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1.  Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids 
PLoS ONE  2014;9(4):e93608.
Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.
doi:10.1371/journal.pone.0093608
PMCID: PMC3973564  PMID: 24695466
2.  Identification of Cross-Species Shared Transcriptional Networks of Diabetic Nephropathy in Human and Mouse Glomeruli 
Diabetes  2012;62(1):299-308.
Murine models are valuable instruments in defining the pathogenesis of diabetic nephropathy (DN), but they only partially recapitulate disease manifestations of human DN, limiting their utility. To define the molecular similarities and differences between human and murine DN, we performed a cross-species comparison of glomerular transcriptional networks. Glomerular gene expression was profiled in patients with early type 2 DN and in three mouse models (streptozotocin DBA/2, C57BLKS db/db, and eNOS-deficient C57BLKS db/db mice). Species-specific transcriptional networks were generated and compared with a novel network-matching algorithm. Three shared human–mouse cross-species glomerular transcriptional networks containing 143 (Human-DBA STZ), 97 (Human-BKS db/db), and 162 (Human-BKS eNOS−/− db/db) gene nodes were generated. Shared nodes across all networks reflected established pathogenic mechanisms of diabetes complications, such as elements of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and vascular endothelial growth factor receptor (VEGFR) signaling pathways. In addition, novel pathways not previously associated with DN and cross-species gene nodes and pathways unique to each of the human–mouse networks were discovered. The human–mouse shared glomerular transcriptional networks will assist DN researchers in selecting mouse models most relevant to the human disease process of interest. Moreover, they will allow identification of new pathways shared between mice and humans.
doi:10.2337/db11-1667
PMCID: PMC3526018  PMID: 23139354
3.  Ca2+ influx through L-type Ca2+ channels and transient receptor potential channels activate pathological hypertrophy signaling 
Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca2+]. These diseases induce cardiac hypertrophy and increases in [Ca2+] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of “hypertrophic” [Ca2+] is not known and is the topic of this study. The role of Ca2+ influx through L-type Ca2+ channels (LTCC), T-type Ca2+ channels (TTCC) and transient receptor potential (TRP) channels on the activation of Calcineurin (Cn) – Nuclear Factor of Activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat (NRVMs) and adult feline (AFVM) ventricular myocytes were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca2+ or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca2+-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca2+ induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca2+ transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca2+ influx through LTCCs is the primary source of Ca2+ to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca2+ entry via LTCCs.
doi:10.1016/j.yjmcc.2012.08.005
PMCID: PMC3472041  PMID: 22921230
L-type calcium channel; Hypertrophy; Nuclear factor of activated T cells; Ventricular myocyte; Transient receptor potential channel
4.  Tongue force and tongue motility are differently affected by unilateral vs bilateral nigrostriatal dopamine depletion in rats 
Behavioural brain research  2012;234(2):343-348.
In addition to its cardinal symptoms of bradykinesia, muscle rigidity, resting tremor and postural disturbances, Parkinson’s disease (PD) also affects orolingual motor function. Orolingual motor deficits can contribute to dysphagia, which increases morbidity and mortality in this population. Previous preclinical studies describing orolingual motor deficits in animal models of PD have focused on unilateral nigrostriatal dopamine (DA) depletion. In this study we compared the effects of unilateral vs bilateral 6-hydroxydopamine (6-OHDA)-induced DA depletion in rats trained to lick water from an isometric force-sensing disc. Rats received either unilateral or bilateral 6-OHDA into the medial forebrain bundle and were tested for four weeks post-lesion. Dependent variables included task engagement (the number of licks per session), tongue force (mean and maximum), and tongue motility (the number of licks per second). While both lesion groups exhibited decreased tongue force output, tongue motility deficits were present in only the group that received unilateral nigrostriatal DA depletion. Task engagement was not significantly diminished by 6-OHDA. Analysis of striatal DA tissue content revealed that DA depletion was ~97% in the unilateral group and ~90% in the bilateral group. These results suggest that while nigrostriatal DA depletion affects tongue force output, deficits in tongue motility may instead result from a functional imbalance in neural pathways affecting this midline structure.
doi:10.1016/j.bbr.2012.07.003
PMCID: PMC3422433  PMID: 22796604
Parkinson’s disease; orolingual; oromotor; isometric; operant; behavioral; nigrostriatal; dysarthria; dysphagia; force
5.  Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells 
PLoS ONE  2013;8(9):e73086.
