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1.  TrAmplification of Human Dental Follicle Cells by piggyBac Transposon - Mediated Reversible Immortalization System 
PLoS ONE  2015;10(7):e0130937.
Dental follicle cells (DFCs) are the precursor cells of periodontium. Under certain differentiation conditions, DFCs can be induced to differentiate into chondrogenic, osteogenic and adipogenic cells. However, DFCs has limited lifespan in vitro, so it’s difficult to harvest enough cells for basic research and translational application. pMPH86 is a piggyBac transposon-mediated vector which contains SV40 T-Ag cassette that can be removed by flippase recognition target (FRT) recombinase. Here we demonstrated the pMPH86 can effectively amplify human DFCs through reversible immortalization. The immortalized DFCs (iDFCs) exhibit higher proliferate activity, which can be reversed to its original level before immortalization when deimmortalized by FLP recombinase. The iDFCs and deimmortalized DFCs (dDFCs) express most DFC markers and maintain multiple differentiation potential in vitro as they can be induced by BMP9 to differentiate into chondrogenic, osteogenic and adipogenic cells evidenced by gene expression and protein marker. We also proved telomerase activity of iDFCs are significantly increased and maintained at a high level, while the telomerase activity of primary DFCs was relatively low and decreased with every passage. After SV40 T-Ag was removed to deimmortalize the cells, telomerase activity was reduced to its original level before immortalization and decreased with passages just the same as primary DFCs. These results suggest that piggyBac immortalization system could be a potential strategy to amplify primary cells, which is critical for regenerative research and further clinical application.
doi:10.1371/journal.pone.0130937
PMCID: PMC4501788  PMID: 26172849
2.  Global Transcriptomic Profiling of Cardiac Hypertrophy and Fatty Heart Induced by Long-Term High-Energy Diet in Bama Miniature Pigs 
PLoS ONE  2015;10(7):e0132420.
A long-term high-energy diet affects human health and leads to obesity and metabolic syndrome in addition to cardiac steatosis and hypertrophy. Ectopic fat accumulation in the heart has been demonstrated to be a risk factor for heart disorders, but the molecular mechanism of heart disease remains largely unknown. Bama miniature pigs were fed a high-fat, high-sucrose diet (HFHSD) for 23 months. These pigs developed symptoms of metabolic syndrome and showed cardiac steatosis and hypertrophy with a greatly increased body weight (2.73-fold, P<0.01), insulin level (4.60-fold, P<0.01), heart weight (1.82-fold, P<0.05) and heart volume (1.60-fold, P<0.05) compared with the control pigs. To understand the molecular mechanisms of cardiac steatosis and hypertrophy, nine pig heart cRNA samples were hybridized to porcine GeneChips. Microarray analyses revealed that 1,022 genes were significantly differentially expressed (P<0.05, ≥1.5-fold change), including 591 up-regulated and 431 down-regulated genes in the HFHSD group relative to the control group. KEGG analysis indicated that the observed heart disorder involved the signal transduction-related MAPK, cytokine, and PPAR signaling pathways, energy metabolism-related fatty acid and oxidative phosphorylation signaling pathways, heart function signaling-related focal adhesion, axon guidance, hypertrophic cardiomyopathy and actin cytoskeleton signaling pathways, inflammation and apoptosis pathways, and others. Quantitative RT-PCR assays identified several important differentially expressed heart-related genes, including STAT3, ACSL4, ATF4, FADD, PPP3CA, CD74, SLA-8, VCL, ACTN2 and FGFR1, which may be targets of further research. This study shows that a long-term, high-energy diet induces obesity, cardiac steatosis, and hypertrophy and provides insights into the molecular mechanisms of hypertrophy and fatty heart to facilitate further research.
doi:10.1371/journal.pone.0132420
PMCID: PMC4498776  PMID: 26161779
3.  Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa 
PLoS ONE  2015;10(6):e0131755.
Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production.
doi:10.1371/journal.pone.0131755
PMCID: PMC4485900  PMID: 26121034
4.  Arsenic Primes Human Bone Marrow CD34+ Cells for Erythroid Differentiation 
Arsenic trioxide exhibits therapeutic effects on certain blood malignancies, at least partly by modulating cell differentiation. Previous in vitro studies in human hematopoietic progenitor cells have suggested that arsenic may inhibit erythroid differentiation. However, these effects were all observed in the presence of arsenic compounds, while the concomitant cytostatic and cytotoxic actions of arsenic might mask a prodifferentiating activity. To eliminate the potential impacts of the cytostatic and cytotoxic actions of arsenic, we adopted a novel protocol by pretreating human bone marrow CD34+ cells with a low, noncytotoxic concentration of arsenic trioxide, followed by assaying the colony forming activities in the absence of the arsenic compound. Bone marrow specimens were obtained from chronic myeloid leukemia patients who achieved complete cytogenetic remission. CD34+ cells were isolated by magnetic-activated cell sorting. We discovered that arsenic trioxide enhanced the erythroid colony forming activity, which was accompanied by a decrease in the granulomonocytic differentiation function. Moreover, in erythroleukemic K562 cells, we showed that arsenic trioxide inhibited erythrocyte maturation, suggesting that arsenic might have biphasic effects on erythropoiesis. In conclusion, our data provided the first evidence showing that arsenic trioxide could prime human hematopoietic progenitor cells for enhanced erythroid differentiation.
