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2.  Conditionally Immortalized Mouse Embryonic Fibroblasts Retain Proliferative Activity without Compromising Multipotent Differentiation Potential 
PLoS ONE  2012;7(2):e32428.
Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications.
PMCID: PMC3285668  PMID: 22384246
3.  Insulin-like Growth Factor 2 (IGF-2) Potentiates BMP-9-Induced Osteogenic Differentiation and Bone Formation 
Journal of Bone and Mineral Research  2010;25(11):2447-2459.
Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self-renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP-9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin-like growth factor 2 (IGF-2) on BMP-9-induced bone formation. We have found that endogenous IGF-2 expression is low in MSCs. Expression of IGF-2 can potentiate BMP-9-induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF-2 has been shown to augment BMP-9-induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF-2 enhances BMP-9-induced endochondral ossification, whereas IGF-2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF-2 effect on BMP-9-induced ALP activity and matrix mineralization. Mechanistically, IGF-2 is further shown to enhance the BMP-9-induced BMPR-Smad reporter activity and Smad1/5/8 nuclear translocation. PI3-kinase (PI3K) inhibitor LY294002 abolishes the IGF-2 potentiation effect on BMP-9-mediated osteogenic signaling and can directly inhibit BMP-9 activity. These results demonstrate that BMP-9 crosstalks with IGF-2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP-9 and IGF-2 may be explored as an effective bone-regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture. © 2010 American Society for Bone and Mineral Research.
PMCID: PMC3179288  PMID: 20499340
BMP-9; bone formation; fracture healing; IGF-2; osteoblastic differentiation
4.  The CCN proteins: important signaling mediators in stem cell differentiation and tumorigenesis 
Histology and histopathology  2010;25(6):795-806.
The CCN proteins contain six members, namely CCN1 to CCN6, which are small secreted cysteine-rich proteins. The CCN proteins are modular proteins, containing up to four functional domains. Many of the CCN members are induced by growth factors, cytokines, or cellular stress. The CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues. The CCN proteins can integrate and modulate the signals of integrins, BMPs, VEGF, Wnts, and Notch. The involvement of integrins in mediating CCN signaling may provide diverse context-dependent responses in distinct cell types. CCN1 and CCN2 play an important role in development, angiogenesis and cell adhesion, whereas CCN3 is critical to skeletal and cardiac development. CCN4, CCN5 and CCN6 usually inhibit cell growth. Mutations of Ccn6 are associated with the progressive pseudorheumatoid dysplasia and spondyloepiphyseal dysplasia tarda. In stem cell differentiation, CCN1, CCN2, and CCN3 play a principal role in osteogenesis, chondrogenesis, and angiogenesis. Elevated expression of CCN1 is associated with more aggressive phenotypes of human cancer, while the roles of CCN2 and CCN3 in tumorigenesis are tumor type-dependent. CCN4, CCN5 and CCN6 function as tumor suppressors. Although CCN proteins may play important roles in fine-tuning other major signaling pathways, the precise function and mechanism of action of these proteins remain undefined. Understanding of the biological functions of the CCN proteins would not only provide insight into their roles in numerous cellular processes but also offer opportunities for developing therapeutics by targeting CCN functions.
PMCID: PMC2922104  PMID: 20376786
CCN family; Chondrogenesis; Osteogenesis; Stem Cell Differentiation; Tumorigenesis
5.  Defective Osteogenic Differentiation in the Development of Osteosarcoma 
Sarcoma  2011;2011:325238.
Osteosarcoma (OS) is associated with poor prognosis due to its high incidence of metastasis and chemoresistance. It often arises in areas of rapid bone growth in long bones during the adolescent growth spurt. Although certain genetic conditions and alterations increase the risk of developing OS, the molecular pathogenesis is poorly understood. Recently, defects in differentiation have been linked to cancers, as they are associated with high cell proliferation. Treatments overcoming these defects enable terminal differentiation and subsequent tumor inhibition. OS development may be associated with defects in osteogenic differentiation. While early regulators of osteogenesis are unable to bypass these defects, late osteogenic regulators, including Runx2 and Osterix, are able to overcome some of the defects and inhibit tumor propagation through promoting osteogenic differentiation. Further understanding of the relationship between defects in osteogenic differentiation and tumor development holds tremendous potential in treating OS.
