prediction, or forecasting of other situations. Here, with suspension adapted Chinese hamster ovary (CHO) cells as the object of study, a serum-free medium for the culture of CHO cells in suspension was optimized by this method. Support vector machine based on genetic algorithm was used to predict the growth rate of CHO and prove the results from the trial designs. Experimental results indicated that ZnSO4, transferrin, and bovine serum albumin (BSA) were important ones. The same conclusion was arrived at when the support vector regression model analyzed the experimental results. With the methods mentioned, the influence of 7 medium supplements on the growth of CHO cells in suspension was evaluated efficiently.
PMCID: PMC4212526  PMID: 25379507
15.  Increased methylation at differentially methylated region of GNAS in infants born to gestational diabetes 
BMC Medical Genetics  2014;15:108.
Offspring of pregnancy complicated with gestational diabetes (GDM) are at high risk for metabolic diseases. The mechanisms behind the association of intrauterine exposure to GDM and high risk of health problems in later life remain largely unknown. The aim of this study was to clarify the alteration in methylation levels at differentially methylated regions (DMRs) of GNAS and IGF2 in fetuses of GDM women and to explore the possible mechanisms linking maternal GDM with high risk of metabolic diseases in later life of GDM offspring.
Lymphocytes were isolated from umbilical cord blood of infants born to 87 women with GDM and 81 women with normal pregnancy. Genomic DNA was extracted and DNA methylation levels of GNAS and IGF2 DMRs were determined by Massarray quantitative methylation analysis.
The methylation levels were detected in 7 CpG sites of GNAS DMRs and 6 sites of IGF2 DMRs. Methylation levels were significantly higher at sites 4, 5 and 7 of GNAS DMR in GDM compared to normal pregnancy (P = 0.007, 0.008 and 0.008, respectively). The methylation level at site 4 of GNAS was significantly correlated with the presence of GDM (P = 0.003), the methylation levels at site 5 and 7 were significantly correlated with the presence of GDM (P = 0.002 for both) and gestational age (P = 0.027 for both). There was no significant difference in any sites of IGF2 DMR (P > 0.05 for all).
We concluded maternal GDM-induced hypermethylation at GNAS DMR and this condition may be among the mechanisms associating maternal GDM with increased risk of metabolic diseases in later life of offspring.
PMCID: PMC4411875  PMID: 25269528
Gestational diabetes mellitus; Methylation; DNA; DMR; GNAS; IGF2
16.  MyD88 controls human metapneumovirus-induced pulmonary immune responses and disease pathogenesis 
Virus research  2013;176(0):10.1016/j.virusres.2013.06.014.
Human metapneumovirus (hMPV) is a common cause of lung and airway infections in infants and young children. Recently, we and others have shown that hMPV infection induces Toll-like receptor (TLR)-dependent cellular signaling. However, the contribution of TLR-mediated signaling in host defenses against pulmonary hMPV infection and associated disease pathogenesis has not been elucidated. In this study, mice deficient in MyD88, a common adaptor of TLRs, was used to investigate the contribution of TLRs to in vivo pulmonary response to hMPV infection. MyD88−/− mice have significantly reduced pulmonary inflammation and associated disease compared with wild-type (WT) C57BL/6 mice after intranasal infection with hMPV. hMPV-induced cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and isolated lung conventional dendritic cells (cDC) are also significantly impaired by MyD88 deletion. In addition, we found that MyD88 is required for the recruitment of DC, T cells, and other immune cells to the lungs, and for the functional regulation of DC and T cells in response to hMPV infection. Taken together, our data indicate that MyD88-mediated pathways are essential for the pulmonary immune and pathogenic responses to this viral pathogen.
PMCID: PMC3819944  PMID: 23845303
hMPV; MyD88; Cytokines/chemokines; Pulmonary immune response
17.  Disrupted Functional Brain Connectivity and Its Association to Structural Connectivity in Amnestic Mild Cognitive Impairment and Alzheimer’s Disease 
PLoS ONE  2014;9(5):e96505.
Although anomalies in the topological architecture of whole-brain connectivity have been found to be associated with Alzheimer’s disease (AD), our understanding about the progression of AD in a functional connectivity (FC) perspective is still rudimentary and few study has explored the function-structure relations in brain networks of AD patients. By using resting-state functional MRI (fMRI), this study firstly investigated organizational alternations in FC networks in 12 AD patients, 15 amnestic mild cognitive impairment (aMCI) patients, and 14 age-matched healthy aging subjects and found that all three groups exhibit economical small-world network properties. Nonetheless, we found a decline of the optimal architecture in the progression of AD, represented by a more localized modular organization with less efficient local information transfer. Our results also show that aMCI forms a boundary between normal aging and AD and represents a functional continuum between healthy aging and the earliest signs of dementia. Moreover, we revealed a dissociated relationship between the overall FC and structural connectivity (SC) in AD patients. In this study, diffusion tensor imaging tractography was used to map the structural network of the same individuals. The decreased FC-SC coupling may be indicative of more stringent and less dynamic brain function in AD patients. Our findings provided insightful implications for understanding the pathophysiological mechanisms of brain dysfunctions in aMCI and AD patients and demonstrated that functional disorders can be characterized by multimodal neuroimaging-based metrics.