doi:10.1155/2015/751013
PMCID: PMC4480244  PMID: 26170775
5.  Hyperpigmentation Results in Aberrant Immune Development in Silky Fowl (Gallus gallus domesticus Brisson) 
PLoS ONE  2015;10(6):e0125686.
The Silky Fowl (SF) is known for its special phenotypes and atypical distribution of melanocytes among internal organs. Although the genes associated with melanocyte migration have been investigated substantially, there is little information on the postnatal distribution of melanocytes in inner organs and the effect of hyperpigmentation on the development of SF. Here, we analyzed melanocyte distribution in 26 tissues or organs on postnatal day 1 and weeks 2, 3, 4, 6, 10, and 23. Except for the liver, pancreas, pituitary gland, and adrenal gland, melanocytes were distributed throughout the body, primarily around blood vessels. Interaction between melanocytes and the tissue cells was observed, and melanin was transported by filopodia delivery through engulfed and internalized membrane-encapsulated melanosomes. SFs less than 10 weeks old have lower indices of spleen, thymus, and bursa of Fabricius than White Leghorns (WLs). The expression levels of interferon-γ and interlukin-4 genes in the spleen, and serum antibody levels against H5N1 and infectious bursal disease virus were lower in SF than in WL. We also found immune organ developmental difference between Black-boned and non-Black- boned chickens from SFs and WLs hybrid F2 population. However, degeneration of the thymus and bursa of Fabricius occurred later in SF than in WL after sexual maturity. Analysis of apoptotic cells and apoptosis-associated Bax and Bcl-2 proteins indicated that apoptosis is involved in degeneration of the thymus and bursa of Fabricius. Therefore, these results suggest that hyperpigmentation in SF may have a close relationship with immune development in SF, which can provide an important animal model to investigate the roles of melanocyte.
doi:10.1371/journal.pone.0125686
PMCID: PMC4457905  PMID: 26047316
6.  Impact of polymorphisms of the GGCX gene on maintenance warfarin dose in Chinese populations: Systematic review and meta-analysis 
Meta Gene  2015;5:43-54.
The meta-analysis was conducted to investigate the impact of gamma-glutamyl carboxylase (GGCX) on maintenance warfarin dose. 8 studies were included, focusing on the impact of GGCX single nucleotide polymorphisms (SNPs) on mean daily warfarin dose (MDWD). GGCX (rs699664; AA versus GG, GA versus GG, A versus GG) and GGCX (rs12714145; GA versus GG, AA versus GG, A versus GG) showed no significant differences on mean daily warfarin dose (MDWD). This meta-analysis was the first to report the relationship between GGCX SNPs and MDWD in Chinese populations. No evidence could be found in the relationship between SNPs of GGCX (rs699664 and rs12714145) and maintenance warfarin dose.
doi:10.1016/j.mgene.2015.05.003
PMCID: PMC4473094  PMID: 26106580
CI, confidence interval; SD, standard deviation; SNPs, single nucleotide polymorphisms; GGCX, gamma-glutamyl carboxylase; CYP2C9, cytochrome P450 complex subunit 2C9; VKORC1, vitamin K epoxide reductase complex subunit 1; CYP4F2, cytochrome P450 complex subunit 4F2; EPHX1, epoxide hydro-lase 1 INR, International Normalized Ratio; MDWD, mean daily warfarin dose; WMD, weight mean difference; Warfarin; GGCX; Gene polymorphisms; Systematic review; Meta-analysis; Chinese
7.  Study on Environmental Causes and SNPs of MTHFR, MS and CBS Genes Related to Congenital Heart Disease 
PLoS ONE  2015;10(6):e0128646.
Purpose
Congenital heart diseases (CHD) are among the most common birth defects in China. Environmental causes and folate metabolism changes may alter susceptibility to CHD. The aim of this study is to evaluate the relevant risk-factors of children with CHD and their mothers.
Methods
138 children with CHD and 207 normal children for controls were recruited. Their mothers were also enlisted in this study and interviewed following a questionnaire about their pregnant history and early pregnancy situation. Five single nucleotide polymorphisms (SNPs) in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS) and cystathionine β-synthase (CBS) of mothers and children were genotyped.
