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3.  Protective effect of epimedium combined with oligomeric proanthocyanidins on exercise-induced renal ischemia-reperfusion injury of rats 
Objective: This paper studied the protective effect and mechanism of epimedium combined with oligomeric proanthocyanidins on exercise-induced renal ischemia-reperfusion injury of rats. Methods: In the experiment, the rats were given exhaustive swimming training and then their blood urea nitrogen (BUN) and other biochemical indexes were measured after they were given gastric perfusion with 6.01 g/kg doze of epimedium and 50 mg/kg doze of oligomeric proanthocyanidins for 56 days. Results: The result indicated that 8 weeks of over training led to ischemia-reperfusion injury of rats. Moreover, their kidney tissues were significantly changed pathologically and renal functions drastically damaged. BUN and serum creatinine increased and EOM group (P < 0.05), OPCOM group (P < 0.05) and EOPCOM group (P < 0.01) were lower than OM group. EOPCOM group was lower than OPCOM group. SOD activity decreased, EOM group (P < 0.05), OPCOM group (P < 0.05), EOPCOM group (P < 0.01) higher than OM group, and EOPCOM group (P < 0.05) higher than OPCOM group. The content of MDA increased, EOM group (P < 0.05), OPCOM group (P < 0.05), EOPCOM group (P < 0.01) lower than OM group, and EOPCOM group (P < 0.05) lower than OPCOM group. Conclusion: Both epimedium and oligomeric proanthocyanidins can boost SOD activity, clean oxygen radicals, clean and alleviate peroxidation of lipids, which exert protection on exercise-induced renal ischemia-reperfusion. The two combined yield a much better result.
PMCID: PMC4307546  PMID: 25664099
Epimedium; oligomeric proanthocyanidins; exercise-induced ischemia-reperfusion
4.  The anterior esophageal region of Schistosoma japonicum is a secretory organ 
Parasites & Vectors  2014;7(1):565.
The esophagus of blood-feeding schistosomes has been largely neglected although its posterior portion was designated as a gland decades ago. However, we recently showed it plays a pivotal role in blood processing. It is clearly demarcated into anterior and posterior compartments, both surrounded by a mass of cell bodies. Feeding movies revealed that erythrocytes accumulate in the anterior compartment before entering the posterior, indicating that a distinct process is executed there. We therefore investigated ultrastructural aspects and possible functions of the anterior region.
The heads of adult Schistosoma japonicum were detached and prepared for both transmission and scanning electron microscopy to define the detailed ultrastructure of the anterior esophagus. Cryosections of heads were also prepared for immunocytochemistry and confocal microscopy to define the pattern of intrinsic host antibody binding in the anterior esophageal lining.
The anterior syncytial lining of the esophagus is highly extended by long, thin corrugations of cytoplasm projecting towards the lumen. Strikingly in the male worm, the tips of the corrugations are further expanded by numerous threads of cytoplasm, producing a spaghetti-like appearance in the central lumen. Flattened, pitted cytoplasmic plates are interspersed in the tangled mass of threads. Abundant, morphologically distinct light vesicles of varied size and contents are manufactured in the cell bodies, from where they traffic through cytoplasmic connections to the corrugations and out to the tips. Clusters of vesicles accumulate in expanded tips in males, together with occasional mitochondria whilst females have more mitochondria but fewer vesicles. The membranous contents of light vesicles are secreted mainly from the tips, but also from the sides of the corrugations. They coat the surfaces and then form organised self-adherent membrane figures when shed into the lumen. Host antibody binds strongly in a characteristic pattern to the anterior esophageal lining indicating that the secretions are highly immunogenic.
We suggest that the anterior esophageal region is an independent secretory organ. The contents of light vesicles are released into the esophageal lumen via the tips of corrugation to interact with incoming blood. Our immediate task is to establish their composition and role in blood processing.
PMCID: PMC4269844  PMID: 25490864
Esophagus; Vesicle; Transmission electron microscopy; Scanning electron microscopy; Antibody localisation; Schistosoma japonicum
5.  Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines 
PLoS ONE  2014;9(12):e114389.
Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.