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.
doi:10.1371/journal.pone.0073086
PMCID: PMC3760886  PMID: 24019898
6.  Pancreaticoduodenectomy Combined with Vascular Resection and Reconstruction for Patients with Locally Advanced Pancreatic Cancer: A Multicenter, Retrospective Analysis 
PLoS ONE  2013;8(8):e70340.
Objective
The aim of this study was to present the therapeutic outcome of patients with locally advanced pancreatic cancer treated with pancreatoduodenectomy combined with vascular resection and reconstruction in addition to highlighting the mortality/morbidity and main prognostic factors associated with this treatment.
Materials and Methods
We retrospectively analyzed the clinical and pathological data of a total of 566 pancreatic cancer patients who were treated with PD from five teaching hospitals during the period of December 2006–December 2011. This study included 119 (21.0%) patients treated with PD combined with vascular resection and reconstruction. We performed a detailed statistical analysis of various factors, including postoperative complications, operative mortality, survival rate, operative time, pathological type, and lymph node metastasis.
Results
The median survival time of the 119 cases that received PD combined with vascular resection was 13.3 months, and the 1-, 2-, and 3-year survival rates were 30.3%, 14.1%, and 8.1%, respectively. The postoperative complication incidence was 23.5%, and the mortality rate was 6.7%. For the combined vascular resection group, complications occurred in 28 cases (23.5%). For the group without vascular resection, complications occurred in 37 cases (8.2%). There was significant difference between the two groups (p = 0.001). The degree of tumor differentiation and the occurrence of complications after surgery were independent prognostic factors that determined the patients’ long-term survival.
Conclusions
Compared with PD without vascular resection, PD combined with vascular resection and reconstruction increased the incidence of postoperative complications. However, PD combined with vascular resection and reconstruction could achieve the complete removal of tumors without significantly increasing the mortality rate, and the median survival time was higher than that of patients who underwent palliative treatment. In addition, the two independent factors affecting the postoperative survival time were the degree of tumor differentiation and the presence or absence of postoperative complications.
doi:10.1371/journal.pone.0070340
PMCID: PMC3732270  PMID: 23936411
7.  Identification and Expression Profile Analysis of Odorant Binding Proteins in the Oriental Fruit Fly Bactrocera dorsalis 
Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs) function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna) of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs (BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10) are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies.
doi:10.3390/ijms140714936
PMCID: PMC3742281  PMID: 23867609
odorant binding protein; olfaction; tissue expression pattern; Bactrocera dorsalis
8.  Ginseng saponin metabolite 20(S)-protopanaxadiol inhibits tumor growth by targeting multiple cancer signaling pathways 
Oncology Reports  2013;30(1):292-298.
Plant-derived active constituents and their semi-synthetic or synthetic analogs have served as major sources of anticancer drugs. 20(S)-protopanaxadiol (PPD) is a metabolite of ginseng saponin of both American ginseng (Panax quinquefolius L.) and Asian ginseng (Panax ginseng C.A. Meyer). We previously demonstrated that ginsenoside Rg3, a glucoside precursor of PPD, exhibits anti-proliferative effects on HCT116 cells and reduces tumor size in a xenograft model. Our subsequent study indicated that PPD has more potent antitumor activity than that of Rg3 in vitro although the mechanism underlying the anticancer activity of PPD remains to be defined. Here, we investigated the mechanism underlying the anticancer activity of PPD in human cancer cells in vitro and in vivo. PPD was shown to inhibit growth and induce cell cycle arrest in HCT116 cells. The in vivo studies indicate that PPD inhibits xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced when the animals were treated with PPD (30 mg/kg body weight) for 3 weeks. When the expression of previously identified Rg3 targets, A kinase (PRKA) anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein α (PITPNA), was analyzed, PPD was shown to inhibit the expression of PITPNA while upregulating AKAP8L expression in HCT116 cells. Pathway-specific reporter assays indicated that PPD effectively suppressed the NF-κB, JNK and MAPK/ERK signaling pathways. Taken together, our results suggest that the anticancer activity of PPD in colon cancer cells may be mediated through targeting NF-κB, JNK and MAPK/ERK signaling pathways, although the detailed mechanisms underlying the anticancer mode of PPD action need to be fully elucidated.
doi:10.3892/or.2013.2438
PMCID: PMC3729206  PMID: 23633038
ginseng; ginseng metabolites; 20(S)-protopanaxadiol; colorectal cancer; signaling pathway; natural products
9.  Insights into the increasing virulence of the swine-origin pandemic H1N1/2009 influenza virus 
Scientific Reports  2013;3:1601.