PMCID: PMC4013022  PMID: 24806295
18.  Lipid modulatory activities of Cichorium glandulosum Boiss et Huet are mediated by multiple components within hepatocytes 
Scientific Reports  2014;4:4715.
To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of non-alcoholic fatty liver disease (NAFLD), we have made use of Cichorium glandulosum Boiss et Huet (CG), a traditional Chinese herbal medicine that has been proven to be effective in treating hepatic diseases. Here, we report that the extract of CG effectively reduced lipid accumulation under conditions of lipid overloading in vivo and in vitro (in a rat high-fat diet model and a hepG2 cell model of free fatty acid treatment). CG extract also protected hepatocytes from injury and inflammation to aid its lipid-lowering properties (in a rat high-fat diet model and a L02 cell model of acetaminophen treatment). Serum chemistry analysis accompanied by in vitro drug screening confirmed that CG-4, CG-10 and CG-14 are the lipo-effective components of CG. Western blotting analysis revealed that these components can regulate key lipid targets at the molecular level, including CD36, FATP5 and PPAR-α, thus the lipid oxidation and lipid absorption pathways. Finally, we adopted the experimental design and statistical method to calculate the best combination proportion (CG-4: CG-10: CG-14 = 2.065: 1.782: 2.153) to optimize its therapeutic effect.
PMCID: PMC4010933  PMID: 24797163
19.  Spectrum of LKB1, EGFR, and KRAS Mutations in Chinese Lung Adenocarcinomas 
Somatic LKB1 mutations are found in lung adenocarcinomas at different frequencies in Caucasian and East Asian (Japanese and Korean) populations. This study was designed to characterize the frequency of LKB1 mutations, their relationship to EGFR and KRAS mutations, and their associated clinicopathologic characteristics in Chinese patients.
Two hundred thirty-nine lung adenocarcinomas consecutively collected from October 2007 to July 2009 were dissected into 3 to 4 small (3 mm) pieces for histopathological analyses of tumor content. Genomic DNA and/or cDNA from 86 samples with more than 70% tumor content were used for sequencing of LKB1 (exons 1–9), EGFR (exons 18–21), and KRAS (exon 2). LKB1 germline mutation status was determined by sequencing of genomic DNA from matched histologically distant lung tissues that are histologically normal.
6.9% of lung adenocarcinomas harbored LKB1 somatic mutations. A total of 10.5% of patients had an LKB1 germline polymorphism, F354L. Interestingly, in two of these patients, tumors displayed loss of heterozygosity at this allele. EGFR kinase domain and KRAS mutations were found in 66.3% and 2.3% of Chinese lung adenocarcinomas, respectively. Concurrent LKB1 and EGFR somatic mutations were observed in one patient. Both KRAS-mutant tumors harbored LKB1 mutations.
These data provide important clinical and molecular characteristics of lung adenocarcinomas from Chinese patients.
PMCID: PMC4009449  PMID: 20559149
Chinese lung adenocarcinoma; LKB1; EGFR; KRAS; Mutation
21.  The RNA-Binding Protein QKI Suppresses Cancer-Associated Aberrant Splicing 
PLoS Genetics  2014;10(4):e1004289.
Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.
Author Summary
Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicates that splicing program is frequently deregulated during tumorigenesis, and cancer cells favor to produce protein isoforms that can promote growth and survival. Lung cancer is one of the most common cancers and the leading cause of cancer-related death worldwide. Although a number of lung cancer-related splicing events have been detected in several genome-wide analyses, much less is known about how aberrant splicing takes place in lung cancer and how it contributes to tumor development. In this study, we characterized the RNA-binding protein QKI as a new critical regulator of alternative splicing in lung cancer and as a potential marker for prognosis. Genome-wide analysis of QKI-dependent splicing by RNA-Seq identified some cancer-associated splicing changes as its targets. Our results demonstrate that QKI-5 inhibits cancer cell proliferation and prevents inappropriate activation of the Notch signaling pathway by regulating its key target, NUMB. We further showed that QKI-5 represses the inclusion of NUMB alternative exon through competing with a core splicing factor SF1. In summary, our data indicate that down-regulation of QKI causes aberrant splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated repression of Notch signaling.
PMCID: PMC3983035  PMID: 24722255
22.  Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome 
Cancer letters  2013;342(1):36-42.
Germline mutations are responsible for familial cancer syndromes which account for approximately 5–10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22 years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.