PMCID: PMC4259319  PMID: 25486532
6.  The effect of loaded deep inhale training on mild and moderate COPD smokers 
Objective: To research the therapeutic effect of loaded deep inhale training on mild and moderate COPD smokers. Design: 30 mild and moderate COPD smokers were divided into the observation group and the control group at random. The observation group underwent loaded deep inhale training in the morning and in the evening twice for 30 minutes each time for 3 months. The control group did regular aerobics like jogging twice a day for 30 minutes as well for 3 months. The power of respiratory muscles and pulmonary function parameters of each group were measured and compared before and three months after the training. Results: After 3 months of hard training, pulmonary function parameters of the observation group was impressively improved compared with the control group and before training. Conclusions: Loaded deep inhale exercise has a remarkable effect on improving pulmonary function of mild and moderate COPD.
PMCID: PMC4238494  PMID: 25419402
Loaded; inhale training; smokers; COPD; therapeutic effect
7.  Unraveling the Role of KIAA1199, a Novel Endoplasmic Reticulum Protein, in Cancer Cell Migration 
Cell migration is a critical determinant of cancer metastasis, and a better understanding of the genes involved will lead to the identification of novel targets aimed at preventing cancer dissemination. KIAA1199 has been shown to be upregulated in human cancers, yet its role in cancer progression was hitherto unknown.
Clinical relevance was assessed by examining KIAA1199 expression in human cancer specimens. In vitro and in vivo studies were employed to determine the function of KIAA1199 in cancer progression. Cellular localization of KIAA1199 was microscopically determined. SNAP-tag pull-down assays were used to identify binding partner(s) of KIAA1199. Calcium levels were evaluated using spectrofluorometric and fluorescence resonance energy transfer analyses. Signaling pathways were dissected by Western blotting. Student t test was used to assess differences. All statistical tests were two-sided.
KIAA1199 was upregulated in invasive breast cancer specimens and inversely associated with patient survival rate. Silencing of KIAA1199 in MDA-MB-435 cancer cells resulted in a mesenchymal-to-epithelial transition that reduced cell migratory ability in vitro (75% reduction; P < .001) and decreased metastasis in vivo (80% reduction; P < .001). Gain-of-function assays further demonstrated the role of KIAA1199 in cell migration. KIAA1199-enhanced cell migration required endoplasmic reticulum (ER) localization, where it forms a stable complex with the chaperone binding immunoglobulin protein (BiP). A novel ER-retention motif within KIAA1199 that is required for its ER localization, BiP interaction, and enhanced cell migration was identified. Mechanistically, KIAA1199 was found to mediate ER calcium leakage, and the resultant increase in cytosolic calcium ultimately led to protein kinase C alpha activation and cell migration.
KIAA1199 serves as a novel cell migration–promoting gene and plays a critical role in maintaining cancer mesenchymal status.
PMCID: PMC3776264  PMID: 23990668
8.  Pooled Analysis of Performance of Liquid Based Cytology in Population-Based Cervical Cancer Screening Studies in China 
Cancer cytopathology  2013;121(9):473-482.
Liquid based cytology (LBC) has been widely used for cervical cancer screening. Despite numerous studies and systematic reviews, few large studies have focused on biopsy-confirmed cervical lesions and controversy remains about its diagnostic accuracy. The aim of our study was to assess LBC for detecting biopsy-confirmed cervical intraepithelial neoplasia (CIN) and cancer.
We performed a pooled analysis of LBC using data from 13 population-based, cross-sectional, cervical-cancer screening studies performed in China from 1999 to 2008. Participants (n = 26782) received LBC and HPV testing. Screen-positive women were referred for colposcopy and biopsy. We analyzed the accuracy of LBC for detecting biopsy-confirmed CIN2 or worse lesion (CIN2+) as well as CIN3 or worse lesion (CIN3+).
Of 25830 women included in the analysis, CIN2+ was found in 107/2612(4.1%) with ASC, 142/923 (15.4%) with LSIL, 512/784 (65.3%) with HSIL, 29/30 (96.7%) with SCC, 4/27(14.8%) with AGC, and 0.4% (85/21454) with normal cytology results. No invasive cancers had ASC, AGC or cytological normal slides. The overall sensitivity, specificity, PPV, NPV and accuracy of LBC for detecting CIN2+ were 81.0%, 95.4%, 38.3%, 99.3 % and 94.9% respectively. Although HC2 was more sensitive than LBC, the specificity, PPV and overall accuracy of LBC were higher than those of HC2, at 85.2%, 18.6% and 85.5%, respectively.