Results
There were significant differences in the gender of children, occupation of mothers, family history with CHD, history of abortion, history of adverse pregnancy, early pregnancy health, fetus during pregnancy, pesticide exposure and drug exposure in CHD group and control group ( P < 0.05). Logistic regression analyses showed that after adjustment for above factors, MTHFR rs1801131 were significantly associated with their offspring CHD risk in mothers. Compared with the mothers whose MTHFR were rs1801131 AA and AC genotypes, the mothers who got a mutation of MTHFR rs1801131 CC genotypes had a 267% increase in risk of given birth of a CHD children (OR=3.67,95%CI=1.12-12.05). Meanwhile, MTHFR rs1801131 were significantly associated with CHD susceptibility in children (OR = 1.42, 95% CI = 1.00-2.44 in additive model).
Conclusions
Besides mothers’ social and fertility characteristics, our results suggested that the genetic variants in folate metabolism pathway might be one of the most related risk-factors of CHD. MTHFR rs1801131 were identified as loci in Chinese population that were involved in CHD.
doi:10.1371/journal.pone.0128646
PMCID: PMC4452709  PMID: 26035828
8.  Toxic Effects of Maternal Zearalenone Exposure on Uterine Capacity and Fetal Development in Gestation Rats 
Reproductive Sciences  2014;21(6):743-753.
The objectives of this study were to determine the effects of high-dose and early gestational exposure to zearalenone (ZEN) in female Sprague-Dawley (SD) rats, to correlate the maternal uterus with the fetus, and to explore the development and malformation of fetuses. Pregnant female SD rats were fed diets containing 0.3, 48.5, 97.6, or 146.0 mg/kg ZEN on gestational days (GDs) 0 through 7. All the females survived until GD 20, at which point a cesarean section was performed to harvest the organs, blood, and fetuses. The results indicated that exposure to ZEN during early gestation can impact the maternal reproductive capability. Delayed fetal development was directly linked to maternal toxicity. The toxic effects of ZEN caused early deaths more frequently than late deaths, and the deleterious effects lasted through the end of pregnancy.
doi:10.1177/1933719113512533
PMCID: PMC4016724  PMID: 24357638
zearalenone; uterine capacity; developmental toxicity; teratogenesis; rat
9.  Urine-derived stem cells for potential use in bladder repair 
Engineered bladder tissues, created with autologous bladder cells seeded on biodegradable scaffolds, are being developed for use in patients who need cystoplasty. However, in individuals with organ damage from congenital disorders, infection, irradiation, or cancer, abnormal cells obtained by biopsy from the compromised tissue could potentially contaminate the engineered tissue. Thus, an alternative cell source for construction of the neo-organ would be useful. Although other types of stem cells have been investigated, autologous mesenchymal stem cells (MSCs) are most suitable to use in bladder regeneration. These cells are often used as a cell source for bladder repair in three ways - secreting paracrine factors, recruiting resident cells, and trans-differentiation, inducing MSCs to differentiate into bladder smooth muscle cells and urothelial cells. Adult stem cell populations have been demonstrated in bone marrow, fat, muscle, hair follicles, and amniotic fluid. These cells remain an area of intense study, as their potential for therapy may be applicable to bladder disorders. Recently, we have found stem cells in the urine and the cells are highly expandable, and have self-renewal capacity and paracrine properties. As a novel cell source, urine-derived stem cells (USCs) provide advantages for cell therapy and tissue engineering applications in bladder tissue repair because they originate from the urinary tract system. Importantly, USCs can be obtained via a noninvasive, simple, and low-cost approach and induced with high efficiency to differentiate into bladder cells.
doi:10.1186/scrt458
PMCID: PMC4055102  PMID: 25157812
10.  Cyclophilin J Is a Novel Peptidyl-Prolyl Isomerase and Target for Repressing the Growth of Hepatocellular Carcinoma 
PLoS ONE  2015;10(5):e0127668.
Cyclophilin J (CYPJ) is a new member of the peptidyl-prolyl cis/trans-isomerase (PPIase) identified with upregulated expression in human glioma. However, the biological function of CYPJ remained unclear. We aimed to study the role of CYPJ in hepatocellular carcinoma (HCC) carcinogenesis and its therapeutic potential. We determined the expression of CYPJ in HCC/adjacent normal tissues using Western blot, Northern blot and semi-quantitative RT-PCR, analyzed the biochemical characteristics of CYPJ, and resolved the 3D-structure of CYPJ/Cyclosporin A (CsA) complex. We also studied the roles of CYPJ in cell cycle, cyclin D1 regulation, in vitro and in vivo tumor growth. We found that CYPJ expression was upregulated in over 60% HCC tissues. The PPIase activity of CYPJ could be inhibited by the widely used immunosuppressive drug CsA. CYPJ was found expressed in the whole cell of HCC with preferential location at the cell nucleus. CYPJ promoted the transition of cells from G1 phase to S phase in a PPIase-dependent manner by activating cyclin D1 promoter. CYPJ overexpression accelerated liver cell growth in vitro (cell growth assay, colony formation) and in vivo (xenograft tumor formation). Inhibition of CYPJ by its inhibitor CsA or CYPJ-specific RNAi diminished the growth of liver cancer cells in vitro and in vivo. In conclusion, CYPJ could facilitate HCC growth by promoting cell cycle transition from G1 to S phase through the upregulation of cyclin D1. Suppression of CYPJ could repress the growth of HCC, which makes CYPJ a potential target for the development of new strategies to treat this malignancy.