PMCID: PMC3061279  PMID: 21437219
6.  Mesenchymal Progenitor Cells and Their Orthopedic Applications: Forging a Path towards Clinical Trials 
Stem Cells International  2010;2010:519028.
Mesenchymal progenitor cells (MPCs) are nonhematopoietic multipotent cells capable of differentiating into mesenchymal and nonmesenchymal lineages. While they can be isolated from various tissues, MPCs isolated from the bone marrow are best characterized. These cells represent a subset of bone marrow stromal cells (BMSCs) which, in addition to their differentiation potential, are critical in supporting proliferation and differentiation of hematopoietic cells. They are of clinical interest because they can be easily isolated from bone marrow aspirates and expanded in vitro with minimal donor site morbidity. The BMSCs are also capable of altering disease pathophysiology by secreting modulating factors in a paracrine manner. Thus, engineering such cells to maximize therapeutic potential has been the focus of cell/gene therapy to date. Here, we discuss the path towards the development of clinical trials utilizing BMSCs for orthopaedic applications. Specifically, we will review the use of BMSCs in repairing critical-sized defects, fracture nonunions, cartilage and tendon injuries, as well as in metabolic bone diseases and osteonecrosis. A review of of the United States National Institute of Health was performed, and ongoing clinical trials will be discussed in addition to the sentinel preclinical studies that paved the way for human investigations.
PMCID: PMC3017936  PMID: 21234334
7.  Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma 
PLoS ONE  2010;5(12):e14182.
Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.
Methodology/Principal Findings
Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma.
Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors.
PMCID: PMC2995727  PMID: 21152068
8.  Mesenchymal stem cells: Molecular characteristics and clinical applications 
World Journal of Stem Cells  2010;2(4):67-80.
Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.
PMCID: PMC3097925  PMID: 21607123
Mesenchymal stem cells; Bone marrow stem cell; Mesenchymal stromal cell; Autoimmune disease; Cell-based therapy; Autologous transplant; Therapeutic application
9.  Retinoic Acids Potentiate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells 
PLoS ONE  2010;5(7):e11917.
As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs).
Methodology/Principal Findings
Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARα and RXRα into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRα or RARα significantly increases trabecular bone and osteoid matrix formation.
Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.
PMCID: PMC2912873  PMID: 20689834
10.  A Comprehensive Analysis of the Dual Roles of BMPs in Regulating Adipogenic and Osteogenic Differentiation of Mesenchymal Progenitor Cells 
Stem Cells and Development  2008;18(4):545-558.
Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARγ2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARγ2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARγ2 knockdown or mouse embryonic fibroblasts derived from PPARγ2−/− mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARγ2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.
PMCID: PMC3132950  PMID: 18616389
11.  Therapeutic Implications of PPARγ in Human Osteosarcoma 
PPAR Research  2010;2010:956427.
Osteosarcoma (OS) is the most common nonhematologic malignancy of bone in children and adults. Although dysregulation of tumor suppressor genes and oncogenes, such as Rb, p53, and the genes critical to cell cycle control, genetic stability, and apoptosis have been identified in OS, consensus genetic changes that lead to OS development are poorly understood. Disruption of the osteogenic differentiation pathway may be at least in part responsible for OS tumorigenesis. Current OS management involves chemotherapy and surgery. Peroxisome proliferator-activated receptor (PPAR) agonists and/or retinoids can inhibit OS proliferation and induce apoptosis and may inhibit OS growth by promoting osteoblastic terminal differentiation. Thus, safe and effective PPAR agonists and/or retinoid derivatives can be then used as adjuvant therapeutic drugs for OS therapy. Furthermore, these agents have the potential to be used as chemopreventive agents for the OS patients who undergo the resection of the primary bone tumors in order to prevent local recurrence and/or distal pulmonary metastasis.