The results indicate that performance of LBC can effectively predict a risk of existing CIN2+ and may be a good screening tool for cervical cancer prevention in a developing country.
PMCID: PMC3775994  PMID: 23907807
Pool analysis; liquid-based cytology; Population-based; cervical cancer; screening
9.  Sympathetic Denervation-Induced MSC Mobilization in Distraction Osteogenesis Associates with Inhibition of MSC Migration and Osteogenesis by Norepinephrine/adrb3 
PLoS ONE  2014;9(8):e105976.
The sympathetic nervous system regulates bone formation and resorption under physiological conditions. However, it is still unclear how the sympathetic nerves affect stem cell migration and differentiation in bone regeneration. Distraction osteogenesis is an ideal model of bone regeneration due to its special nature as a self-engineering tissue. In this study, a rat model of mandibular distraction osteogenesis with transection of cervical sympathetic trunk was used to demonstrate that sympathetic denervation can deplete norepinephrine (NE) in distraction-induced bone callus, down-regulate β3-adrenergic receptor (adrb3) in bone marrow mesenchymal stem cells (MSCs), and promote MSC migration from perivascular regions to bone-forming units. An in vitro Transwell assay was here used to demonstrate that NE can inhibit stroma-derived factor-1 (SDF-1)-induced MSC migration and expression of the migration-related gene matrix metalloproteinase-2 (MMP-2) and downregulate that of the anti-migration gene tissue inhibitor of metalloproteinase-3 (TIMP-3). Knockdown of adrb3 using siRNA abolishes inhibition of MSC migration. An in vitro osteogenic assay was used to show that NE can inhibit the formation of MSC bone nodules and expression of the osteogenic marker genes alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor-2 (RUNX2), but knockdown of adrb3 by siRNA can abolish such inhibition of the osteogenic differentiation of MSCs. It is here concluded that sympathetic denervation-induced MSC mobilization in rat mandibular distraction osteogenesis is associated with inhibition of MSC migration and osteogenic differentiation by NE/adrb3 in vitro. These findings may facilitate understanding of the relationship of MSC mobilization and sympathetic nervous system across a wide spectrum of tissue regeneration processes.
PMCID: PMC4140837  PMID: 25144690
10.  Multi-Cultural Association of the Serotonin Transporter Gene (SLC6A4) with Substance Use Disorder 
Neuropsychopharmacology  2013;38(9):1737-1747.
A number of studies have reported associations between the serotonin transporter gene (SLC6A4) and alcohol, heroin, cocaine, or methamphetamine abuse. Other studies have yielded contrary results. There are a number of reasons for non-replication, including inadequate statistical power, population stratification, and poor phenotype definition. This study was to test the association using a meta-analytic approach across a variety of racial and ethnic populations. Using the genotype data of 55 studies (7999 cases, 8264 controls, and 676 families or parent-offspring trios) published in the past 15 years, we have conducted comprehensive meta-analyses to examine the associations of the 5-HTTLPR and STin2 polymorphisms with substance use disorder. The meta-analyses support the associations of 5-HTTLPR with alcohol, heroin, cocaine, and methamphetamine dependence and abuse (eg, the smallest P-values were 0.0058 with odds ratio (OR)=0.54 (0.35, 0.84); 0.0024 with OR=0.77 (0.66, 0.91); 0.018 with OR=1.38 (1.06, 1.81); and 0.028 with OR=0.46 (0.23, 0.92) for alcohol, heroin, cocaine, and methamphetamine dependence/abuse, respectively). When all the phenotypes are combined, the P-value was 0.0006 with OR=0.86 (0.78, 0.94) in the combined European, Asian, and Mexican populations and P-value was 0.0028 with OR=1.41 (1.13, 1.78) in the African populations. Evidence of significant associations was also identified in other subgroup analyses regarding differently combined substance and populations. The effect sizes of 5-HTTLPR were comparable among the European, Asian, and Mexican populations, however, the risk allele was more frequent in Asians than in Europeans and Mexicans. The opposite directions of risk allele in African population might be driven by the opposite directions of risk allele in cocaine dependence. This meta-analysis supports that the association of the SLC6A4 gene with substance use disorder varies depending on substances with different risk allele frequencies in the multi-cultural populations. Further studies using larger sample size are warranted.