doi:10.1371/journal.pone.0127668
PMCID: PMC4447340  PMID: 26020957
11.  Expansion on matrix deposited by nonchondrogenic urine stem cells strengthens repeated passage bone marrow stromal cells’ chondrogenic capacity 
Cell and tissue research  2014;356(2):391-403.
Human urine-derived stem cells (hUSCs) are a newly found stem cell with a potential for therapeutic application in urology. The aim of this study was to investigate whether hUSCs contributed to cartilage regeneration. Despite the characterization with multi-lineage differentiation capacities, in terms of osteogenesis, adipogenesis, and myogenesis, hUSCs did not show the ability to differentiate into chondrocytes. Human bone marrow stromal cells (hBMSCs) are a tissue-specific stem cell for endochondral bone formation; however, repeated passage hBMSCs have less capacity for chondrogenic differentiation. Our findings showed that extracellular matrix deposited by hUSCs (UECM) could greatly recharge repeated passage hBMSCs toward chondrogenic differentiation, which might be explained by trophic factors released by hUSCs that were immobilized in UECM. We also found that ECM from repeated passage hBMSCs (BECM) had a limited rejuvenation effect; the Wnt11-mediated noncanonical signaling pathway might be responsible for UECM-mediated hBMSC rejuvenation and subsequent chondrogenic differentiation. Our data indicated that commercially available UECM from young and healthy donors might provide a simple and promising approach for autologous hBMSC rejuvenation. This study also provides an excellent model to investigate the effect of trophic factors released by stem cells on tissue regeneration without interference by stem cell differentiation.
doi:10.1007/s00441-014-1801-4
PMCID: PMC4128485  PMID: 24705582
Decellularized stem cell matrix; Monolayer expansion; Urine-derived stem cell; Bone marrow stromal cell; Endochondral bone formation
12.  Lipid metabolism and inflammation modulated by Vitamin D in liver of diabetic rats 
Background
In recent years, much evidence suggested that vitamin D plays an important role in decreasing the risk of type 2 diabetes. The purpose of this study was to investigate whether 1, 25 (OH) 2D3 can modulate inflammation and lipid metabolism in type 2 diabetic rat liver.
Methods
Type 2 diabetes was induced in SD rat with high-fat and high-sugar diets and multiple low-dose streptozotocin. The levels of serum calcium, phosphorus, glucose, TC, TG, AST, ALT and hepatic TG were determined. H & E staining were performed to assess the effects of vitamin D treatment on pathological changes in the liver tissues. Immunohistology, real-time PCR and Western blot were used to evaluate the expressions of NF-κ B, MCP-1, ICAM-1, TGF-β1, PPAR-α and CPT-1.
Results
The administration of 1, 25 (OH) 2D3 reduced liver weight. Compared to DM rats, 1, 25 (OH) 2D3-treated DM rats had lower liver weight. Moreover, compared to healthy or 1, 25 (OH) 2D3-treated DM rats, DM rats had increased hepatic transcription factors (NF-κ B), monocyte chemoattractant protein −1 (MCP-1), intercellular adhesion molecule −1 (ICAM-1), transforming growth factor-β1 (TGF-β1) expressions, but had fewer hepatic PPAR- α and CPT-1 expressions.
Conclusions
1, 25 (OH) 2D3 significantly modulated the liver inflammation and lipid metabolism in diabetic rat models, which may be caused by its regulations on hepatic signaling NF-κ B pathway and PPAR- α.
doi:10.1186/s12944-015-0030-5
PMCID: PMC4406219  PMID: 25899686
Diabetes-induced liver injury; Vitamin D; NF-κ B; PPAR-α
13.  Association of Polymorphisms in HLA Antigen Presentation-Related Genes with the Outcomes of HCV Infection 
PLoS ONE  2015;10(4):e0123513.