Pandemic H1N1/2009 viruses have been stabilized in swine herds, and some strains display higher pathogenicity than the human-origin isolates. In this study, high-throughput RNA sequencing (RNA-seq) is applied to explore the systemic transcriptome responses of the mouse lungs infected by swine (Jia6/10) and human (LN/09) H1N1/2009 viruses. The transcriptome data show that Jia6/10 activates stronger virus-sensing signals, such as the toll-like receptor, RIG-I like receptor and NOD-like receptor signalings, as well as a stronger NF-κB and JAK-STAT singals, which play significant roles in inducing innate immunity. Most cytokines and interferon-stimulated genes show higher expression lever in Jia/06 infected groups. Meanwhile, virus Jia6/10 activates stronger production of reactive oxygen species, which might further promote higher mutation rate of the virus genome. Collectively, our data reveal that the swine-origin pandemic H1N1/2009 virus elicits a stronger innate immune reaction and pro-oxidation stimulation, which might relate closely to the increasing pathogenicity.
doi:10.1038/srep01601
PMCID: PMC3615340  PMID: 23549303
10.  Hyperphosphorylation of the Cardiac Ryanodine Receptor at Serine 2808 Is Not Involved in Cardiac Dysfunction after Myocardial Infarction 
Circulation Research  2012;110(6):831-840.
Rationale
Abnormal behavior of the cardiac ryanodine receptor (RyR2) has been linked to cardiac arrhythmias and heart failure (HF) after Myocardial infarction (MI). It has been proposed that protein kinase A (PKA) hyperphosphorylation of the RyR2 at a single residue, Ser-2808 is a critical mediator of RyR dysfunction, depressed cardiac performance and HF after MI.
Objectives
We used a mouse model (RyRS2808A) in which PKA hyperphosphorylation of the RyR2 at Ser-2808 is prevented to determine whether loss of PKA phosphorylation at this site averts post MI cardiac pump dysfunction.
Methods and Results
MI was induced in WT and S2808A mice. Myocyte and cardiac function were compared in WT and S2808A animals before and after MI. The effects of the PKA activator Isoproterenol (Iso) on L-type Ca2+ current (ICaL), contractions and [Ca2+]I transients were also measured. Both WT and S2808A mice had depressed pump function after MI and were no differences between groups. MI size was also identical in both groups. L type Ca2+ current, contractions, Ca2+ transients and SR Ca2+ load were also not significantly different in WT versus S2808A myocytes either before or after MI. Iso effects on Ca2+ current, contraction, Ca2+ transients and SR Ca2+ load were identical in WT and S2808A myocytes before and after MI at both low and high concentrations.
Conclusions
These results strongly support the idea that PKA phosphorylation of RyR-S2808 is irrelevant to the development of cardiac dysfunction after MI, at least in the mice used in this study.
doi:10.1161/CIRCRESAHA.111.255158
PMCID: PMC3322671  PMID: 22302785
Myocardial infarction; ryanodine receptor; heart failure; PKA hyperphosphorylation
11.  Draft Genome Sequence of Streptomyces bottropensis ATCC 25435, a Bottromycin-Producing Actinomycete 
Genome Announcements  2013;1(2):e00019-13.