PMCID: PMC2825651  PMID: 20182546
12.  Transcription Factor Ets1 Regulates Expression of Thioredoxin-Interacting Protein and Inhibits Insulin Secretion in Pancreatic β-Cells 
PLoS ONE  2014;9(6):e99049.
Long-term activation of extracellular-regulated kinase (ERK1/2) pathway has been shown to cause glucotoxicity and inhibit insulin gene expression in β-cells. Transcription factor Ets1 is activated by ERK1/2-mediated phosphorylation at the Thr38 residue. We hypothesize that Ets1 plays an important role in mediating ERK1/2 induced glucotoxicity in β-cells. We determined the role of Ets1 in Min6 cells and isolated mouse islets using overexpression and siRNA mediated knockdown of Ets1. The results show that Ets1 was localized in insulin-staining positive cells but not in glucagon-staining positive cells. Overexpression of Ets1 reduced glucose-stimulated insulin secretion in primary mouse islets. Overexpression of Ets1 in Min6 β-cells and mouse islets increased expression of thioredoxin-interacting protein (TXNIP). Conversely, knockdown of Ets1 by siRNA reduced expression of TXNIP in Min6 cells. Ets1 was associated with the txnip promoter in min6 cells and transfection of 293 cells with Ets1 and p300 synergistically increased txnip promoter reporter activity. Moreover, overexpression of Ets1 inhibited Min6 cell proliferation. Our results suggest that Ets1, by promoting TXNIP expression, negatively regulates β-cell function. Thus, over-activation of Ets1 may contribute to diet-induced β-cell dysfunction.
PMCID: PMC4045976  PMID: 24897113
13.  Accumulated hippocampal formaldehyde induces age-dependent memory decline 
Age  2012;35(3):583-596.
Aging is an important factor in memory decline in aged animals and humans and in Alzheimer’s disease and is associated with the impairment of hippocampal long-term potentiation (LTP) and down-regulation of NR1/NR2B expression. Gaseous formaldehyde exposure is known to induce animal memory loss and human cognitive decline; however, it is unclear whether the concentrations of endogenous formaldehyde are elevated in the hippocampus and how excess formaldehyde affects LTP and memory formation during the aging process. In the present study, we report that hippocampal formaldehyde accumulated in memory-deteriorating diseases such as age-related dementia. Spatial memory performance was gradually impaired in normal Sprague–Dawley rats by persistent intraperitoneal injection with formaldehyde. Furthermore, excess formaldehyde treatment suppressed the hippocampal LTP formation by blocking N-methyl-d-aspartate (NMDA) receptor. Chronic excess formaldehyde treatment over a period of 30 days markedly decreased the viability of the hippocampus and down-regulated the expression of the NR1 and NR2B subunits of the NMDA receptor. Our results indicate that excess endogenous formaldehyde is a critical factor in memory loss in age-related memory-deteriorating diseases.
PMCID: PMC3636394  PMID: 22382760
Alzheimer’s disease (AD); Aging; Endogenous formaldehyde; Long-term potentiation (LTP); Long-term memory (LTM); NMDA receptor
14.  Yes-associated protein (YAP) increases chemosensitivity of hepatocellular carcinoma cells by modulation of p53 
Cancer Biology & Therapy  2013;14(6):511-520.
The yes-associated protein (YAP) transcription co-activator has been reported either as an oncogene candidate or a tumor suppressor. Liver tissue chips revealed that about 51.4% human hepatocellular carcinoma (HCC) samples express YAP and 32.9% HCC samples express phosphorylated YAP. In this study, we found that chemotherapy increased YAP protein expression and nuclear translocation in HepG2 cells, as well as p53 protein expression and nuclear translocation. However, little is known about YAP functions during chemotherapy. Our results show that overexpression of YAP increases chemosensitivity of HepG2 cells during chemotherapy. Dominant negative transfection of Flag-S94A (TEAD binding domain mutant) or Flag-W1W2 (WW domain mutant) to HepG2 cells decreases p53 expression/ nuclear translocation and chemosensitivity when compared with control HepG2 cells. Furthermore, rescue transfection of Flag-5SA-S94A or Flag-5SA-W1W2, respectively to HepG2 cells regains p53 expression/nuclear translocation and chemosensitivity. These results indicate that YAP promotes chemosensitivity by modulating p53 during chemotherapy and both TEAD and WW binding domains are required for YAP-mediated p53 function. ChIP assay results also indicated that YAP binds directly to the p53 promoter to improve its expression. In addition, p53 could positively feedback YAP expression through binding to the YAP promoter. Taken together, our current data indicate that YAP functions as a tumor suppressor that enhances apoptosis by modulating p53 during chemotherapy.