Antigen-presentation genes play a vital role in the pathogenesis of HCV infection. However, the relationship of variants of these genes with spontaneous outcomes of HCV infection has not been fully investigated. To explore novel loci in the Chinese population, 34 tagging-SNPs in 9 candidate genes were genotyped for their associations with the outcomes of HCV infection. The distributions of different genotypes and haplotypes were compared among 773 HCV-negative controls, 246 subjects with HCV natural clearance, and 218 HCV persistent carriers recruited from hemodialysis patients and intravenous drug users. Our study implicated that TAP2, HLA-DOA, HLA-DOB, and tapasin loci were novel candidate regions for susceptibility to HCV infection and viral clearance in the Chinese population. Logistic regression analyses showed that TAP2 rs1800454 A (OR = 1.48, P = 0.002) and HLA-DOB rs2071469 G (OR = 1.23, P = 0.048) were significantly associated with increased susceptibility to establishment of HCV infection. However, high-risk behavior exposure and age were stronger predictors of HCV infection. Mutation of tapasin rs9277972 T (OR = 1.57, P =0.043) increased the risk of HCV chronicity, and HLA-DOA rs3128935 C (OR = 0.62, P = 0.019) increased the chance of viral resolution. With regards to the effect of rs3128925, interactions were found with high-risk behavior (P = 0.013) and age (P = 0.035). The risk effect of rs3128925 T for persistent HCV infection was higher in injecting drug users (vs. dialysis patients) and in subjects ≥ 40 years old (vs. < 40 years old).
doi:10.1371/journal.pone.0123513
PMCID: PMC4395248  PMID: 25874709
14.  Continuing Treatment with Salvia miltiorrhiza Injection Attenuates Myocardial Fibrosis in Chronic Iron-Overloaded Mice 
PLoS ONE  2015;10(4):e0124061.
Iron overload cardiomyopathy results from iron accumulation in the myocardium that is closely linked to iron-mediated myocardial fibrosis. Salvia miltiorrhiza (SM, also known as Danshen), a traditional Chinese medicinal herb, has been widely used for hundreds of years to treat cardiovascular diseases. Here, we investigated the effect and potential mechanism of SM on myocardial fibrosis induced by chronic iron overload (CIO) in mice. Kunming male mice (8 weeks old) were randomized to six groups of 10 animals each: control (CONT), CIO, low-dose SM (L-SM), high-dose SM (H-SM), verapamil (VRP) and deferoxamine (DFO) groups. Normal saline was injected in the CONT group. Mice in the other five groups were treated with iron dextran at 50 mg/kg per day intraperitoneally for 7 weeks, and those in the latter four groups also received corresponding daily treatments, including 3 g/kg or 6 g/kg of SM, 100 mg/kg of VRP, or 100 mg/kg of DFO. The iron deposition was estimated histologically using Prussian blue staining. Myocardial fibrosis was determined by Masson’s trichrome staining and hydroxyproline (Hyp) quantitative assay. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and protein expression levels of type I collagen (COL I), type I collagen (COL III), transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase-9 (MMP-9) were analyzed to investigate the mechanisms underlying the effects of SM against iron-overloaded fibrosis. Treatment of chronic iron-overloaded mice with SM dose-dependently reduced iron deposition levels, fibrotic area percentage, Hyp content, expression levels of COL I and COL III, as well as upregulated the expression of TGF- β1 and MMP-9 proteins in the heart. Moreover, SM treatment decreased MDA content and increased SOD activity. In conclusion, SM exerted activities against cardiac fibrosis induced by CIO, which may be attributed to its inhibition of iron deposition, as well as collagen metabolism and oxidative stress.
doi:10.1371/journal.pone.0124061
PMCID: PMC4388639  PMID: 25850001
15.  Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture 
PLoS ONE  2015;10(4):e0123276.
As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway.
doi:10.1371/journal.pone.0123276
PMCID: PMC4388686  PMID: 25849723
16.  Protective effects of calcitriol on diabetic nephropathy are mediated by down regulation of TGF-β1 and CIP4 in diabetic nephropathy rat 
Objective: To explore the protective effects of calcitriol on diabetic nephropathy by modulating the expressions of transforming growth factor-beta 1 (TGF-β1) and Cdc42 interacting protein-4 (CIP4). Methods: Streptozotocin-induced diabetic nephropathy rats (n=36) were randomly divided into control group (control-H, control-M, control-L) and calcitriol group (calcitriol-H, calcitriol-M, calcitriol-L). The expression of TGF-β1 gradually decreased in control-H, control-M and control-L subgroups by injection of different virus vectors. Peanut oil and calcitriol were given to control and calcitriol group, respectively. The expressions of TGF-β1 and CIP4 in kidney, the pathology, and the renal function and lipid profiles were compared between control and calcitriol treatment groups. Results: In the control group, the higher level of TGF-β1 was associated with more severe glomerular pathology (P<0.05). There is a positive correlation between the expression of CIP4 and TGF-β1. Control-H subgroup had significant more severe kidney disease, higher levels of cholesterol, triglyceride, blood glucose, blood urea nitrogen (BUN) and creatinine (Cr) than control-M and control-L subgroups. After calcitriol treatment, the expression of TGF-β1 and CIP4 were significantly decreased compared to the corresponding control subgroups (all P<0.05). Renal fibrosis and pathological changes were markedly improved. The levels of cholesterol, triglyceride, blood glucose, BUN and Cr were significantly reduced (P<0.05). Conclusion: Calcitriol may protect diabetic nephropathy from fibrosis via inhibition of TGF-β1 and CIP4.