PMCID: PMC3717550  PMID: 23518607
addiction & substance abuse; alcohol & alcoholism; behavioral science; genetic association; meta-analysis; serotonin; meta-analysis; association; serotonin transporter; addiction; common genetic risk
11.  Role of non-coding RNAs in pancreatic cancer: The bane of the microworld 
Our understanding of the mechanisms underlying the development of pancreatic cancer has been greatly advanced. However, the molecular events involved in the initiation and development of pancreatic cancer remain inscrutable. None of the present medical technologies have been proven to be effective in significantly improving early detection or reducing the mortality/morbidity of this disease. Thus, a better understanding of the molecular basis of pancreatic cancer is required for the identification of more effective diagnostic markers and therapeutic targets. Non-coding RNAs (ncRNAs), generally including microRNAs and long non-coding RNAs, have recently been found to be deregulated in many human cancers, which provides new opportunities for identifying both functional drivers and specific biomarkers of pancreatic cancer. In this article, we review the existing literature in the field documenting the significance of aberrantly expressed and functional ncRNAs in human pancreatic cancer, and discuss how oncogenic ncRNAs may be involved in the genetic and epigenetic networks regulating functional pathways that are deregulated in this malignancy, particularly of the ncRNAs’ role in drug resistance and epithelial-mesenchymal transition biological phenotype, with the aim of analyzing the feasibility of clinical application of ncRNAs in the diagnosis and treatment of pancreatic cancer.
PMCID: PMC4110572  PMID: 25071335
MicroRNAs; Long non-coding RNAs; Pancreatic cancer; Diagnosis; Treatment
12.  Associations of the 5-hydroxytryptamine (Serotonin) Receptor 1B Gene (HTR1B) with Alcohol, Cocaine, and Heroin Abuse 
Abnormal serotonergic pathways are implicated in numerous neuropsychiatric disorders including alcohol and drug dependence (abuse). The human 5-hydroxytryptamine (serotonin) receptor 1B, encoded by the HTR1B (5-HT1B) gene, is a presynaptic serotonin autoreceptor that plays an important role in regulating serotonin synthesis and release. Although there was evidence of associations of the HTR1B gene variants in the etiologies of substance use disorders, negative findings were also reported. To clarify the roles of commonly-reported single nucleotide polymorphisms (SNPs) of the HTR1B gene underlying alcohol and drug dependence (abuse), we performed a meta-analysis based on the available genotype data from individual candidate gene-based association studies. Evidence of association was found between the functional SNP -161A>T (rs130058) and alcohol, cocaine, and heroin dependence (e.g., P = 0.03 and odds ratio = 1.2 (1.02, 1.42) in the combined European, Asian, African, and Hispanic populations). SNP -261T>G (rs11568817) also showed evidence of association but with different directions in Europeans and non-Europeans (e.g., P = 0.0018 with odds ratio = 1.42 (1.14, 1.76) and P = 0.01 with odds ratio = 0.5 (0.3, 0.85), respectively). This meta-analysis supports the associations of HTR1B -261T>G and -161A>T with alcohol and drug abuse and further investigations are warranted in larger samples.
PMCID: PMC4089973  PMID: 23335468
Substance Use Disorder; Addiction; Meta-analysis; Association; Linkage Disequilibrium
13.  Rho/ROCK Signal Cascade Mediates Asymmetric Dimethylarginine-Induced Vascular Smooth Muscle Cells Migration and Phenotype Change 
BioMed Research International  2014;2014:683707.
Asymmetric dimethylarginine (ADMA) induces vascular smooth muscle cells (VSMCs) migration. VSMC phenotype change is a prerequisite of migration. RhoA and Rho-kinase (ROCK) mediate migration of VSMCs. We hypothesize that ADMA induces VSMC migration via the activation of Rho/ROCK signal pathway and due to VSMCs phenotype change. ADMA activates Rho/ROCK signal pathway that interpreted by the elevation of RhoA activity and phosphorylation level of a ROCK substrate. Pretreatment with ROCK inhibitor, Y27632 completely reverses the induction of ADMA on ROCK and in turn inhibits ADMA-induced VSMCs migration. When the Rho/ROCK signal pathway has been blocked by pretreatment with Y27632, the induction of ERK signal pathway by ADMA is completely abrogated. Elimination of ADMA via overexpression of dimethylarginine dimethylaminohydrolase 2 (DDAH2) and L-arginine both blocks the effects of ADMA on the activation of Rho/ROCK and extra cellular signal-regulated kinase (ERK) in VSMCs. The expression of differentiated phenotype relative proteins was reduced and the actin cytoskeleton was disassembled by ADMA, which were blocked by Y27632, further interpreting that ADMA inducing VSMCs migration via Rho/ROCK signal pathway is due to its effect on the VSMCs phenotype change. Our present study may help to provide novel insights into the therapy and prevention of atherosclerosis.