PMCID: PMC3813567  PMID: 23760493
YAP; p53; hepatocellular carcinoma
15.  (−)-Epigallocatechin-3-gallate induces Du145 prostate cancer cell death via downregulation of inhibitor of DNA binding 2, a dominant negative helix-loop-helix protein 
Cancer science  2009;101(3):707-712.
(−)-Epigallocatechin-3-gallate (EGCG) is one of the major polyphenol components in green tea. It effectively induces apoptosis in prostate cancer cells. The anticancer effect of this reagent is appealing because it is a natural component of a popular daily beverage that has proven harmless for thousands of years, making it a good candidate chemopreventive agent. EGCG suppresses cell growth and causes cell death, but the mechanisms are not well characterized, especially in androgen-independent prostate cancer cells. In the present study, using Affymetrix genechip Hu133 2.0, we analyzed the gene expression patterns of the androgen-independent prostate cancer cell line Du145, treated with or without EGCG, and found 40 genes whose expression levels were altered (>twofold, either upregulated or downregulated, P < 0.01) upon treatment with EGCG. These gene products are involved in the functions of transcription, RNA processing, protein folding, phosphorylation, protein degradation, cell motility, and ion transport. Among them, inhibitor of DNA binding 2 (ID2), known as a dominant anti-retinoblastoma (Rb) helix-loop-helix protein, was found to be downregulated fourfold by EGCG treatment. Forced expression of ID2 in Du145 cells reduced apoptosis and increased cell survival in the presence of EGCG, and knockdown ID2 expression in Du145 cells using a morpholino oligonucleotide specific for ID2 mimicked the apoptosis effect generated by EGCG treatment, although it was milder. To our knowledge, this is the first report indicating that ID2 is one of the critical factors in the signaling pathway of Du145 cell death induced by EGCG.
PMCID: PMC4040217  PMID: 20002680
16.  Marine Compound Catunaregin Inhibits Angiogenesis through the Modulation of Phosphorylation of Akt and eNOS in vivo and in vitro 
Marine Drugs  2014;12(5):2790-2801.
Angiogenesis is the formation of blood vessels from pre-existing vasculature. Excessive or uncontrolled angiogenesis is a major contributor to many pathological conditions whereas inhibition of aberrant angiogenesis is beneficial to patients with pathological angiogenesis. Catunaregin is a core of novel marine compound isolated from mangrove associate. The potential anti-angiogenesis of catunaregin was investigated in human umbilical vein endothelial cells (HUVECs) and zebrafish. HUVECs were treated with different concentrations of catunaregin in the presence or absence of VEGF. The angiogenic phenotypes including cell invasion cell migration and tube formation were evaluated following catunaregin treatment in HUVECs. The possible involvement of AKT, eNOS and ERK1/2 in catunaregin-induced anti-angiogenesis was explored using Western blotting. The anti-angiogenesis of catunaregin was further tested in the zebrafish embryo neovascularization and caudal fin regeneration assays. We found that catunaregin dose-dependently inhibited angiogenesis in both HUVECs and zebrafish embryo neovascularization and zebrafish caudal fin regeneration assays. In addition, catunaregin significantly decreased the phosphorylation of Akt and eNOS, but not the phosphorylation of ERK1/2. The present work demonstrates that catunaregin exerts the anti-angiogenic activity at least in part through the regulation of the Akt and eNOS signaling pathways.