PMCID: PMC4466921  PMID: 26097534
Diabetic nephropathy; calcitriol; transforming growth factor beta 1 (TGF-β1); Cdc42 interacting protein-4 (CIP4)
17.  Abbreviated Half-Lives and Impaired Fuel Utilization in Carnitine Palmitoyltransferase II Variant Fibroblasts 
PLoS ONE  2015;10(3):e0119936.
Carnitine palmitoyltransferase II (CPT II) deficiency is one of the most common causes of fatty acid oxidation metabolism disorders. However, the molecular mechanism between CPT2 gene polymorphisms and metabolic stress has not been fully clarified. We previously reported that a number of patients show a thermal instable phenotype of compound hetero/homozygous variants of CPT II. To understand the mechanism of the metabolic disorder resulting from CPT II deficiency, the present study investigated CPT II variants in patient fibroblasts, [c.1102 G>A (p.V368I)] (heterozygous), [c.1102 G>A (p.V368I)] (homozygous), and [c.1055 T>G (p.F352C)] (heterozygous) + [c.1102 G>A (p.V368I)] (homozygous) compared with fibroblasts from healthy controls. CPT II variants exerted an effect of dominant negative on the homotetrameric proteins that showed thermal instability, reduced residual enzyme activities and a short half-life. Moreover, CPT II variant fibroblasts showed a significant decrease in fatty acid β-oxidation and adenosine triphosphate generation, combined with a reduced mitochondrial membrane potential, resulting in cellular apoptosis. Collectively, our data indicate that the CPT II deficiency induces an energy crisis of the fatty acid metabolic pathway. These findings may contribute to the elucidation of the genetic factors involved in metabolic disorder encephalopathy caused by the CPT II deficiency.
doi:10.1371/journal.pone.0119936
PMCID: PMC4364069  PMID: 25781464
18.  A preliminary investigation of EZSCAN™ screening for impaired glucose tolerance and diabetes in a patient population 
EZSCAN™ is a non-invasive technology that evaluates sweat gland dysfunction using electrochemical skin conductance measurements, providing an opportunity to determine the risk of impaired glucose tolerance (IGT) and diabetes mellitus (DM). This study was conducted with the aims of detecting IGT and DM and investigating the efficacy and cut-off points of the EZSCAN test in a patient population. The traditional serum and plasma glucose tests were used as comparators. In this cross-sectional study, 270 previously undiagnosed patients (180 women and 90 men) with a high risk of glucose metabolism disorders (≥45 years old) were enrolled. All patients underwent an oral glucose tolerance test (OGTT) and hemoglobin A1c (HbA1c), fasting plasma glucose (FPG) and EZSCAN tests. Forty (14.8%) patients had newly diagnosed DM (NDM), 79 (29.3%) had IGT and 151 (55.9%) had normal glucose tolerance. The EZSCAN values of these groups were 48±11, 47±11 and 34±13%, respectively. For all patients, the correlation coefficient of EZSCAN was 0.462 with the OGTT (P<0.001), 0.182 with the FPG test (P<0.001) and 0.379 with the HbA1c test (P<0.001). The EZSCAN cut-off point for the detection of IGT was 37% [sensitivity, 82%; specificity, 62%; area under the curve (AUC), 0.778], and the cut-off point for NDM was 50% (sensitivity, 53%; specificity, 59%; AUC, 0.528). This study demonstrated that the non-invasive EZSCAN system is an effective screening tool for the detection of glucose dysfunction in the population tested, and that its performance in detecting previously undiagnosed IGT is superior to its performance in detecting DM.
doi:10.3892/etm.2015.2358
PMCID: PMC4471801  PMID: 26136878
EZSCAN™; impaired glucose tolerance; diabetes; Chinese
19.  Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery 
Stem cell research  2013;12(2):467-480.
The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.
doi:10.1016/j.scr.2013.12.004
PMCID: PMC3966181  PMID: 24434629
Nuclear reprogramming; Induced pluripotent stem cells; Myocytes; Cardiac; Muscular Dystrophy; Duchenne; Cell differentiation
20.  Cell-Based Screening Identifies the Active Ingredients from Traditional Chinese Medicine Formula Shixiao San as the Inhibitors of Atherosclerotic Endothelial Dysfunction 
PLoS ONE  2015;10(2):e0116601.
