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1.  Transcriptional regulation in pluripotent stem cells by methyl CpG-binding protein 2 (MeCP2) 
Human Molecular Genetics  2013;23(4):1045-1055.
Rett syndrome (RTT) is one of the most prevalent female mental disorders. De novo mutations in methyl CpG-binding protein 2 (MeCP2) are a major cause of RTT. MeCP2 regulates gene expression as a transcription regulator as well as through long-range chromatin interaction. Because MeCP2 is present on the X chromosome, RTT is manifested in an X-linked dominant manner. Investigation using murine MeCP2 null models and post-mortem human brain tissues has contributed to understanding the molecular and physiological function of MeCP2. In addition, RTT models using human induced pluripotent stem cells derived from RTT patients (RTT-iPSCs) provide novel resources to elucidate the regulatory mechanism of MeCP2. Previously, we obtained clones of female RTT-iPSCs that express either wild-type or mutant MECP2 due to the inactivation of one X chromosome. Reactivation of the X chromosome also allowed us to have RTT-iPSCs that express both wild-type and mutant MECP2. Using these unique pluripotent stem cells, we investigated the regulation of gene expression by MeCP2 in pluripotent stem cells by transcriptome analysis. We found that MeCP2 regulates genes encoding mitochondrial membrane proteins. In addition, loss of function in MeCP2 results in de-repression of genes on the inactive X chromosome. Furthermore, we showed that each mutation in MECP2 affects a partly different set of genes. These studies suggest that fundamental cellular physiology is affected by mutations in MECP2 from early development, and that a therapeutic approach targeting to unique forms of mutant MeCP2 is needed.
doi:10.1093/hmg/ddt500
PMCID: PMC3900111  PMID: 24129406
2.  Non-stochastic reprogramming from a privileged somatic cell state 
Cell  2014;156(4):649-662.
SUMMARY
Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming.
doi:10.1016/j.cell.2014.01.020
PMCID: PMC4318260  PMID: 24486105
3.  Engineering a Blood-Retinal Barrier With Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium: Transcriptome and Functional Analysis 
To develop a culture model for drug development and tissue-engineering human retina, retinal pigment epithelia (RPE) were derived from human embryonic stem cells (hESCs), and their barrier properties were compared with those of a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). It was found that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study also identified a panel of genes to monitor further maturation of RPE.
Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood-retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue-engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). A serum-free medium (SFM-1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC-derived RPE. In SFM-1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription-polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study identified a panel of genes to monitor the maturation of RPE.
doi:10.5966/sctm.2012-0134
PMCID: PMC3697821  PMID: 23734062
Retinal pigmented epithelium; Blood-retinal barrier; Claudins; Embryonic stem cells
4.  Study of the molecular variation in pre-eclampsia placenta based on micro-Raman spectroscopy 
Purpose
Study of the molecular variation in pre-eclampsia placenta based on micro-Raman spectroscopy.
Methods
Five pregnant women with pre-eclampsia from Nanfang hospital were selected as study group whose average age is 28.5 years and 38 ± 2 weeks gestation. The same period of healthy pregnant women, whose average age is 27.6 years and pregnant 39 ± 1 weeks, as control group (n = 5). The normal and pre-eclamptic placental tissues are detected by micro-Raman spectroscopy with the spectrum resolution of 1 cm−1.
Results
We find that the protein structure of α-helix, β-pleated sheet and β-turn is overlying in pre-eclamptic placenta, which lead to a disorder of protein structure. The Raman peaks assigned to tryptophan indole ring and phenylalanine in pre-eclamptic placental tissue are more higher than that in normal tissue.
Conclusions
Results suggest that the ordered structures of the main chain in protein molecules are reduced significantly, and the amino acid of side chains is damaged obviously. And a principal component analysis is used to classify the Raman spectra between normal and pre-eclamptic placental tissues. This study presents that Raman spectroscopy has a great potential on the mechanism research and diagnosis of placental lesions.
doi:10.1007/s00404-014-3282-9
PMCID: PMC4186689  PMID: 24866887
Molecular variation; Pre-eclampsia placenta; Micro-Raman spectroscopy; Principal component analysis; Scores plots
5.  ZNF217 is associated with poor prognosis and enhances proliferation and metastasis in ovarian cancer 
ZNF217 is an alternatively spliced Kruppel-like transcription factor that has recently been implicated to play a role in human carcinogenesis. Here, we used immunohistochemistry (IHC) to show that ZNF217 protein is overexpressed in nearly 60% of ovarian tumor samples. The disease-free survival time was shorter in patients with positive ZNF217 expression than in ZNF217-negative patients (P=0.042). Fluorescence in situ hybridization (FISH) analysis showed ZNF217 genomic amplification in the poorly differentiated tumors, suggesting that ZNF217 is associated with the progression of ovarian cancer. Invasion was enhanced in HO-8910 cells stably transfected with constructs carrying full-length ZNF217 relative to cells transfected with the empty vector. To confirm our findings in vivo, we performed a tumorigenicity assay in nude mice inoculated with the HO-8910 overexpressing ZNF217 cells. As expected, tumors grown in the ZNF217 group were more invasive and prone to metastasis than those formed control groups. Based on these clinical and laboratory observations, we conclude that ZNF217 may contribute to ovarian cancer invasion and metastasis, and associated with worse clinical outcomes.