PMCID: PMC4052316  PMID: 24824025
anti-angiogenesis; catunaregin; VEGF; zebrafish; HUVECs
17.  Is Gestational Hypertension Protective against Perinatal Mortality in Twin Pregnancies? 
PLoS ONE  2014;9(4):e94865.
Pregnancy-induced or gestational hypertension is a common pregnancy complication. Paradoxically, gestational hypertension has been associated with a protective effect against perinatal mortality in twin pregnancies in analytic models (logistic regression) without accounting for survival time. Whether this effect is real remains uncertain. This study aimed to validate the impact of gestational hypertension on perinatal mortality in twin pregnancies using a survival analysis approach.
This was a retrospective cohort study of 278,821 twin pregnancies, using the U.S. 1995–2000 matched multiple birth dataset (the largest dataset available for multiple births). Cox proportional hazard models were applied to estimate the adjusted hazard ratios (aHR) of perinatal death (stillbirth and neonatal death) comparing gestational hypertensive vs. non-hypertensive pregnancies controlling for maternal characteristics and twin cluster-level dependence.
Comparing births in gestational hypertensive vs. non-hypertensive twin pregnancies, perinatal mortality rates were significantly lower (1.20% vs. 3.38%), so were neonatal mortality (0.72% vs. 2.30%) and stillbirth (0.48% vs. 1.10%) rates. The aHRs (95% confidence intervals) were 0.34 (0.31–0.38) for perinatal death, 0.31 (0.27–0.34) for neonatal death, and 0.45 (0.38–0.53) for stillbirth, respectively. The protective effect of gestational hypertension against perinatal death became weaker over advancing gestational age; the aHRs in very preterm (<32 weeks), mild preterm (32–36 weeks) and term (37+ weeks) births were 0.29, 0.48 and 0.76, respectively. The largest risk reductions in neonatal mortality were observed for infections and immaturity-related conditions.
Gestational hypertension appears to be beneficial for fetal survival in twin pregnancies, especially in those ending more prematurely or for deaths due to infections and immaturity-related conditions. Prospective studies are required to rule out the possibility of unmeasured confounders.
PMCID: PMC3986365  PMID: 24733364
18.  GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts 
Endocrine-Related Cancer  2014;21(2):355-369.
Cancer-associated fibroblasts (CAFs) are crucial co-mediators of breast cancer progression. Estrogen is the predominant driving force in the cyclic regulation of the mammary extracellular matrix, thus potentially affecting the tumor-associated stroma. Recently, a third estrogen receptor, estrogen (G-protein-coupled) receptor (GPER), has been reported to be expressed in breast CAFs. In this study, GPER was detected by immunohistochemical analysis in stromal fibroblasts of 41.8% (59/141) of the primary breast cancer samples. GPER expression in CAFs isolated from primary breast cancer tissues was confirmed by immunostaining and RT-PCR analyses. Tamoxifen (TAM) in addition to 17β-estradiol (E2) and the GPER agonist G1 activated GPER, resulting in transient increases in cell index, intracellular calcium, and ERK1/2 phosphorylation. Furthermore, TAM, E2, and G1 promoted CAF proliferation and cell-cycle progression, both of which were blocked by GPER interference, the selective GPER antagonist G15, the epidermal growth factor receptor (EGFR) inhibitor AG1478, and the ERK1/2 inhibitor U0126. Importantly, TAM as well as G1 increased E2 production in breast CAFs via GPER/EGFR/ERK signaling when the substrate of E2, testosterone, was added to the medium. GPER-induced aromatase upregulation was probably responsible for this phenomenon, as TAM- and G1-induced CYP19A1 gene expression was reduced by GPER knockdown and G15, AG1478, and U0126 administration. Accordingly, GPER-mediated CAF-dependent estrogenic effects on the tumor-associated stroma are conceivable, and CAF is likely to contribute to breast cancer progression, especially TAM resistance, via a positive feedback loop involving GPER/EGFR/ERK signaling and E2 production.
PMCID: PMC3959763  PMID: 24481325
CAF; GPER; tamoxifen resistance; breast cancer
19.  Analysis of Individual and Combined Effects of Ochratoxin A and Zearalenone on HepG2 and KK-1 Cells with Mathematical Models 
Toxins  2014;6(4):1177-1192.