PMCID: PMC4119924  PMID: 25121106
Molecular cancer research : MCR  2011;9(10):1305-1318.
Oxidative stress caused by high levels of reactive oxygen species (ROS) has been correlated with prostate cancer (PCa) aggressiveness. Expression of membrane-type 1-matrix metalloproteinase (MT1-MMP), which has been implicated in cancer invasion and metastasis, is associated with advanced PCa. We demonstrate here that MT1-MMP plays a key role in eliciting oxidative stress in PCa cancer cells. Stable MT1-MMP expression in less invasive LNCaP prostate cancer cells with low endogenous MT1-MMP increased activity of ROS, whereas MT1-MMP knockdown in DU145 cells with high endogenous MT1-MMP decreased ROS. Expression of MT1-MMP increased oxidative DNA damage in LNCaP and in DU145 cells, indicating MT1-MMP-mediated induction of ROS caused oxidative stress. MT1-MMP expression promoted a more aggressive phenotype in LNCaP cells that was dependent on elaboration of ROS. Blocking ROS activity using the ROS scavenger, N-acetylcysteine (NAC), abrogated MT1-MMP-mediated increase in cell migration and invasion. MT1-MMP-expressing LNCaP cells displayed an enhanced ability to grow in soft agar that required increased ROS. Employing cells expressing MT1-MMP mutant cDNAs, we demonstrated that ROS activation entails cell surface MT1-MMP proteolytic activity. Induction of ROS in PCa cells expressing MT1-MMP required adhesion to extracellular matrix (ECM) proteins and was impeded by anti-β1 integrin antibodies. These results highlight a novel mechanism of malignant progression in PCa cells that involves β1 integrin-mediated adhesion, in concert with MT1-MMP proteolytic activity, to elicit oxidative stress and induction of a more invasive phenotype.
PMCID: PMC4013752  PMID: 21849471
MT1-MMP; reactive oxygen species (ROS); oxidative stress; cell invasion; prostate cancer
15.  7-difluoromethoxyl-5,4′-di-n-octyl genistein inhibits ovarian cancer stem cell characteristics through the downregulation of FOXM1 
Oncology Letters  2014;8(1):295-300.
7-Difluoromethoxyl-5,4′-di-n-octylgenistein (DFOG) is a novel synthetic genistein analogue that possesses anti-cancer activity in a variety of cancers, including ovarian cancer. The objective of the present study was to investigate whether DFOG inhibits the self-renewal capacity of ovarian cancer stem-like cells (OCSLCs) and to identify its potential mechanism of action. It was found that the sphere-forming cells (SFCs) of the SKOV3 cell line exhibited a self-renewal capacity and high tumorigenicity, indicating that they possessed the properties of ovarian cancer stem cells (OCSCs). It was also shown for the first time that DFOG preferentially inhibited proliferation, self-renewal capacity and expression of stem cell markers [cluster of differentiation (CD)133, CD44 and aldehyde dehydrogenase 1 (ALDH1)] in the SFCs derived from the SKOV3 cells. These effects were accompanied by the downregulation of forkhead box M1 (FOXM1) expression. Overexpression of FOXM1 rescued the DFOG-induced downregulation of FOXM1, CD133, CD44 and ALDH1 protein expression. It also inhibited the self-renewal capacity of the SFCs derived from the SKOV3 cells. Thus, DFOG appears to inhibit the characteristics of OCSLCs by downregulating FOXM1 expression.
PMCID: PMC4063643  PMID: 24959264
ovarian cancer; cancer stem cells; therapeutic action; 7-difluoromethoxyl-5; 4′-di-n-octylgenistein
16.  Feasibility of Low-Dose Contrast Medium High Pitch CT Angiography for the Combined Evaluation of Coronary, Head and Neck Arteries 
PLoS ONE  2014;9(3):e90268.
To evaluate the image quality and radiation dose of combined heart, head, and neck CT angiography (CTA) using prospectively electrocardiography (ECG)-triggered high-pitch spiral scan protocol, compared with single coronary CTA.