PMCID: PMC4097264  PMID: 25031722
Ovarian cancer; ZNF217 gene; gene expression; proliferation and invasion; tumor metastasis
6.  Correction: Evolutionary History of Triticum petropavlovskyi Udacz. et Migusch. Inferred from the Sequences of the 3-Phosphoglycerate Kinase Gene 
PLoS ONE  2014;9(1):10.1371/annotation/51159241-2192-46c3-a4f1-a09f590e1f08.
doi:10.1371/annotation/51159241-2192-46c3-a4f1-a09f590e1f08
PMCID: PMC3882131
7.  Comparative Pharmacokinetics of Naringin in Rat after Oral Administration of Chaihu-Shu-Gan-San Aqueous Extract and Naringin Alone 
Metabolites  2013;3(4):867-880.
Chaihu-Shu-Gan-San (CSGS), a traditional Chinese medicine (TCM) formula containing seven herbal medicines, has been used in the clinical treatment of gastritis, peptic ulcer, irritable bowel syndrome and depression in China. In order to explore the interaction between naringin and other constituents in CSGS, the pharmacokinetic difference of naringin in rats after oral administration of CSGS aqueous extract and naringin alone was investigated. The pharmacokinetic parameters of naringin in rats were achieved by quantification of its aglycone, naringenin by LC-MS/MS method. The double peaks phenomenon was observed in both serum profiles of rats after orally administered CSGS aqueous extract and naringin alone. However, the T1/2β was significantly decreased in rats given CSGS aqueous extract compared with naringin alone, and the mean residence time (MRT) and the area under the serum concentration–time curve (AUC0-τ) were higher than those of naringin, which indicated that naringin in CSGS had higher bioavailability, longer term efficacy and somewhat faster metabolism and excretion than those of naringin. The results suggested that certain ingredients co-exist in CSGS could influence pharmacokinetic behavior of naringin. This also provides a reference for human studies.
doi:10.3390/metabo3040867
PMCID: PMC3937833  PMID: 24958255
pharmacokinetics; naringin; Chaihu-Shu-Gan-San; LC-MS/MS
8.  Evolutionary History of Triticum petropavlovskyi Udacz. et Migusch. Inferred from the Sequences of the 3-Phosphoglycerate Kinase Gene 
PLoS ONE  2013;8(8):e71139.
Single- and low-copy genes are less likely to be subject to concerted evolution. Thus, they are appropriate tools to study the origin and evolution of polyploidy plant taxa. The plastid 3-phosphoglycerate kinase gene (Pgk-1) sequences from 44 accessions of Triticum and Aegilops, representing diploid, tetraploid, and hexaploid wheats, were used to estimate the origin of Triticum petropavlovskyi. Our phylogenetic analysis was carried out on exon+intron, exon and intron sequences, using maximum likelihood, Bayesian inference and haplotype networking. We found the D genome sequences of Pgk-1 genes from T. petropavlovskyi are similar to the D genome orthologs in T. aestivum, while their relationship with Ae. tauschii is more distant. The A genome sequences of T. petropavlovskyi group with those of T. polonicum, but its Pgk-1 B genome sequences to some extent diverge from those of other species of Triticum. Our data do not support for the origin of T. petropavlovskyi either as an independent allopolyploidization event between Ae. tauschii and T. polonicum, or as a monomendelian mutation in T. aestivum. We suggest that T. petropavlovskyi originated via spontaneous introgression from T. polonicum into T. aestivum. The dating of this introgression indicates an age of 0.78 million years; a further mutation event concerning the B genome occurred 0.69 million years ago.
doi:10.1371/journal.pone.0071139
PMCID: PMC3747190  PMID: 23990932
9.  Low Immunogenicity of Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Derived from Less Immunogenic Somatic Cells 
PLoS ONE  2013;8(7):e69617.