Ochratoxin A (OTA) and Zearalenone (ZEA) are widespread mycotoxins that contaminate foodstuffs simultaneously, but sufficient data regarding their mixed toxicities are lacking. This study aims to analyze the style of combined effects of OTA and ZEA on cells of their target organs. For this purpose, cytotoxicity was determined in HepG2 and KK-1 cells treated with single and combined forms of OTA and ZEA. Furthermore, we have analyzed the data using two mathematical models based on the concepts of concentration addition (CA) and independent addition (IA). By analyzing data with nonlinear regression, toxins applied singly showed classic sigmoid dose-response curves in HepG2 cells whereas in KK-1 cells hormetic responses were observed. Exposure to equieffective mixtures of OTA and ZEA showed additive effects, irrespective of different nonlinear regression models used. Our results demonstrate that IA is an appropriate concept to account for mixture effects of OTA and ZEA. The results in ROS generation indicate a departure from additivity to antagonism or synergism at different concentrations, probably due to potential interaction during ROS production. This study shows that a risk assessment of mycotoxins should account for mixture effects, and prediction models are valuable tools for mixture assessment.
PMCID: PMC4014727  PMID: 24674935
ochratoxin A; zearalenone; combined effect; concentration addition; independent addition; risk assessment
20.  Aurora-A is a novel predictor of poor prognosis in patients with resected lung adenocarcinoma 
The Aurora-A (Aur-A) gene, a key regulator of mitosis, has been proved as an oncogene in a variety of cancers. The Aur-A overexpression has been proved correlated with aggressiveness of cancer cells. However, the frequency of Aur-A protein overexpression, as well as its association with clinicopathologic parameters and prognosis remain unclear in lung adenocarcinoma (ADC). This study tried to clarify the clinical significance of Aur-A in patients with resected lung ADC.
Patients and methods
A total of 142 informative patients with surgically resected lung ADC and 20 normal lung tissues were enrolled. Western blot and immunohistochemistry (IHC) were utilized to assess protein expression of Aur-A.
The expression of Aur-A was elevated in most of tumor tissues compared with the adjacent tissues by western blot. The IHC results showed that Aur-A protein was over-expressed in 98 of 142 (69.0%) tumor sections, while Aur-A was low-expressed in all normal lung sections. A positive correlation between Aur-A overexpression rate and ascending pathologic stages was observed (P<0.05). Kaplan-Meier analysis demonstrated that patients with Aur-A high expression had significantly inferior survival compared to those with Aur-A low expression. Both overall survival (OS) and disease-free survival (DFS) of positive overexpression patients were shorter than the negative group (P=0.036, P=0.041, respectively). Multivariate analysis confirmed that Aur-A expression, as an independent and significant factor for both DFS and OS, could predict a poor prognosis in patients with resected lung ADC (P=0.022, P=0.049, respectively).
Aur-A was overexpressed in lung ADC and overexpression of Aur-A might be a novel predictor for poor prognosis and potential therapeutic target in lung ADC.
PMCID: PMC4000897  PMID: 24826057
Aurora-A (Aur-A); lung adenocarcinoma (ADC); prognosis
21.  Allogeneic Bone Marrow Co-Cultured with Human Islets Significantly Improves Islet Survival and Function in vivo 
Transplantation  2013;95(6):801-809.
A significant barrier to islet transplantation is the rapid loss of human islet function in vivo. The present study evaluates whether bone marrow (BM) could be used to support human islet survival and function in vivo.
We co-cultured human islets and BM for three weeks prior to transplantation into the left subrenal capsule of diabetic SCID mice.
The co-cultured human islets prior to transplantation demonstrated improved viability, increased size and migration capacity in vitro. After 4 months, animals transplanted with pre-cultured BM /human islets exhibited euglycemia and detectable human insulin levels (157μU/ml), while no human insulin was detected in the islet only transplantation group. Furthermore, the removal of the transplants on day 126 resulted in hyperglycemia indicating that the reduction of blood glucose was dependent on the transplants. Diabetic mice transplanted with BM/islets demonstrated the longest survival period (130 days vs 40 days for those with islet only transplants). The transplanted BM/islets showed signs of vascularization and migration from the renal capsule into medulla.