Materials and Methods
151 consecutive patients were prospectively included and randomly divided into three groups. Group 1 (n = 47) underwent combined heart, neck, and head CTA using prospectively ECG-triggered high-pitch spiral (Flash) scan protocol with a single-phase intravenous injection of iodinated contrast and saline flush; Group 2 (n = 51) underwent single coronary CTA with Flash scan protocol; and Group 3 (n = 53) underwent single coronary CTA with prospective sequence scan protocol. All patients were examined on a dual source CT (Definition FLASH). The image quality was determined for each CT study.
Patients of scanning protocol Group 1, 2, and 3 showed no significant differences in age, sex, heart rates, and BMI. Evaluation of coronary artery image quality showed comparable results in the three scanning protocol groups on a per patient-based analysis. In group 1, image quality was found to be sufficient to be diagnostic in all arterial segments of carotid arteries. The mean dose-length product (DLP) for group 1 was 256.3±24.5 mGy×cm and was significantly higher in comparison with group 2 (93.4±19.9 mGy×cm; p < 0.001). However, there was no significant difference of DLP between group 1 and group 3 (254.1±69.9 mGy×cm).
The combined heart, neck, and head arteries scan using prospectively electrocardiography (ECG)-triggered high-pitch spiral scan protocol in 1 single examination resulted in an excellent opacification of the aorta, the carotid arteries, and the coronary arteries and provided a good image quality with low radiation dose.
PMCID: PMC3940874  PMID: 24595301
17.  Insight into Insulin Secretion from Transcriptome and Genetic Analysis of Insulin-Producing Cells of Drosophila 
Genetics  2014;197(1):175-192.
Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to the hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and messenger RNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing β-cells of the mammalian pancreas. The genes in common encode ILPs and proteins that control insulin metabolism, storage, secretion, β-cell proliferation, and some not previously linked to insulin production or β-cell function. Among these novelties is unc-104, a kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.
PMCID: PMC4012477  PMID: 24558258
insulin; pancreas; Drosophila; Unc-104; kinesin; Rab1; Golgi; ER; RNA-seq; laser microdissection; transport
18.  Development of a High-Throughput Three-Dimensional Invasion Assay for Anti-Cancer Drug Discovery 
PLoS ONE  2013;8(12):e82811.
The lack of three-dimensional (3-D) high-throughput (HT) screening assays designed to identify anti-cancer invasion drugs is a major hurdle in reducing cancer-related mortality, with the key challenge being assay standardization. Presented is the development of a novel 3-D invasion assay with HT potential that involves surrounding cell-collagen spheres within collagen to create a 3-D environment through which cells can invade. Standardization was achieved by designing a tooled 96-well plate to create a precisely designated location for the cell-collagen spheres and by using dialdehyde dextran to inhibit collagen contraction, maintaining uniform size and shape. This permits automated readout for determination of the effect of inhibitory compounds on cancer cell invasion. Sensitivity was demonstrated by the ability to distinguish varying levels of invasiveness of cancer cell lines, and robustness was determined by calculating the Z-factor. A Z-factor of 0.65 was obtained by comparing the effects of DMSO and anti-β1-integrin antibody, an inhibitory reagent, on the invasion of Du145 cancer cells, suggesting this novel assay is suitable for large scale drug discovery. As proof of principle, the NCI Diversity Compound Library was screened against human invasive cancer cells. Nine compounds exhibiting high potency and low toxicity were identified, including DX-52-1, a compound previously reported to inhibit cell migration, a critical determinant of cancer invasion. The results indicate that this innovative HT platform is a simple, precise, and easy to replicate 3-D invasion assay for anti-cancer drug discovery.