A series of bottromycin antibiotics have been isolated and identified from Streptomyces bottropensis strain ATCC 25435. Here, a draft genome sequence of S. bottropensis ATCC 25435 is presented. The genome carries an intact biosynthetic gene cluster for bottromycin antibiotics, which provides insight into the combinatorial biosynthesis of bottromycin antibiotics.
doi:10.1128/genomeA.00019-13
PMCID: PMC3622962  PMID: 23516178
12.  BMP9 signaling in stem cell differentiation and osteogenesis 
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and play a critical role in skeletal development, bone formation and stem cell differentiation. Disruptions in BMP signaling result in a variety of skeletal and extraskeletal anomalies. BMP9 is a poorly characterized member of the BMP family and is among the most osteogenic BMPs, promoting osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. Recent findings from various in vivo and molecular studies strongly suggest that the mechanisms governing BMP9-mediated osteoinduction differ from other osteogenic BMPs. Many signaling pathways with diverse functions have been found to play a role in BMP9-mediated osteogenesis. Several of these pathways are also critical in the differentiation of other cell lineages, including adipocytes and chondrocytes. While BMP9 is known to be a potent osteogenic factor, it also influences several other pathways including cancer development, angiogenesis and myogenesis. Although BMP9 has been demonstrated as one of the most osteogenic BMPs, relatively little is known about the specific mechanisms responsible for these effects. BMP9 has demonstrated efficacy in promoting spinal fusion and bony non-union repair in animal models, demonstrating great translational promise. This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed work which may help us to further elucidate these pathways.
PMCID: PMC3636726  PMID: 23671813
BMP; BMP9; bone regeneration; IGF; osteogenesis; TGF-β; Wnt; signal transduction; mesenchymal stem cells
13.  A Caveolae Targeted L-type Ca2+ Channel Antagonist Inhibits Hypertrophic Signaling Without Reducing Cardiac Contractility 
Circulation Research  2012;110(5):669-674.
Rationale
The source of Ca2+ to activate pathological cardiac hypertrophy is not clearly defined. Ca2+ influx through the L-type Ca2+ channels (LTCCs) determines “contractile” Ca2+, which is not thought to be the source of “hypertrophic” Ca2+. However, some LTCCs are housed in caveolin-3 (Cav-3) enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown.
Objective
To test the idea that LTCCs in Cav-3 containing signaling domains are a source of Ca2+ to activate the calcineurin-nuclear factor of activated T cells (Cn-NFAT) signaling cascade that promotes pathological hypertrophy.
Methods and Results
We developed reagents that targeted Ca2+ channel blocking Rem proteins to Cav-3 containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current, almost all of the Ca2+ influx induced NFAT nuclear translocation, but did not reduce myocyte contractility.
Conclusions
We provide proof of concept that Ca2+ influx through LTCCs within caveolae signaling domains can activate “hypertrophic” signaling, and this Ca2+ influx can be selectively blocked without reducing cardiac contractility.
doi:10.1161/CIRCRESAHA.111.264028
PMCID: PMC3324037  PMID: 22302787
Caveolae; L-Type Calcium Channel; Hypertrophy; Contractility; NFAT
14.  The Impact of Environmental Heterogeneity and Life Stage on the Hindgut Microbiota of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae) 
PLoS ONE  2013;8(2):e57169.
Gut microbiota has diverse ecological and evolutionary effects on its hosts. However, the ways in which it responds to environmental heterogeneity and host physiology remain poorly understood. To this end, we surveyed intestinal microbiota of Holotrichia parallela larvae at different instars and from different geographic regions. Bacterial 16S rRNA gene clone libraries were constructed and clones were subsequently screened by DGGE and sequenced. Firmicutes and Proteobacteria were the major phyla, and bacteria belonging to Ruminococcaceae, Lachnospiraceae, Enterobacteriaceae, Desulfovibrionaceae and Rhodocyclaceae families were commonly found in all natural populations. However, bacterial diversity (Chao1 and Shannon indices) and community structure varied across host populations, and the observed variation can be explained by soil pH, organic carbon and total nitrogen, and the climate factors (e.g., mean annual temperature) of the locations where the populations were sampled. Furthermore, increases in the species richness and diversity of gut microbiota were observed during larval growth. Bacteroidetes comprised the dominant group in the first instar; however, Firmicutes composed the majority of the hindgut microbiota during the second and third instars. Our results suggest that the gut's bacterial community changes in response to environmental heterogeneity and host's physiology, possibly to meet the host's ecological needs or physiological demands.