Our results suggest that BM pre-cultured with human islets may enhance the survival and function of transplanted islets, thus significantly improving the therapeutic efficacy of islet transplantation for type 1 diabetes.
PMCID: PMC3604119  PMID: 23416682
Allogeneic Bone Marrow; Human Islet; Diabetes
22.  The genomes of four tapeworm species reveal adaptations to parasitism 
Nature  2013;496(7443):57-63.
Tapeworms cause debilitating neglected diseases that can be deadly and often require surgery due to ineffective drugs. Here we present the first analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115-141 megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have species-specific expansions of non-canonical heat shock proteins and families of known antigens; specialised detoxification pathways, and metabolism finely tuned to rely on nutrients scavenged from their hosts. We identify new potential drug targets, including those on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.
PMCID: PMC3964345  PMID: 23485966
HSP70; parasitism; Cestoda; cysticercosis; echinococcosis; Platyhelminthes
24.  Genistein-Inhibited Cancer Stem Cell-Like Properties and Reduced Chemoresistance of Gastric Cancer 
Genistein, the predominant isoflavone found in soy products, has exerted its anticarcinogenic effect in many different tumor types in vitro and in vivo. Accumulating evidence in recent years has strongly indicated the existence of cancer stem cells in gastric cancer. Here, we showed that low doses of genistein (15 μM), extracted from Millettia nitida Benth var hirsutissima Z Wei, inhibit tumor cell self-renewal in two types of gastric cancer cells by colony formation assay and tumor sphere formation assay. Treatment of gastric cancer cells with genistein reduced its chemoresistance to 5-Fu (fluorouracil) and ciplatin. Further results indicated that the reduced chemoresistance may be associated with the inhibition of ABCG2 expression and ERK 1/2 activity. Furthermore, genistein reduced tumor mass in the xenograft model. Together, genistein inhibited gastric cancer stem cell-like properties and reduced its chemoresistance. Our results provide a further rationale and experimental basis for using the genistein to improve treatment of patients with gastric cancer.
PMCID: PMC3975346  PMID: 24573253
genistein; gastric cancer stem cell; chemoresistance
25.  Enhanced surveillance of acute flaccid paralysis following importation of wild poliovirus in Xinjiang Uygur Autonomous Region, China 
BMC Infectious Diseases  2014;14:113.
After being polio free for more than 10 years, an outbreak occurred in China in 2011 in Xinjiang Uygur Autonomous Region (Xinjiang) following the importation of wild poliovirus (WPV) originating from neighboring Pakistan.
To strengthen acute flaccid paralysis (AFP) surveillance in Xinjiang, “zero case daily reporting” and retrospective searching of AFP cases were initiated after the confirmation of the WPV outbreak. To pinpoint all the polio cases in time, AFP surveillance system was expanded to include persons of all ages in the entire population in Xinjiang.
Totally, 578 AFP cases were reported in 2011 in Xinjiang, including 21 WPV cases, 23 clinical compatible polio cases and 534 non-polio AFP cases. Of the 44 polio cases, 27 (61.4%) cases were reported among adults aged 15–53 years. Strengthening AFP surveillance resulted in an increase in the number of non-polio AFP cases in 2011 (148 children < 15 years) compared with 76 cases < 15 years in 2010. The AFP surveillance system in Xinjiang was sensitive enough to detect polio cases, with the AFP incidence of 3.28/100,000 among children < 15 years of age.
Incorporating adult cases into the AFP surveillance system is of potential value to understand the overall characteristics of the epidemic and to guide emergency responses, especially in countries facing WPV outbreak following long-term polio free status. The AFP surveillance system in Xinjiang was satisfactory despite limitations in biological sample collection.
PMCID: PMC3941572  PMID: 24576083
Acute flaccid paralysis; Wild poliovirus; Clinical compatible polio cases; China

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