PMCID: PMC3859626  PMID: 24349367
19.  Early Growth Response Protein 1 Promotes Restenosis by Upregulating Intercellular Adhesion Molecule-1 in Vein Graft 
Objectives. To verify the relationship between Egr-1 and vein graft restenosis and investigate the related mechanisms. Methods. Mouse vein graft models were established in Egr-1 knockout (KO) and wild-type (WT) mice. The vein grafts in the mice were taken for pathological examination and immunohistochemical analysis. The endothelial cells (ECs) were stimulated by using a computer-controlled cyclic stress unit. BrdU staining and PCR were used to detect ECs proliferation activity and Egr-1 and ICAM-1 mRNA expression, respectively. Western-blot analysis was also used to detect expression of Egr-1 and intercellular adhesion molecule-1 (ICAM-1) proteins. Results. The lumens of vein grafts in Egr-1 KO mice were wider than in WT mice. ECs proliferation after mechanical stretch stimulation was suppressed by Egr-1 knockout (P < 0.05). Both in vein grafts and ECs from WT mice after mechanical stretch stimulation, mRNA expression and protein of Egr-1 and ICAM-1 showed increases (P < 0.05). However, ICAM-1 expression was significantly suppressed in ECs from Egr-1 knockout mice (P < 0.05). Conclusions. Egr-1 may promote ECs proliferation and result in vein graft restenosis by upregulating the expression of ICAM-1. As a key factor of vein graft restenosis, it could be a target for the prevention of restenosis after CABG surgery.
PMCID: PMC3872240  PMID: 24386503
20.  8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of β-catenin 
AIM: To evaluate whether 8-bromo-7-methoxychrysin (BrMC), a synthetic analogue of chrysin, inhibits the properties of cancer stem cells derived from the human liver cancer MHCC97 cell line and to determine the potential mechanisms.
METHODS: CD133+ cells were sorted from the MHCC97 cell line by magnetic activated cell sorting, and amplified in stem cell-conditioned medium to obtain the enriched CD133+ sphere forming cells (SFCs). The stem cell properties of CD133+ SFCs were validated by the tumorsphere formation assay in vitro and the xenograft nude mouse model in vivo, and termed liver cancer stem cells (LCSCs). The effects of BrMC on LCSCs in vitro were evaluated by MTT assay, tumorsphere formation assay and transwell chamber assay. The effects of BrMC on LCSCs in vivo were determined using a primary and secondary xenograft model in Balb/c-nu mice. Expressions of the stem cell markers, epithelial-mesenchymal transition (EMT) markers and β-catenin protein were analyzed by western blotting or immunohistochemical analysis.
RESULTS: CD133+ SFCs exhibited stem-like cell properties of tumorsphere formation and tumorigenesis capacity in contrast to the parental MHCC97 cells. We found that BrMC preferentially inhibited proliferation and self-renewal of LCSCs (P < 0.05). Furthermore, BrMC significantly suppressed EMT and invasion of LCSCs. Moreover, BrMC could efficaciously eliminate LCSCs in vivo. Interestingly, we showed that BrMC decreased the expression of β-catenin in LCSCs. Silencing of β-catenin by small interfering RNA could synergize the inhibition of self-renewal of LCSCs induced by BrMC, while Wnt3a treatment antagonized the inhibitory effects of BrMC.
CONCLUSION: BrMC can inhibit the functions and characteristics of LCSCs derived from the liver cancer MHCC97 cell line through downregulation of β-catenin expression.
PMCID: PMC3837267  PMID: 24431896
Liver cancer; Cancer stem cell; 8-bromo-7-methoxychrysin; Self-renewal; β-catenin
21.  Propofol's effect on the sciatic nerve: Harmful or protective? 
Neural Regeneration Research  2013;8(27):2520-2530.
Propofol can inhibit the inflammatory response and reduce the secretion and harmful effects of astrocyte-derived proinflammatory cytokines. In this study, after propofol was injected into the injured sciatic nerve of mice, nuclear factor kappa B expression in the L4–6 segments of the spinal cord in the injured side was reduced, apoptosis was decreased, nerve myelin defects were alleviated, and the nerve conduction block was lessened. The experimental findings indicate that propofol inhibits the inflammatory and immune responses, decreases the expression of nuclear factor kappa B, and reduces apoptosis. These effects of propofol promote regeneration following sciatic nerve injury.
PMCID: PMC4145931  PMID: 25206562
neural regeneration; peripheral nerve injury; propofol; nuclear factor kappa B; sciatic nerve injury; inflammatory response; nerve conduction; immunosuppression; myelin sheath; grant-supported paper; neuroregeneration
22.  Plant-Based Assessment of Inherent Soil Productivity and Contributions to China’s Cereal Crop Yield Increase since 1980 
PLoS ONE  2013;8(9):e74617.
China’s food production has increased 6-fold during the past half-century, thanks to increased yields resulting from the management intensification, accomplished through greater inputs of fertilizer, water, new crop strains, and other Green Revolution’s technologies. Yet, changes in underlying quality of soils and their effects on yield increase remain to be determined. Here, we provide a first attempt to quantify historical changes in inherent soil productivity and their contributions to the increase in yield.
