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1.  Inducible Cardiomyocyte-Specific Gene Disruption Directed by the Rat Tnnt2 Promoter in the Mouse 
We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by co-injection of two transgenes, a “reverse” tetracycline-controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline-responsive promoter (TetO). Here, Tnnt2-rtTA activated TetO-Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte-specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2-rtTA;TetO-Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods.
doi:10.1002/dvg.20573
PMCID: PMC2806493  PMID: 20014345
cardiomyocyte; Cre recombinase; doxycycline; rtTA; Tnnt2
2.  Fog2 is critical for cardiac function and maintenance of coronary vasculature in the adult mouse heart  
The Journal of Clinical Investigation  2009;119(6):1462-1476.
Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of heart disease. While the transcriptional regulator friend of Gata 2 (FOG2) is known to be essential for heart morphogenesis and coronary development, its tissue-specific function has not been previously investigated. Additionally, little is known about the role of FOG2 in the adult heart. Here we used spatiotemporally regulated inactivation of Fog2 to delineate its function in both the embryonic and adult mouse heart. Early cardiomyocyte-restricted loss of Fog2 recapitulated the cardiac and coronary defects of the Fog2 germline murine knockouts. Later cardiomyocyte-restricted loss of Fog2 (Fog2MC) did not result in defects in cardiac structure or coronary vessel formation. However, Fog2MC adult mice had severely depressed ventricular function and died at 8–14 weeks. Fog2MC adult hearts displayed a paucity of coronary vessels, associated with myocardial hypoxia, increased cardiomyocyte apoptosis, and cardiac fibrosis. Induced inactivation of Fog2 in the adult mouse heart resulted in similar phenotypes, as did ablation of the FOG2 interaction with the transcription factor GATA4. Loss of the FOG2 or FOG2-GATA4 interaction altered the expression of a panel of angiogenesis-related genes. Collectively, our data indicate that FOG2 regulates adult heart function and coronary angiogenesis.
doi:10.1172/JCI38723
PMCID: PMC2689123  PMID: 19411759
3.  Knockdown of GRP78 Promotes Apoptosis in Pancreatic Acinar Cells and Attenuates the Severity of Cerulein and LPS Induced Pancreatic Inflammation 
PLoS ONE  2014;9(3):e92389.
Acute pancreatitis (AP) is a potentially lethal disease characterized by inflammation and parenchymal cell death; also, the severity of AP correlates directly with necrosis and inversely with apoptosis. However, mechanisms of regulating cell death in AP remain unclear. The endoplasmic reticulum (ER) chaperone protein GRP78 has anti-apoptotic properties, in addition to modulating ER stress responses. This study used RNA interference (RNAi) approach to investigate the potential role of GRP78 in regulating apoptosis during AP. In vitro models of AP were successfully developed by treating AR42J cells with cerulein or cerulein plus lipoplysaccharide (LPS). There was more pancreatic inflammation and less apoptosis with the cerulein plus LPS treatment. Furthermore, knockdown of GRP78 expression markedly promoted apoptosis and reduced necrosis in pancreatic acinar cells. This was accomplished by enhancing the activation of caspases and inhibiting the activity of X-linked inhibitor of apoptosis protein (XIAP), as well as a receptor interacting protein kinase-1(RIPK1), which is a key mediator of necrosis. This attenuated the severity of pancreatic inflammation, especially after cerulein plus LPS treatment. In conclusion, these findings indicate that GRP78 plays an anti-apoptotic role in regulating the cell death response during AP. Therefore, GRP78 is a potential therapeutic target for AP.
doi:10.1371/journal.pone.0092389
PMCID: PMC3958537  PMID: 24643222
4.  Promotion of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium 
AIM
To investigate the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium (Ti) surface.
METHODS
The chimeric peptide RKLPDAPRGDN (minTBP-1-PRGDN) was synthesized by connecting RKLPDA (minTBP-1) to the N-terminal of PRGDN, the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim- ethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide up-taking methods. The secretion of type I collagen were determined using an ELISA kit.
RESULTS
The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti (1.40±0.03 folds, P=0.003), to promote the proliferation (1.26±0.05 folds, P=0.014) and the synthesis of type I collagen (1.530±0.128, P=0.008). MinTBP-1 at the same concentration could only promote the attachment (1.13±0.04 folds, P=0.020) and proliferation(1.15±0.06 folds, P=0.021), while PRGDN had no significant influence (P>0.05).
CONCLUSION
Our data shows that the novel chimeric peptide minTBP-1-PRGDN could promote the attachment, proliferation and type I collagen synthesis of human keratocytes on the surface of Ti.
doi:10.3980/j.issn.2222-3959.2014.01.04
PMCID: PMC3949453  PMID: 24634858
minTBP-1-PRGDN; titanium; cell attachment; proliferation; type I collagen
5.  Crystal structure of an LRR protein with two solenoids 
Cell Research  2012;23(2):303-305.
doi:10.1038/cr.2012.159
PMCID: PMC3567821  PMID: 23147790
6.  A Model of Two-Way Selection System for Human Behavior 
PLoS ONE  2014;9(1):e81424.
Two-way selection is a common phenomenon in nature and society. It appears in the processes like choosing a mate between men and women, making contracts between job hunters and recruiters, and trading between buyers and sellers. In this paper, we propose a model of two-way selection system, and present its analytical solution for the expectation of successful matching total and the regular pattern that the matching rate trends toward an inverse proportion to either the ratio between the two sides or the ratio of the state total to the smaller group's people number. The proposed model is verified by empirical data of the matchmaking fairs. Results indicate that the model well predicts this typical real-world two-way selection behavior to the bounded error extent, thus it is helpful for understanding the dynamics mechanism of the real-world two-way selection system.
doi:10.1371/journal.pone.0081424
PMCID: PMC3890283  PMID: 24454687
7.  Prioritization of Putative Metabolite identifications in LC-MS/MS Experiments Using a Computational Pipeline 
Proteomics  2013;13(2):248-260.