The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However, whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues, such as umbilical cord mesenchymal cells (UMCs), are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report, we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs), we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly, we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay, reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system, in T cell co-culture system as well. Furthermore, through whole genome expression microarray analysis, we showed that over 70 immune genes, including all members of HLA-I, were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation, thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine.
doi:10.1371/journal.pone.0069617
PMCID: PMC3724937  PMID: 23922758
10.  Rotigotine Transdermal Patch in Parkinson’s Disease: A Systematic Review and Meta-Analysis 
PLoS ONE  2013;8(7):e69738.
Background and Methods
The efficacy and safety of rotigotine transdermal patch in Parkinson’s disease (PD) were studied in some clinical trials. We performed a systematic review and meta-analysis of randomized controlled trials to evaluate the efficacy, tolerability, and safety of rotigotine transdermal patch versus placebo in PD.
Results
Six randomized controlled trials (1789 patients) were included in this meta-analysis. As compared with placebo, the use of rotigotine resulted in greater improvements in Unified Parkinson’s Disease Rating Scale activities of daily living score (weighted mean difference [WMD] –1.69, 95% confidence interval [CI] –2.18 to –1.19), motor score (WMD –3.86, 95% CI –4.86 to –2.86), and the activities of daily living and motor subtotal score (WMD –4.52, 95% CI –5.86 to –3.17). Rotigotine was associated with a significantly higher rate of withdrawals due to adverse events (relative risk [RR] 1.82, 95% CI 1.29–2.59), and higher rates of application site reactions (RR 2.92, 95% CI 2.29–3.72), vomiting (RR 5.18, 95% CI 2.25–11.93), and dyskinesia (RR 2.52, 95% CI 1.47–4.32) compared with placebo. No differences were found in the relative risks of headache, constipation, back pain, diarrhea, or serious adverse events.
Conclusions
Our meta-analysis showed that the use of rotigotine can reduce the symptoms of PD. However, rotigotine was also associated with a higher incidence of adverse events, especially application site reactions, compared with placebo.
doi:10.1371/journal.pone.0069738
PMCID: PMC3720658  PMID: 23936090
11.  RNA sequencing and transcriptomal analysis of human monocyte to macrophage differentiation 
Gene  2013;519(2):279-287.
Monocytes can be differentiated into macrophages in vivo and these cells play an important role in innate and adaptive immune responses. To reveal the global gene transcription change that occurs during monocyte to macrophage differentiation, we performed genome-wide RNA sequencing and analyses in human primary monocytes and monocyte-derived macrophages. We show that 1208 genes (with >twofold differences) were differentially expressed in macrophages compared with monocytes, including 800 upregulated and 408 downregulated genes. Gene ontology, pathway, and protein–protein interaction analyses indicated that the upregulated genes were related to macrophage functions in phagocytosis, metabolic processes, and cell cycle. The majority of downregulated genes comprised genes involved in the inflammatory response and locomotion. Genes encoding transcription regulatory factors, such as FOXO1, RUNX3, NF-κB1, and C/EBP δ, were highly expressed in monocytes and appeared to function in significant transcriptional repression, resulting in slight metabolic activity. Our transcriptome comparison between human monocytes and monocyte-derived macrophages using RNA sequencing revealed novel molecules and pathways associated with the differentiation process. These molecules and pathways may represent candidate targets involved in the pathophysiology of these important immune cells.
doi:10.1016/j.gene.2013.02.015
PMCID: PMC3666862  PMID: 23458880
Monocyte-derived macrophages; Monocytes; RNA-seq; Transcriptome; M-CSF
12.  Integration of 1H NMR and UPLC-Q-TOF/MS for a Comprehensive Urinary Metabonomics Study on a Rat Model of Depression Induced by Chronic Unpredictable Mild Stress 
PLoS ONE  2013;8(5):e63624.
Depression is a type of complex psychiatric disorder with long-term, recurrent bouts, and its etiology remains largely unknown. Here, an integrated approach utilizing 1H NMR and UPLC-Q-TOF/MS together was firstly used for a comprehensive urinary metabonomics study on chronic unpredictable mild stress (CUMS) treated rats. More than twenty-nine metabolic pathways were disturbed after CUMS treatment and thirty-six potential biomarkers were identified by using two complementary analytical technologies. Among the identified biomarkers, nineteen (10, 11, 16, 17, 21–25, and 27–36) were firstly reported as potential biomarkers of CUMS-induced depression. Obviously, this paper presented a comprehensive map of the metabolic pathways perturbed by CUMS and expanded on the multitude of potential biomarkers that have been previously reported in the CUMS model. Four metabolic pathways, including valine, leucine and isoleucine biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; tryptophan metabolism; synthesis and degradation of ketone bodies had the deepest influence in the pathophysiologic process of depression. Fifteen potential biomarkers (1–2, 4–6, 15, 18, 20–23, 27, 32, 35–36) involved in the above four metabolic pathways might become the screening criteria in clinical diagnosis and predict the development of depression. Moreover, the results of Western blot analysis of aromatic L-amino acid decarboxylase (DDC) and indoleamine 2, 3-dioxygenase (IDO) in the hippocampus of CUMS-treated rats indicated that depletion of 5-HT and tryptophan, production of 5-MT and altered expression of DDC and IDO together played a key role in the initiation and progression of depression. In addition, none of the potential biomarkers were detected by NMR and LC-MS simultaneously which indicated the complementary of the two kinds of detection technologies. Therefore, the integration of 1H NMR and UPLC-Q-TOF/MS in metabonomics study provided an approach to identify the comprehensive potential depression-related biomarkers and helpful in further understanding the underlying molecular mechanisms of depression through the disturbance of metabolic pathways.