doi:10.1371/journal.pone.0057169
PMCID: PMC3578786  PMID: 23437336
15.  Wnt signaling in bone formation and its therapeutic potential for bone diseases 
The Wnt signaling pathway plays an important role not only in embryonic development but also in the maintenance and differentiation of the stem cells in adulthood. In particular, Wnt signaling has been shown as an important regulatory pathway in the osteogenic differentiation of mesenchymal stem cells. Induction of the Wnt signaling pathway promotes bone formation while inactivation of the pathway leads to osteopenic states. Our current understanding of Wnt signaling in osteogenesis elucidates the molecular mechanisms of classic osteogenic pathologies. Activating and inactivating aberrations of the canonical Wnt signaling pathway in osteogenesis results in sclerosteosis and osteoporosis respectively. Recent studies have sought to target the Wnt signaling pathway to treat osteogenic disorders. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway. Antibodies against endogenous antagonists, such as sclerostin and dickkopf-1, have demonstrated promising results in promoting bone formation and fracture healing. Lithium, an inhibitor of glycogen synthase kinase 3β, has also been reported to stimulate osteogenesis by stabilizing β catenin. Although manipulating the Wnt signaling pathway has abundant therapeutic potential, it requires cautious approach due to risks of tumorigenesis. The present review discusses the role of the Wnt signaling pathway in osteogenesis and examines its targeted therapeutic potential.
doi:10.1177/1759720X12466608
PMCID: PMC3582304  PMID: 23514963
Wnt signaling; bone formation; osteoporosis; fracture healing; bone tumors
16.  Abnormal methylation of seven genes and their associations with clinical characteristics in early stage non-small cell lung cancer 
Oncology Letters  2013;5(4):1211-1218.
To identify novel abnormally methylated genes in early stage non-small cell lung cancer (NSCLC), we analyzed the methylation status of 13 genes (ALX1, BCL2, FOXL2, HPP1, MYF6, OC2, PDGFRA, PHOX2A, PITX2, RARB, SIX6, SMPD3 and SOX1) in cancer tissues from 101 cases of stage I NSCLC patients and lung tissues from 30 cases of non-cancerous lung disease controls, using methylation-specific PCR (MSP). The methylation frequencies (29.70–64.36%) of 7 genes (MYF6, SIX6, SOX1, RARB, BCL2, PHOX2A and FOLX2) in stage I NSCLC were significantly higher compared with those in non-cancerous lung disease controls (P<0.05). The co-methylation of SIX6 and SOX1, or the co-methyaltion of SIX6, RARB and SOX1 was associated with adenosquamous carcinoma (ADC), and the co-methylation of BCL2, RARB and SIX6 was associated with smoking. A panel of 4 genes (MYF6, SIX6, BCL2 and RARB) may offer a sensitivity of 93.07% and a specificity of 83.33% in the diagnosis of stage I NSCLC. Furthermore, we also detected the expression of 8 pathological markers (VEGF, HER-2, P53, P21, EGFR, CHGA, SYN and EMA) in cancer tissues of stage I NSCLC by immunohistochemistry, and found that high expression levels of p53 and CHGA were associated with the methylation of BCL2 (P=0.025) and PHOX2A (P=0.023), respectively. In this study, among the 7 genes which demonstrated hypermethylation in stage I NSCLC compared with non-cancerous lung diseases, 5 genes (MYF6, SIX6, PHOX2A, FOLX2 and SOX1) were found for the first time to be abonormally methylated in NSCLC. Further study of these genes shed light on the carcinogenesis of NSCLC.
doi:10.3892/ol.2013.1161
PMCID: PMC3629069  PMID: 23599765
DNA methylation; non-small cell lung cancer; stage I; smoking
17.  Establishment and Characterization of the Reversibly Immortalized Mouse Fetal Heart Progenitors 
Objective: Progenitor cell-based cardiomyocyte regeneration holds great promise of repairing an injured heart. Although cardiomyogenic differentiation has been reported for a variety of progenitor cell types, the biological factors that regulate effective cardiomyogenesis remain largely undefined. Primary cardiomyogenic progenitors (CPs) have a limited life span in culture, hampering the CPs' in vitro and in vivo studies. The objective of this study is to investigate if primary CPs isolated from fetal mouse heart can be reversibly immortalized with SV40 large T and maintain long-term cell proliferation without compromising cardiomyogenic differentiation potential.