The assessment was conducted based on data-set derived from 7410 on-farm trials, 8 long-term experiments and an inventory of soil organic matter concentrations of arable land.
Results show that even without organic and inorganic fertilizer addition crop yield from on-farm trials conducted in the 2000s was significantly higher compared with those in the 1980s — the increase ranged from 0.73 to 1.76 Mg/ha for China’s major irrigated cereal-based cropping systems. The increase in on-farm yield in control plot since 1980s was due primarily to the enhancement of soil-related factors, and reflected inherent soil productivity improvement. The latter led to higher and stable yield with adoption of improved management practices, and contributed 43% to the increase in yield for wheat and 22% for maize in the north China, and, 31%, 35% and 22% for early and late rice in south China and for single rice crop in the Yangtze River Basin since 1980.
Thus, without an improvement in inherent soil productivity, the ‘Agricultural Miracle in China’ would not have happened. A comprehensive strategy of inherent soil productivity improvement in China, accomplished through combining engineering-based measures with biological-approaches, may be an important lesson for the developing world. We propose that advancing food security in 21st century for both China and other parts of world will depend on continuously improving inherent soil productivity.
PMCID: PMC3776784  PMID: 24058605
23.  Heme Oxygenase Gene Targeting to Adipocyte Attenuates Adiposity and Vascular Dysfunction in Mice Fed a High Fat Diet 
Hypertension  2012;60(2):467-475.
We examined the hypothesis that adipocyte dysfunction in mice fed a high fat (HF) diet can be prevented by lentiviral-mediated and adipocyte specific-targeting delivery of the human heme oxygenase-1 (aP2-HO-1). A bolus intracardial injection of aP2-HO-1 resulted in expression of human HO-1 for up to 9.5 months. Transduction of aP2-HO-1 increased human HO-1 expression in fat tissues without affecting murine HO-1. In mice fed a HF diet, aP2-HO-1 transduction attenuated the increases in body weight, blood glucose, blood pressure and inflammatory cytokines as well as the content of both visceral and subcutaneous fat. Transduction of aP2-HO-1 increased the numbers of adipocytes of small cell size (p<0.05), insulin sensitivity (p<0.05),adiponectin levels as well as vascular relaxation to acetylcholine compared to HF mice administered the aP2-Green Fluorescent Protein (aP2-GFP). Adipocytes of mice fed a HF diet expressed high levels of PPARγ, aP2, C/EBP and Wnt5b proteins and displayed marked increases in Peg1/Mest (p<0.03). Transduction of aP2-HO-1 lowered the elevated levels of these proteins and increased Shh, Wnt10b and β-catenin (p<0.05). Inhibition of HO activity by administration of tin mesoporphyrin (SnMP) to HF-fed mice transduced with the aP2-HO-1 reversed the decrease in Peg 1/Mest, TNFα and MCP-1 levels. Collectively, this novel study demonstrates that adipocyte-specific overexpression of HO-1 attenuates HF-mediated adiposity and vascular dysfunction, increases insulin sensitivity and improves adipocyte function by increasing adiponectin, Shh and WNT10b and decreasing inflammatory cytokines.
PMCID: PMC3423899  PMID: 22753217
HO-1; adiposity; Wnt 10b; Peg1/Mest; lentivirus
24.  Improved Myocardial Perfusion in Chronic Diabetic Mice by the Up-Regulation of pLKB1 and AMPK Signaling 
Journal of cellular biochemistry  2010;109(5):1033-1044.
Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up-regulating pAMPK-pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild-type and streptozotocin (STZ)-treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO-1, 3-nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time-dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L-NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK-3 expression. Cobalt protoporphyrin treatment to induce HO-1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up-regulation of HO-1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK-3, and peNOS levels and a decrease in myocardial superoxide and 3-nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes.
PMCID: PMC3723413  PMID: 20108250
Coronary Microcirculation; Diabetic Cardiomyopathy; Heme-Oxygenase-1; Endothelial Dysfunction; Adiponectin
25.  Correction: Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry 
PLoS ONE  2013;8(6):10.1371/annotation/47607b18-ed69-4a08-8619-60c39bd83a13.
PMCID: PMC3741025

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