doi:10.1371/journal.pone.0063624
PMCID: PMC3656962  PMID: 23696839
13.  Paradoxical effects of streptozotocin-induced diabetes on endothelial dysfunction in stroke-prone spontaneously hypertensive rats 
The Journal of Physiology  2011;589(Pt 21):5153-5165.
Non-technical summary
Elevated blood glucose is generally regarded as one of the risk factors that lead to coronary heart disease in patients with type 2 diabetes. However, our studies show that after inducing short-term damage, high blood glucose subsequently provides paradoxical protection for vessel function of animals with high blood pressure. Vessels can adapt to sustained high blood glucose and produce different stress proteins to counteract, to some extent, the damage brought about by hypertension. The results help us understand part of the basis for vessel adaptation in diabetes. The implication for treatment of diabetes is that if the patients have long-standing diabetes and established cardiovascular disease, the target of blood glucose lowering should be less stringent and reached gradually to avoid abrupt cancellation of the pre-existing adaptations.
Abstract
Although both diabetes and hypertension are risk factors for cardiovascular disease, the role of hyperglycaemia per se in endothelial dysfunction is controversial. This study was designed to examine whether hyperglycaemia, or streptozotocin-induced diabetes, could aggravate endothelial dysfunction in stroke-prone spontaneously hypertensive rats (SHRSP). Hyperglycaemia was induced by streptozotocin in 2-month-old SHRSP and age-matched normotensive Wistar–Kyoto (WKY) rats. The aorta was isolated 8 weeks after induction of hyperglycaemia to record its function and to examine its morphology with transmission electron microscopy. Endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive haem oxygenase (HO-1/HO-2) levels were determined with Western blotting. Aortic endothelial function and production of reactive oxygen species and nitric oxide were assayed after incubation in vitro in hyperglycaemic, hyperosmolar solution. Streptozotocin-induced diabetes of 8 weeks duration did not result in endothelial dysfunction in normotensive WKY rats. In contrast, hyperglycaemic WKY rats showed significantly enhanced endothelium-dependent vasodilatation, which was abrogated by simultaneous blocking of NOS and HO. The enhanced vasodilatation was associated with elevation of vascular eNOS and HO-1. Significant endothelial dysfunction and massive macrophage–monocyte infiltration were found in SHRSP aorta (the ratio of the number of macrophages to endothelial cells in the intima, expressed as a percentage, was 20.9 ± 2.8% in SHRSP versus 1.9 ± 0.5% in WKY rats, P < 0.01), which was attenuated significantly in hyperglycaemic SHRSP (11.3 ± 1.6%, P < 0.01 versus SHRSP). Acute hyperglycaemia (10 min) aggravated endothelial dysfunction in SHRSP, with a marked increase in intracellular reactive oxygen species and NO production. Sustained in vitro incubation in hyperglycaemic/hyperosmolar conditions (addition of an extra 50 mmol L−1 of glucose or mannitol to the usual buffer, to produce a final osmolarity of 350 mosmol L−1) for 5 h enhanced endothelium-dependent vasodilatation, with elevated vessel NO production and upregulation of eNOS/HO-1 proteins. Sustained hyperglycaemia does not aggravate endothelial dysfunction and macrophage infiltration in SHRSP. Hyperglycaemia/hyperosmolarity-induced upregulation of eNOS and HO-1 may play a role in this paradoxical adaptation of endothelial function.
doi:10.1113/jphysiol.2011.213686
PMCID: PMC3225671  PMID: 21930604
14.  The Ability of Pandemic Influenza Virus Hemagglutinins to Induce Lower Respiratory Pathology is Associated with Decreased Surfactant Protein D Binding 
Virology  2011;412(2):426-434.