Methods: Primary cardiomyocytes were isolated from mouse E15.5 fetal heart, and immortalized retrovirally with the expression of SV40 large T antigen flanked with loxP sites. Expression of cardiomyogenic markers were determined by quantitative RT-PCR and immunofluorescence staining. The immortalization phenotype was reversed by using an adenovirus-mediated expression of the Cre reconbinase. Cardiomyogenic differentiation induced by retinoids or dexamethasone was assessed by an α-myosin heavy chain (MyHC) promoter-driven reporter.
Results: We demonstrate that the CPs derived from mouse E15.5 fetal heart can be efficiently immortalized by SV40 T antigen. The conditionally immortalized CPs (iCP15 clones) exhibit an increased proliferative activity and are able to maintain long-term proliferation, which can be reversed by Cre recombinase. The iCP15 cells express cardiomyogenic markers and retain differentiation potential as they can undergo terminal differentiate into cardiomyctes under appropriate differentiation conditions although the iCP15 clones represent a large repertoire of CPs at various differentiation stages. The removal of SV40 large T increases the iCPs' differentiation potential. Thus, the iCPs not only maintain long-term cell proliferative activity but also retain cardiomyogenic differentiation potential.
Conclusions: Our results suggest that the reported reversible SV40 T antigen-mediated immortalization represents an efficient approach for establishing long-term culture of primary cardiomyogenic progenitors for basic and translational research.
doi:10.7150/ijms.6639
PMCID: PMC3691803  PMID: 23801891
Cardiomyogenic progenitors; cardiomyogenic differentiation; cardiomyogenesis; cardiovascular disorders; heart progenitor cells; immortalization
18.  B19, a Novel Monocarbonyl Analogue of Curcumin, Induces Human Ovarian Cancer Cell Apoptosis via Activation of Endoplasmic Reticulum Stress and the Autophagy Signaling Pathway 
Background: The unfolded protein response, autophagy and endoplasmic reticulum (ER) stress-induced apoptosis regulate tumor cell fate and have become novel signaling targets for the development of cancer therapeutic drugs. Curcumin has been used to treat several different cancers, including ovarian cancer, in clinical trials and research; however, the role of ER stress and autophagy in the therapeutic effects of curcumin and new curcumin analogues remains unclear.
Methods: Cell viability was determined using the MTT assay. Apoptosis was detected using flow cytometry with PI/Annexin V-FITC staining. The expression levels of ER stress- and autophagy-related proteins were analyzed by western blotting. The activation of autophagy was detected using immunofluorescence staining.
Results: We demonstrated that B19 induced HO8910 cell apoptosis in a dose-responsive manner. We also determined and that this effect was associated with corresponding increases in a series of key components in the UPR and ER stress-mediated apoptosis pathways, followed by caspase 3 cleavage and activation. We also observed that B19 treatment induced autophagy in HO8910 cells. The inhibition of autophagy using 3-methyladenine (3-MA) increased levels of intracellular misfolded proteins, which enhanced ovarian cancer apoptosis.
Conclusions: Our data indicate that ER stress and autophagy may play a role in the apoptosis that is induced by the curcumin analogue B19 in an epithelial ovarian cancer cell line and that autophagy inhibition can increase curcumin analogue-induced apoptosis by inducing severe ER stress.
doi:10.7150/ijbs.5711
PMCID: PMC3753441  PMID: 23983610
Curcumin analogues; B19; ovarian cancer; apoptosis; ER stress; autophagy.
19.  Modulation of β-Catenin Signaling by the Inhibitors of MAP Kinase, Tyrosine Kinase, and PI3-Kinase Pathways 
Aberrant activation of β-catenin signaling plays an important role in human tumorigenesis. However, molecular mechanisms behind the β-catenin signaling deregulation are mostly unknown because genetic alterations in this pathway only account for a small fraction of tumors. Here, we investigator if other major pathways can regulate β-catenin signaling activity. By employing a panel of chemical activators and/or inhibitors of several cellular signaling pathways, we assess these modulators' effects on luciferase reporter driven by β-catenin/TCF4-responsive elements. We find that lithium-stimulated β-catenin activity is synergistically enhanced by protein kinase C activator PMA. However, β-catenin-regulated transcriptional (CRT) activity is significantly inhibited by casein kinase II inhibitor DRB, MEK inhibitor PD98059, G-proteins and their receptor uncoupling agent suramin, protein tyrosine kinase inhibitor genistein, and PI-3 kinase inhibitor wortmannin, suggesting that these cellular pathways may participate in regulating β-catenin signaling. Interestingly, the Ca++/calmodulin kinase II inhibitor HDBA is shown to activate β-catenin activity at low doses. Furthermore, Wnt3A-stimulated and constitutively activated CRT activities, as well as the intracellular accumulation of β-catenin protein in human colon cancer cells, are effectively suppressed by PD98059, genistein, and wortmannin. We further demonstrate that EGF can activate TCF4/β-catenin activity and induce the tyrosine phosphorylation of β-catenin protein. Thus, our results should provide important insights into the molecular mechanisms underlying Wnt/β-catenin activation. This knowledge should facilitate our efforts to develop efficacious and novel therapeutics by targeting these pathways.