In this study, we performed a phenotypic screening in human endothelial cells exposed to oxidized low density lipoprotein (an in vitro model of atherosclerotic endothelial dysfunction) to identify the effective compounds in Shixiao San. After investigating the suitability and reliability of the cell-based screening method using atorvastatin as the positive control drug, this method was applied in screening Shixiao San and its extracts. The treatment of n-butanol fraction on endothelial cells exhibited stronger healing effects against oxidized low density lipoprotein-induced insult when compared with other fractions. Cell viability, the level of nitric oxide, endothelial nitric oxide synthase and endothelin-1 were measured, respectively. The assays revealed n-butanol fraction significantly elevated the survival ratio of impaired cells in culture. In parallel, n-butanol fraction exhibited the highest inhibition of inflammation. The generation of prostaglandin-2 and adhesion molecule (soluble intercellular adhesion molecule-1) was obviously declined. Furthermore, n-butanol fraction suppressed the production of reactive oxygen species and malondialdehyde, and restored the activity of superoxide dismutase. Compounds identification of the n-butanol fraction was carried out by ultra high liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. The active ingredients including quercetin-3-O-(2G-α-l-rhamnosyl)-rutinoside, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside revealed the ability of anti-atherosclerosis after exposing on endothelial cells. The current work illustrated the pharmacology effect of Shixiao San and clearly indicated the major active components in Shixiao San. More importantly, the proposed cell-based screening method might be particularly suitable for fast evaluating the anti-atherosclerosis efficacy of Traditional Chinese Medicines and screening out the interesting ingredients of Traditional Chinese Medicines.
doi:10.1371/journal.pone.0116601
PMCID: PMC4336328  PMID: 25699522
21.  Dysbiosis gut microbiota associated with inflammation and impaired mucosal immune function in intestine of humans with non-alcoholic fatty liver disease 
Scientific Reports  2015;5:8096.
Non-alcoholic fatty liver disease (NAFLD) has recently been considered to be under the influence of the gut microbiota, which might exert toxic effects on the human host after intestinal absorption and delivery to the liver via the portal vein. In this study, the composition of the gut microbiota in NAFLD patients and healthy subjects was determined via 16S ribosomal RNA Illumina next-generation sequencing. Among those taxa displaying greater than 0.1% average abundance in all samples, five genera, including Alistipes and Prevotella, were significantly more abundant in the gut microbiota of healthy subjects compared to NAFLD patients. Alternatively, Escherichia, Anaerobacter, Lactobacillus and Streptococcus were increased in the gut microbiota of NAFLD patients compared to healthy subjects. In addition, decreased numbers of CD4+ and CD8+ T lymphocytes and increased levels of TNF-α, IL-6 and IFN-γ were detected in the NAFLD group compared to the healthy group. Furthermore, irregularly arranged microvilli and widened tight junctions were observed in the gut mucosa of the NAFLD patients via transmission electron microscopy. We postulate that aside from dysbiosis of the gut microbiota, gut microbiota-mediated inflammation of the intestinal mucosa and the related impairment in mucosal immune function play an important role in the pathogenesis of NAFLD.
doi:10.1038/srep08096
PMCID: PMC4314632  PMID: 25644696
22.  Facile synthesis of fluorescent Au/Ce nanoclusters for high-sensitive bioimaging 
Background
Tumor-target fluorescence bioimaging is an important means of early diagnosis, metal nanoclusters have been used as an excellent fluorescent probe for marking tumor cells due to their targeted absorption. We have developed a new strategy for facile synthesis of Au/Ce nanoclusters (NCs) by doping trivalent cerium ion into seed crystal growth process of gold. Au/Ce NCs have bright fluorescence which could be used as fluorescent probe for bioimaging.
Results
In this study, we synthesized fluorescent Au/Ce NCs through two-step hydrothermal reaction. The concentration range of 25–350 μM, Au/Ce NCs have no obvious cell cytotoxicity effect on HeLa, HepG2 and L02 cells. Furthermore, normal cells (L02) have no obvious absorption of Au/Ce NCs. Characterization of synthesized Au/Ce NCs was done by using TEM, EDS and XPS. Then these prepared Au/Ce NCs were applied for in vitro/in vivo tumor-target bioimaging due to its prolonged fluorescence lifetime and bright luminescence properties.
Conclusions
The glutathione stabilized Au/Ce NCs synthesized through hydrothermal reaction possess stable and bright fluorescence that can be readily utilized for high sensitive fluorescence probe. Our results suggest that Au/Ce NCs are useful candidate for in vitro/in vivo tumor bioimaging in potential clinical application.
doi:10.1186/s12951-015-0071-y
PMCID: PMC4320607  PMID: 25643754
Fluorescence bioimaging; Au/Ce nanoclusters; Probe; Tumor
23.  The BRCA1/2-directed miRNA signature predicts a good prognosis in ovarian cancer patients with wild-type BRCA1/2 
Oncotarget  2014;6(4):2397-2406.