Pandemic influenza viral infections have been associated with viral pneumonia. Chimeric influenza viruses with the hemagglutinin segment of the 1918, 1957, 1968 or 2009 pandemic influenza viruses in the context of a seasonal H1N1 influenza genome were constructed to analyze the role of hemagglutinin (HA) in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the ability of lung surfactant protein D (SP-D) to bind to the HA and the ability of the corresponding chimeric virus to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Viruses expressing the hemagglutinin of pandemic viruses were associated with significant pathology in the lower respiratory tract, including acute inflammation, and showed low binding activity for SP-D. In contrast, the virus expressing the HA of a seasonal influenza strain induced only mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D.
doi:10.1016/j.virol.2011.01.029
PMCID: PMC3060949  PMID: 21334038
15.  Antiatherogenic and Anti-Ischemic Properties of Traditional Chinese Medicine Xinkeshu via Endothelial Protecting Function 
Including herbal medicine, complementary and alternative medicine (CAM) is popular worldwide. The traditional Chinese medicine xinkeshu has been widely used to treat coronary heart disease in China. This study was designed to investigate the protective effect and probable mechanism of xinkeshu tablet to atherosclerotic myocardial ischemia rabbit. Rabbits were divided into four groups (n = 12 each) and fed with different diet for 12 weeks: Control (standard diet), Model (high-cholesterol diet), XKS (high-cholesterol diet with 184.8 mg/kg/d xinkeshu), and Atorvastatin (high-cholesterol diet with 5.0 mg/kg/d atorvastatin). Plasma lipoprotein, ECG, endothelium-dependent vessel relaxation, histomorphological study, and expressions of eNOS and VCAM-1 on coronary arteries were assessed. The findings showed that, similar to atorvastatin, xinkeshu presented significant effects on rescuing endothelium-dependent vessel relaxation, inhibiting atherosclerotic progress, preventing myocardial ischemia, and changing eNOS and VCAM-1 expression. However, xinkeshu showed no lipoprotein lowering effect in hypercholesterolemia rabbits. The results of the present study indicated that xinkeshu exerted potent antiatherogenic and anti-ischemic properties on atherosclerotic myocardial ischemia rabbit. An endothelial protecting effect may be involved in the mechanism other than antihyperlipidemic effect.
doi:10.1155/2012/302137
PMCID: PMC3191825  PMID: 22007259
16.  Differential Changes of Aorta and Carotid Vasodilation in Type 2 Diabetic GK and OLETF Rats: Paradoxical Roles of Hyperglycemia and Insulin 
Experimental Diabetes Research  2011;2012:429020.
We investigated large vessel function in lean Goto-Kakizaki diabetic rats (GK) and Otsuka Long-Evans Tokushima Fatty diabetic rats (OLETF) with possible roles of hyperglycemia/hyperosmolarity and insulin. Both young and old GK showed marked hyperglycemia with normal insulin level and well-preserved endothelium-dependent and endothelium-independent vasodilation in aorta and carotid artery. There were significant elevations in endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive heme oxygenase (HO-1/HO-2) in GK. The endothelium-dependent vasodilation in GK was inhibited partly by NOS blockade and completely by simultaneous blocking of HO and NOS. In contrast, OLETF showed hyperinsulinemia and mild hyperglycemia but significant endothelium dysfunction beginning at early ages with concomitantly reduced eNOS. Insulin injection corrected hyperglycemia in GK but induced endothelium dysfunction and intima hyperplasia. Hyperglycemia/hyperosmolarity in vitro enhanced vessel eNOS/HO. We suggest that hyperinsulinemia plays a role in endothelium dysfunction in obese diabetic OLETF, while hyperglycemia/hyperosmolarity-induced eNOS/HO upregulation participates in the adaptation of endothelium function in lean diabetic GK.