doi:10.7150/ijms.6019
PMCID: PMC3856380  PMID: 24324366
β-catenin; G protein; MEK kinase; PI3 kinase; protein kinase C; tumorigenesis; tyrosine phosphorylation; Wnt signaling.
20.  Draft Genome Sequence of Marine Streptomyces sp. Strain W007, Which Produces Angucyclinone Antibiotics with a Benz[a]anthracene Skeleton 
Journal of Bacteriology  2012;194(6):1628-1629.
A series of angucyclinone antibiotics have been isolated from marine Streptomyces sp. strain W007 and identified. Here, a draft genome sequence of Streptomyces sp. W007 is presented. The genome contains an intact biosynthetic gene cluster for angucyclinone antibiotics, which provides insight into the combinatorial biosynthesis of angucyclinone antibiotics produced by marine streptomycetes.
doi:10.1128/JB.06701-11
PMCID: PMC3294854  PMID: 22374958
21.  Computed tomography-guided permanent brachytherapy for locoregional recurrent gastric cancer 
Background
Locoregional recurrence is the typical pattern of recurrence in gastric cancer, and cannot be removed by surgery in most of the patients. We aimed to evaluate the feasibility and efficacy of computed tomography (CT)-guided brachytherapy for patients with locoregional recurrent gastric cancer.
Materials and methods
We reviewed the case histories of 28 patients with locoregional recurrent gastric cancer that were selected for CT- guided brachytherapy by a multidisciplinary team. The clinical data of the patients including patient characteristics, treatment parameters, short-term effects, and survival data were collected and analyzed.
Results
15-75 125I seeds were implanted into each patient to produce a minimal peripheral dose (MPD) 100-160 Gy. Median day 0 dosimetry was significant for the following: V100 (the volume treated with the prescription dose) 95.8% (90.2-120.5%) and D90 (prescription dose received by at least 90% of the volume) 105.2% (98.0-124.6%) of prescription dose. No serious complications occurred during the study. Two months after brachytherapy, complete response, partial response and progressive disease were observed in 50.0%, 28.6% and 21.4% of patients, respectively. The median survival time was 22.0 ± 5.2 months, and the 1, 2,and 3-year survival rate was 89 ± 6%, 52 ± 10% and 11 ± 7%, respectively. A univariate analysis showed that the tumor size was a significant predictor of overall survival (P = 0.034). Patients with tumors <3 cm had relatively higher complete response rate (66.7%), compared to those with tumors >3 cm (30.8%). The PTV (planning target volume) smaller than 45 cm3 was significantly correlated with achieving complete tumor eradication in the treated region (P = 0.020).
Conclusions
For selected patients with limited locoregional recurrent gastric cancer, CT-guided brachytherapy using 125I seeds implantation can provide a high local control rate, with minimal trauma.
doi:10.1186/1748-717X-7-114
PMCID: PMC3485112  PMID: 22827960
Gastric cancer; Surgery; Locoregional recurrence; Brachytherapy; Iodine-125 seed
22.  β-Adrenergic Stimulation Increases Cav3.1 Activity in Cardiac Myocytes through Protein Kinase A 
PLoS ONE  2012;7(7):e39965.