PMCID: PMC3981830  PMID: 23981578
P53 V157D; PMS2 R20Q; Germline mutation; Familial cancer syndrome; Co-existing
23.  Aqueous Extract of Gracilaria tenuistipitata Suppresses LPS-Induced NF-κB and MAPK Activation in RAW 264.7 and Rat Peritoneal Macrophages and Exerts Hepatoprotective Effects on Carbon Tetrachloride-Treated Rat 
PLoS ONE  2014;9(1):e86557.
In addition to the previous investigations of bioactivity of aqueous extract of the edible Gracilaria tenuistipitata (AEGT) against H2O2-induced DNA damage and hepatitis C virus replication, the purpose of this study is to evaluate the potential therapeutic properties of AEGT against inflammation and hepatotoxicity using lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 cells, primary rat peritoneal macrophages and carbon tetrachloride (CCl4)-induced acute hepatitis model in rats. AEGT concentration-dependently inhibited the elevated RNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2, thereby reducing nitric oxide and prostaglandin E2 levels, respectively. Moreover, AEGT significantly suppressed the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. These inhibitory effects were associated with the suppression of nuclear factor-kappa B activation and mitogen-activated protein kinase phosphorylation by AEGT in LPS-stimulated cells. In addition, we highlighted the hepatoprotective and curative effects of AEGT in a rat model of CCl4-intoxicated acute liver injury, which was evident from reduction in the elevated serum aspartate aminotransferase and alanine aminotransferase levels as well as amelioration of histological damage by pre-treatment or post-treatment of AEGT. In conclusion, the results demonstrate that AEGT may serve as a potential supplement in the prevention or amelioration of inflammatory diseases.
PMCID: PMC3903563  PMID: 24475143
24.  An improved ChIP-seq peak detection system for simultaneously identifying post-translational modified transcription factors by combinatorial fusion, using SUMOylation as an example 
BMC Genomics  2014;15(Suppl 1):S1.
Post-translational modification (PTM) of transcriptional factors and chromatin remodelling proteins is recognized as a major mechanism by which transcriptional regulation occurs. Chromatin immunoprecipitation (ChIP) in combination with high-throughput sequencing (ChIP-seq) is being applied as a gold standard when studying the genome-wide binding sites of transcription factor (TFs). This has greatly improved our understanding of protein-DNA interactions on a genomic-wide scale. However, current ChIP-seq peak calling tools are not sufficiently sensitive and are unable to simultaneously identify post-translational modified TFs based on ChIP-seq analysis; this is largely due to the wide-spread presence of multiple modified TFs. Using SUMO-1 modification as an example; we describe here an improved approach that allows the simultaneous identification of the particular genomic binding regions of all TFs with SUMO-1 modification.
Traditional peak calling methods are inadequate when identifying multiple TF binding sites that involve long genomic regions and therefore we designed a ChIP-seq processing pipeline for the detection of peaks via a combinatorial fusion method. Then, we annotate the peaks with known transcription factor binding sites (TFBS) using the Transfac Matrix Database (v7.0), which predicts potential SUMOylated TFs. Next, the peak calling result was further analyzed based on the promoter proximity, TFBS annotation, a literature review, and was validated by ChIP-real-time quantitative PCR (qPCR) and ChIP-reChIP real-time qPCR. The results show clearly that SUMOylated TFs are able to be pinpointed using our pipeline.
A methodology is presented that analyzes SUMO-1 ChIP-seq patterns and predicts related TFs. Our analysis uses three peak calling tools. The fusion of these different tools increases the precision of the peak calling results. TFBS annotation method is able to predict potential SUMOylated TFs. Here, we offer a new approach that enhances ChIP-seq data analysis and allows the identification of multiple SUMOylated TF binding sites simultaneously, which can then be utilized for other functional PTM binding site prediction in future.
PMCID: PMC4046823  PMID: 24564277
25.  Phyto-SERM Constitutes from Flemingia macrophylla 
The methanolic extract of Flemingia macrophylla roots exhibited significant estrogenic activity in the transgenic plant assay system which was comparable to the activity of soybean extract. Utilizing estrogenic activity-guided fractionation, one new compound, fleminigin, together with 23 known compounds were isolated from F. macrophylla roots’ methanolic extract. The structure of the new compound was identified based on intensive spectroscopic analysis and the full spectral data for one of the isolated compounds, flemichin E, was introduced for the first time in the current investigation. The estrogenic and anti-estrogenic activities of the isolated compounds were evaluated revealing that the isolated isoflavonoids may act as partial estrogen agonists, as well as antagonists. Additionally, the anti-inflammatory and the cytotoxic activities of the isolated compounds were studied. These results suggested the potential applications of F. macrophylla extract and its isolated compounds as selective estrogen receptor modulators (SERMs).
PMCID: PMC3759874  PMID: 23896592
Flemingia macrophylla; menopausal; phytoestrogen; fleminigin; flemichin E; pER8:GUS

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