doi:10.1155/2012/429020
PMCID: PMC3184433  PMID: 21977022
17.  Integrative Analysis of the Caenorhabditis elegans Genome by the modENCODE Project 
Gerstein, Mark B. | Lu, Zhi John | Van Nostrand, Eric L. | Cheng, Chao | Arshinoff, Bradley I. | Liu, Tao | Yip, Kevin Y. | Robilotto, Rebecca | Rechtsteiner, Andreas | Ikegami, Kohta | Alves, Pedro | Chateigner, Aurelien | Perry, Marc | Morris, Mitzi | Auerbach, Raymond K. | Feng, Xin | Leng, Jing | Vielle, Anne | Niu, Wei | Rhrissorrakrai, Kahn | Agarwal, Ashish | Alexander, Roger P. | Barber, Galt | Brdlik, Cathleen M. | Brennan, Jennifer | Brouillet, Jeremy Jean | Carr, Adrian | Cheung, Ming-Sin | Clawson, Hiram | Contrino, Sergio | Dannenberg, Luke O. | Dernburg, Abby F. | Desai, Arshad | Dick, Lindsay | Dosé, Andréa C. | Du, Jiang | Egelhofer, Thea | Ercan, Sevinc | Euskirchen, Ghia | Ewing, Brent | Feingold, Elise A. | Gassmann, Reto | Good, Peter J. | Green, Phil | Gullier, Francois | Gutwein, Michelle | Guyer, Mark S. | Habegger, Lukas | Han, Ting | Henikoff, Jorja G. | Henz, Stefan R. | Hinrichs, Angie | Holster, Heather | Hyman, Tony | Iniguez, A. Leo | Janette, Judith | Jensen, Morten | Kato, Masaomi | Kent, W. James | Kephart, Ellen | Khivansara, Vishal | Khurana, Ekta | Kim, John K. | Kolasinska-Zwierz, Paulina | Lai, Eric C. | Latorre, Isabel | Leahey, Amber | Lewis, Suzanna | Lloyd, Paul | Lochovsky, Lucas | Lowdon, Rebecca F. | Lubling, Yaniv | Lyne, Rachel | MacCoss, Michael | Mackowiak, Sebastian D. | Mangone, Marco | McKay, Sheldon | Mecenas, Desirea | Merrihew, Gennifer | Miller, David M. | Muroyama, Andrew | Murray, John I. | Ooi, Siew-Loon | Pham, Hoang | Phippen, Taryn | Preston, Elicia A. | Rajewsky, Nikolaus | Rätsch, Gunnar | Rosenbaum, Heidi | Rozowsky, Joel | Rutherford, Kim | Ruzanov, Peter | Sarov, Mihail | Sasidharan, Rajkumar | Sboner, Andrea | Scheid, Paul | Segal, Eran | Shin, Hyunjin | Shou, Chong | Slack, Frank J. | Slightam, Cindie | Smith, Richard | Spencer, William C. | Stinson, E. O. | Taing, Scott | Takasaki, Teruaki | Vafeados, Dionne | Voronina, Ksenia | Wang, Guilin | Washington, Nicole L. | Whittle, Christina M. | Wu, Beijing | Yan, Koon-Kiu | Zeller, Georg | Zha, Zheng | Zhong, Mei | Zhou, Xingliang | Ahringer, Julie | Strome, Susan | Gunsalus, Kristin C. | Micklem, Gos | Liu, X. Shirley | Reinke, Valerie | Kim, Stuart K. | Hillier, LaDeana W. | Henikoff, Steven | Piano, Fabio | Snyder, Michael | Stein, Lincoln | Lieb, Jason D. | Waterston, Robert H.
Science (New York, N.Y.)  2010;330(6012):1775-1787.
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
doi:10.1126/science.1196914
PMCID: PMC3142569  PMID: 21177976
18.  Biphasic effects of sodium danshensu on vessel function in isolated rat aorta 
Acta Pharmacologica Sinica  2010;31(4):421-428.
Aim:
To investigate the effects of sodium danshensu on vessel function in isolated rat aortic ring.
Methods:
Thoracic aortae from normal rats were isolated and equilibrated in organ bath with Krebs-Henseleit buffer and ring tension was recorded. Effects of sodium danshensu on basal tonus of the vessel and its effects on vessel contraction and relaxation with or without endothelium were observed.
Results:
In thoracic arteries under basal tonus, sodium danshensu (0.3–3 g/L) produced a dose-dependent transient contraction. In phenylephrine-precontracted thoracic arteries with or without endothelium, low concentration (0.1–0.3 g/L) of sodium danshensu produced a weak contraction, while high concentrations (1–3 g/L) produced a pronounced vasodilator after a transient vasocontraction. Pre-incubation with sodium danshensu could inhibit vessel contraction induced by phenylephrine and potassium chloride in a concentration-dependent way. Sodium danshensu inhibited phenylephrine- and CaCl2-induced vasoconstriction in Ca2+-free medium. Pre-incubation with tetraethylammonium, a non-selective K+ channel blocker, and apamin, a small-conductance calcium-activated K+ channel blocker partially antagonized the relaxation response induced by sodium danshensu. However, iberiotoxin (big-conductance calcium-sensitive K+ channel blocker), barium chloride (inward rectifier K+ channel blocker), and glibencalmide (ATP-sensitive K+ channel blocker) had no influence on the vasodialtion effect of sodium danshensu.