Ovarian cancer patients carrying alterations (i.e., germline mutations, somatic mutations, hypermethylations and/or deletions) of BRCA1 or BRCA2 (BRCA1/2) have a better prognosis than BRCA1/2 alteration non-carriers. However, patients with wild-type BRCA1/2 may also have a favorable prognosis as a result of other mechanisms that remain poorly elucidated, such as the deregulation of miRNAs. We therefore sought to identify BRCA1/2-directed miRNA signatures that have prognostic value in ovarian cancer patients with wild-type BRCA1/2 and study how the deregulation of miRNAs impacts the prognosis of patients treated with platinum-based chemotherapy. By analyzing multidimensional datasets of ovarian cancer patients from the TCGA data portal, we identified three miRNAs (hsa-miR-146a, hsa-miR-148a and hsa-miR-545) that target BRCA1/2 and were associated with overall survival and progression-free survival in patients with wild-type BRCA1/2. By analyzing the expression profiles and Gene Ontology functional enrichment, we found that carriers of BRCA1/2 alterations and patients with miRNA deregulation shared a common mechanism, regulation of the DNA repair-related pathways, that affects the prognosis of ovarian cancer patients. Our work highlights that a proportion of patients with wild-type BRCA1/2 ovarian cancers benefit from platinum-based chemotherapy and that the patients who benefit could be predicted from BRCA1/2-directed miRNA signature.
PMCID: PMC4385859  PMID: 25537514
miRNAs; BRCA1; BRCA2; Prognosis; Ovarian Cancer
24.  Metformin and lung cancer risk of patients with type 2 diabetes mellitus: A meta-analysis 
Biomedical Reports  2015;3(2):235-241.
The association between metformin and the lung cancer risk of patients with type 2 diabetes mellitus (T2DM) remains controversial. Therefore, the present meta-analysis on epidemiological studies was performed to explore this issue. A comprehensive literature search was conducted for all the potential studies addressing metformin use and lung cancer risk by utilizing Pubmed, CBM and ISI Web of Science using the Mesh terms: ‘Metformin,’ or ‘biguanides’ and ‘lung cancer,’ or ‘neoplasms’. The reference lists were also inspected. Eight observational studies, including 17,997 lung cancer patients, were eventually selected, which contained seven case-control and one cohort study. Compared to other antidiabetic agents, metformin was significantly associated with the 16% reduction of lung cancer risk in type 2 diabetic patients [relative risk (RR)=0.84; 95% confidence interval (95% CI), 0.73-0.97]. In the sensitivity analysis by separately excluding the study with a high weight or lower quality, the results did not materially change. Subsequently, subgroup analysis was performed on the type of study design, unadjusted or adjusted hazard ratio, quality of enrolled studies, duration of treatment, country and control drugs. The magnitude of lung cancer risk reduction was strengthened when compared to sulfonylureas (RR=0.79; 95% CI, 0.83-0.9), without significant heterogeneity (Q-value=2.98, P=0.085). In conclusion, the present analysis supported that the use of metformin significantly decreased the risk of lung cancer among patients with T2DM. However, further studies are required to confirm these findings.
doi:10.3892/br.2015.417
PMCID: PMC4448013  PMID: 26075077
metformin; type 2 diabetes mellitus; lung cancer; risk; meta-analysis
25.  Microinjection of calcitonin in midbrain periaqueductal gray attenuates hyperalgesia in a chronic constriction injury rat model 
Objective(s):
As heat, pain is one of the most common clinical symptoms. Generally, calcitonin (CT) is prescribed as an analgesic agent for the treatment of pain, especially for the pain caused by osteoporosis or primary and metastatic bone tumor. However, the detailed mechanism remains unknown.
Materials and Methods:
In this study, chronic constriction injury (CCI) rat model was created, and hot plate test and von frey filaments test were employed to evaluate thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT), respectively. Immunohistochemistry staining and western blot analyses were used to assess the distribution and expression of calcitonin receptor (CT-R) in the midbrain periaqueductal gray (PAG), which was a pivotal site in the modulatory system for the endogenous pain.
Results:
The TWL and MWT before the surgery (19.6±1.19 sec) were significantly longer than that at day 2 (12.5±1.60 sec), and day 14 (7.75±0.89 sec) (P< 0.01; P< 0.01), respectively. The TWL and MWT at day 14 were significantly increased compared to that at day 7 after microinjection of salmon calcitonin (sCT) with different doses. Interestingly, the expression of CT-R was up-regulated in neuropathic pain. Furthermore, the expression of CT-R was significantly up-regulated and algesia was remarkably relieved when CT was microinjected into PAG.
Conclusion:
These results suggested that an increased CT-R might be associated with hyperalgesia in CCI rat, and CT had a potent antinociceptive effect by the up-regulation of CT-R in the PAG. Thus, it might provide a potential approach for the treatment of bone pain.
PMCID: PMC4366746  PMID: 25810879
Analgesia; Calcitonin; Calcitonin receptor; Pain; Periaqueductal gray

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