The T-type Ca2+ channel (TTCC) plays important roles in cellular excitability and Ca2+ regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN) and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs) from Cav3.1 double transgenic (TG) mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca2+ current (ICa-T(3.1)). ICa-T(3.1) was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO) and a cyclic AMP analog (dibutyryl-cAMP) significantly increased ICa-T(3.1) as well as ICa-L in TG-VMs at both physiological and room temperatures. The ISO effect on ICa-L and ICa-T in TG myocytes was blocked by H89, a PKA inhibitor. ICa-T was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of ICa-T in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. ICa-T in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR) system. In conclusion, β-adrenergic stimulation increases ICa-T(3.1) in cardiomyocytes, which is mediated by the cAMP/PKA pathway. The upregulation of ICa-T(3.1) by the β-adrenergic system could play important roles in cellular functions involving Cav3.1.
doi:10.1371/journal.pone.0039965
PMCID: PMC3396630  PMID: 22808078
23.  Single-base resolution maps of cultivated and wild rice methylomes and regulatory roles of DNA methylation in plant gene expression 
BMC Genomics  2012;13:300.
Background
DNA methylation plays important biological roles in plants and animals. To examine the rice genomic methylation landscape and assess its functional significance, we generated single-base resolution DNA methylome maps for Asian cultivated rice Oryza sativa ssp. japonica, indica and their wild relatives, Oryza rufipogon and Oryza nivara.
Results
The overall methylation level of rice genomes is four times higher than that of Arabidopsis. Consistent with the results reported for Arabidopsis, methylation in promoters represses gene expression while gene-body methylation generally appears to be positively associated with gene expression. Interestingly, we discovered that methylation in gene transcriptional termination regions (TTRs) can significantly repress gene expression, and the effect is even stronger than that of promoter methylation. Through integrated analysis of genomic, DNA methylomic and transcriptomic differences between cultivated and wild rice, we found that primary DNA sequence divergence is the major determinant of methylational differences at the whole genome level, but DNA methylational difference alone can only account for limited gene expression variation between the cultivated and wild rice. Furthermore, we identified a number of genes with significant difference in methylation level between the wild and cultivated rice.
Conclusions
The single-base resolution methylomes of rice obtained in this study have not only broadened our understanding of the mechanism and function of DNA methylation in plant genomes, but also provided valuable data for future studies of rice epigenetics and the epigenetic differentiation between wild and cultivated rice.
doi:10.1186/1471-2164-13-300
PMCID: PMC3447678  PMID: 22747568
Cultivated and wild rice; Methylomes; Transcriptional termination regions (TTRs); Gene expression
24.  Inhibition of CLIC4 Enhances Autophagy and Triggers Mitochondrial and ER Stress-Induced Apoptosis in Human Glioma U251 Cells under Starvation 
PLoS ONE  2012;7(6):e39378.
CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.
doi:10.1371/journal.pone.0039378
PMCID: PMC3382619  PMID: 22761775
25.  Repair of the Injured Adult Heart Involves New Myocytes Potentially Derived From Resident Cardiac Stem Cells 
Circulation Research  2011;108(10):1226-1237.
Rationale
The ability of the adult heart to generate new myocytes after injury is not established.
Objective
Our purpose was to determine if the adult heart has the capacity to generate new myocytes after injury, and to gain insight into their source.
Methods and Results
Cardiac injury was induced in the adult feline heart by infusing Isoproterenol (ISO) for 10 days via minipumps and then animals were allowed to recover for 7 or 28 days. Cardiac function was measured with echocardiography and proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU; 7 day minipump infusion). BrdU was infused for 7 days before euthanasia at Days 10, 17, and 38 or during injury and animals euthanized at Day 38. ISO caused reduction in cardiac function with evidence of myocyte loss from necrosis. During this injury phase there was a significant increase in the number of proliferative cells in the atria and ventricle, but there was no increase in BrdU+ myocytes. cKit+ cardiac progenitor cells were BrdU labeled during injury. During the first seven days of recovery there was a significant reduction in cellular proliferation (BrdU incorporation) but a significant increase in BrdU+ myocytes. There was modest improvement in cardiac structure and function during recovery. At day 38, overall cell proliferation was not different than control but increased numbers of BrdU+ myocytes were found when BrdU was infused during injury.
Conclusions
These studies suggest that ISO injury activates cardiac progenitor cells that can differentiate into new myocytes during cardiac repair.
doi:10.1161/CIRCRESAHA.110.239046
PMCID: PMC3322670  PMID: 21454756
Cardiac regeneration; catecholamine injury; cardiac progenitor cells

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