Conclusion:
Sodium danshensu showed a biphasic effects on vessel tension. While low dosage of sodium danshensu produced small contraction possibly through transient enhancement of Ca2+ influx, high dosage produced significant vasodilation mainly through promoting the opening of non-selective K+ channels and small-conductance calcium-sensitive K+ channels in the vascular smooth muscle cells.
doi:10.1038/aps.2010.24
PMCID: PMC4007672  PMID: 20228827
isolated aorta rings; sodium danshensu; vessel contraction and relaxation
19.  Characterization and analyses of multidrug resistance-associated protein 1 (MRP1/ABCC1) polymorphisms in Chinese population 
Pharmacogenetics and genomics  2009;19(3):206-216.
Multidrug resistance (MDR) is one of the major obstacles for successful cancer chemotherapy. Over-expression of ATP-binding cassette (ABC) transporters such as MRP1/ABCC1 has been suggested to cause MDR. In this study, we explored the distribution frequencies of four common single nucleotide polymorphisms (SNPs) of MRP1/ABCC1 in a mainland Chinese population and investigated whether these SNPs affect the expression and function of the MRP1/ABCC1. We found that the allelic frequencies of Cys43Ser (128G>C), Thr73Ile (218C>T), Arg723Gln (2168G>A) and Arg1058Gln (3173G>A) in mainland Chinese were 0.5%, 1.4%, 5.8% and 0.5%, respectively. These four SNPs were recreated by site-directed mutagenesis and tested for their effect on MRP1/ABCC1 expression and MDR function in HEK293 and CHO-K1 cells lines. We found that none of these mutations had any effect on MRP1/ABCC1 expression and trafficking, but that Arg723Gln mutation significantly reduced MRP1/ABCC1-mediated resistance to daunorubicin, doxorubicin, etoposide, vinblastine and vincristine. The Cys43Ser mutation did not affect all tested drugs resistance. On the other hand, the Thr73Ile mutation reduced resistance to methotrexate and etoposide while the Arg1058Gln mutation increased the response of two anthracycline drugs and etoposide in HEK293 and CHO-K1 cells as well as vinblastine and methotrexate in CHO-K1 cells. We conclude that the allelic frequency of the Arg723Gln mutation is relatively higher than other SNPs in mainland Chinese population and therefore this mutation significantly reduces MRP1/ABCC1 activity in MDR.
doi:10.1097/FPC.0b013e328323f680
PMCID: PMC2667206  PMID: 19214144
ABC transporter; MDR; Multidrug resistance-associated protein 1 (MRP1/ABCC1); drug resistance; genetic polymorphism
20.  Genome-Wide Identification of Binding Sites Defines Distinct Functions for Caenorhabditis elegans PHA-4/FOXA in Development and Environmental Response 
PLoS Genetics  2010;6(2):e1000848.
Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.
Author Summary
The C. elegans transcription factor PHA-4 is a member of the highly conserved FOXA family of transcription factors. These factors act as master regulators of organ development by controlling how genes are turned off and on as tissues are formed. Additionally they regulate genes in response to nutrient levels and control both longevity and survival of the organism. However, the extent to which these factors control similar or distinct gene targets for each of these functions is unknown. For this reason, we have used the technique of chromatin immunoprecipitation followed by deep sequencing (ChIP–Seq), to define the target binding sites of PHA-4 on a genome-wide scale, when it is either functioning as an organ identity regulator or in response to environmental stress. Our data clearly demonstrate distinct sets of biologically relevant target genes for the transcription factor PHA-4 under these two different conditions. Not only have we defined PHA-4 targets, but we established an experimental ChIP–Seq pipeline to facilitate the identification of binding sites for many transcription factors in the future.
doi:10.1371/journal.pgen.1000848
PMCID: PMC2824807  PMID: 20174564
21.  Aberrant Expression of ID2 protein and its correlation with EBV-LMP1 and P16(INK4A) in Classical Hodgkin Lymphoma in China 
BMC Cancer  2008;8:379.
Background
The relationships between the expression of ID2, EBV-LMP1 and P16(INK4A) in Chinese classical Hodgkin lymphoma are unknown and need exploring.
Methods
Samples of classical Hodgkin lymphoma from 60 Chinese patients were analyzed for the expression of ID2, EBV-LMP1 and p16(INK4A) proteins by immunohistochemistry.
Results
ID2 protein was expressed in 83.3% of this group of classical Hodgkin lymphoma, staining strongly in both cytoplasm and nucleus of the Hodgkin and Reed-Sternberg (HRS) cells. EBV-LMP1 and P16(INK4A) were overexpressed in 85.0% and 71.7% of Hodgkin lymphoma, respectively. EBV-LMP1 was noted in the cytoplasm, membrane and nucleus of HRS cells; P16(INK4A) was in the nucleus and cytoplasm. Microscopically, ID2, EBV-LMP1 and P16(INK4A) staining distinguished the HRS cells from the complex background of lymphocytes. ID2 was positively correlated with EBV-LMP1(P < 0.01), but P16(INK4A) was inversely related to EBV-LMP1 (P < 0.05).
Conclusion
It is suggested that ID2, EBV-LMP1 and P16(INK4A) could play an important role in the evolution of classical Hodgkin lymphoma, and be considered as potential adjunct markers to identify HRS cells in diagnosis.
doi:10.1186/1471-2407-8-379
PMCID: PMC2625365  PMID: 19099554
22.  Bcl-2–related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury 
Journal of Clinical Investigation  2005;115(4):1039-1048.
Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11–induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11–induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11– and VEGF-induced cytoprotection.
doi:10.1172/JCI200523004
PMCID: PMC1070412  PMID: 15841185
23.  Phosphorylation of the MAPKKK Regulator Ste50p in Saccharomyces cerevisiae: a Casein Kinase I Phosphorylation Site Is Required for Proper Mating Function1  
Eukaryotic Cell  2003;2(5):949-961.
The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.
doi:10.1128/EC.2.5.949-961.2003
PMCID: PMC219381  PMID: 14555477
24.  Paradoxical effects of streptozotocin-induced diabetes on endothelial dysfunction in stroke-prone spontaneously hypertensive rats 
The Journal of Physiology  2011;589(21):5153-5165.
Non-technical summary
Elevated blood glucose is generally regarded as one of the risk factors that lead to coronary heart disease in patients with type 2 diabetes. However, our studies show that after inducing short-term damage, high blood glucose subsequently provides paradoxical protection for vessel function of animals with high blood pressure. Vessels can adapt to sustained high blood glucose and produce different stress proteins to counteract, to some extent, the damage brought about by hypertension. The results help us understand part of the basis for vessel adaptation in diabetes. The implication for treatment of diabetes is that if the patients have long-standing diabetes and established cardiovascular disease, the target of blood glucose lowering should be less stringent and reached gradually to avoid abrupt cancellation of the pre-existing adaptations.
Abstract
Although both diabetes and hypertension are risk factors for cardiovascular disease, the role of hyperglycaemia per se in endothelial dysfunction is controversial. This study was designed to examine whether hyperglycaemia, or streptozotocin-induced diabetes, could aggravate endothelial dysfunction in stroke-prone spontaneously hypertensive rats (SHRSP). Hyperglycaemia was induced by streptozotocin in 2-month-old SHRSP and age-matched normotensive Wistar–Kyoto (WKY) rats. The aorta was isolated 8 weeks after induction of hyperglycaemia to record its function and to examine its morphology with transmission electron microscopy. Endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive haem oxygenase (HO-1/HO-2) levels were determined with Western blotting. Aortic endothelial function and production of reactive oxygen species and nitric oxide were assayed after incubation in vitro in hyperglycaemic, hyperosmolar solution. Streptozotocin-induced diabetes of 8 weeks duration did not result in endothelial dysfunction in normotensive WKY rats. In contrast, hyperglycaemic WKY rats showed significantly enhanced endothelium-dependent vasodilatation, which was abrogated by simultaneous blocking of NOS and HO. The enhanced vasodilatation was associated with elevation of vascular eNOS and HO-1. Significant endothelial dysfunction and massive macrophage–monocyte infiltration were found in SHRSP aorta (the ratio of the number of macrophages to endothelial cells in the intima, expressed as a percentage, was 20.9 ± 2.8% in SHRSP versus 1.9 ± 0.5% in WKY rats, P < 0.01), which was attenuated significantly in hyperglycaemic SHRSP (11.3 ± 1.6%, P < 0.01 versus SHRSP). Acute hyperglycaemia (10 min) aggravated endothelial dysfunction in SHRSP, with a marked increase in intracellular reactive oxygen species and NO production. Sustained in vitro incubation in hyperglycaemic/hyperosmolar conditions (addition of an extra 50 mmol L−1 of glucose or mannitol to the usual buffer, to produce a final osmolarity of 350 mosmol L−1) for 5 h enhanced endothelium-dependent vasodilatation, with elevated vessel NO production and upregulation of eNOS/HO-1 proteins. Sustained hyperglycaemia does not aggravate endothelial dysfunction and macrophage infiltration in SHRSP. Hyperglycaemia/hyperosmolarity-induced upregulation of eNOS and HO-1 may play a role in this paradoxical adaptation of endothelial function.
doi:10.1113/jphysiol.2011.213686
PMCID: PMC3225671  PMID: 21930604

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