One of the major bottle-necks in current LC-MS based metabolomic investigations is metabolite identification. An often-used approach is to first look up metabolites from databases through peak mass, followed by verification of the obtained putative identifications using MS/MS data. However, the mass-based search may provide inappropriate putative identifications when the observed peak is from isotopes, fragments or adducts. In addition, a large fraction of peaks is often left with multiple putative identifications. To differentiate these putative identifications, manual verification of metabolites through comparison between biological samples and authentic compounds is necessary. However, such experiments are laborious especially when multiple putative identifications are encountered. It is desirable to use computational approaches to obtain more reliable putative identifications and prioritize them before performing experimental verification of the metabolites. In this paper, a computational pipeline is proposed to assist metabolite identification with improved metabolome coverage and prioritization capability. Multile publicly available software tools and databases, along with in-house developed algorithms, are utilized to fully exploit the information acquired from LC-MS/MS experiments.. The pipeline is successfully applied to identify metabolites on the basis of LC-MS as well as MS/MS data. Using accurate masses, retention time values, MS/MS spectra and metabolic pathways/networks, more appropriate putative identifications are retrieved and prioritized to guide subsequent metabolite verification experiments.
doi:10.1002/pmic.201200306
PMCID: PMC3719968  PMID: 23307777
metabolomics; ion annotation; isotopic pattern analysis; pathway and network analysis; spectral interpretation; spectral matching
8.  Analysis of Intervention Strategies for Inhalation Exposure to Polycyclic Aromatic Hydrocarbons and Associated Lung Cancer Risk Based on a Monte Carlo Population Exposure Assessment Model 
PLoS ONE  2014;9(1):e85676.
It is difficult to evaluate and compare interventions for reducing exposure to air pollutants, including polycyclic aromatic hydrocarbons (PAHs), a widely found air pollutant in both indoor and outdoor air. This study presents the first application of the Monte Carlo population exposure assessment model to quantify the effects of different intervention strategies on inhalation exposure to PAHs and the associated lung cancer risk. The method was applied to the population in Beijing, China, in the year 2006. Several intervention strategies were designed and studied, including atmospheric cleaning, smoking prohibition indoors, use of clean fuel for cooking, enhancing ventilation while cooking and use of indoor cleaners. Their performances were quantified by population attributable fraction (PAF) and potential impact fraction (PIF) of lung cancer risk, and the changes in indoor PAH concentrations and annual inhalation doses were also calculated and compared. The results showed that atmospheric cleaning and use of indoor cleaners were the two most effective interventions. The sensitivity analysis showed that several input parameters had major influence on the modeled PAH inhalation exposure and the rankings of different interventions. The ranking was reasonably robust for the remaining majority of parameters. The method itself can be extended to other pollutants and in different places. It enables the quantitative comparison of different intervention strategies and would benefit intervention design and relevant policy making.
doi:10.1371/journal.pone.0085676
PMCID: PMC3885750  PMID: 24416436
9.  LC-MS Based Serum Metabolomics for Identification of Hepatocellular Carcinoma Biomarkers in Egyptian Cohort 
Journal of proteome research  2012;11(12):5914-5923.
Although hepatocellular carcinoma (HCC) has been subjected to continuous investigation and its symptoms are well known, early-stage diagnosis of this disease remains difficult and the survival rate after diagnosis is typically very low (3–5%). Early and accurate detection of metabolic changes in the sera of patients with liver cirrhosis can help improve the prognosis of HCC and lead to a better understanding of its mechanism at the molecular level, thus providing patients with in-time treatment of the disease. In this study, we compared metabolite levels in sera of 40 HCC patients and 49 cirrhosis patients from Egypt by using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from cirrhotic controls are selected by statistical methods. Putative metabolite identifications for these ions are obtained through mass-based database search. The identities of some of the putative identifications are verified by comparing their MS/MS fragmentation patterns and retention times with those from authentic compounds. Finally, the serum samples are reanalyzed for quantitation of selected metabolites along with other metabolites previously selected as candidate biomarkers of HCC. This quantitation was performed using isotope dilution by selected reaction monitoring (SRM) on a triple quadrupole linear ion trap (QqQLIT) coupled to UPLC. Statistical analysis of the UPLC-QTOF data identified 274 monoisotopic ion masses with statistically significant differences in ion intensities between HCC cases and cirrhotic controls. Putative identifications were obtained for 158 ions by mass based search against databases. We verified the identities of selected putative identifications including glycholic acid (GCA), glycodeoxycholic acid (GDCA), 3beta, 6beta-dihydroxy-5beta-cholan-24-oic acid, oleoyl carnitine, and Phe-Phe. SRM-based quantitation confirmed significant differences between HCC and cirrhotic controls in metabolite levels of bile acid metabolites, long chain carnitines and small peptide. Our study provides useful insight into appropriate experimental design and computational methods for serum biomarker discovery using LC-MS/MS based metabolomics. This study has led to the identification of candidate biomarkers with significant changes in metabolite levels between HCC cases and cirrhotic controls. This is the first MS-based metabolic biomarker discovery study on Egyptian subjects that led to the identification of candidate metabolites that discriminate early stage HCC from patients with liver cirrhosis.
doi:10.1021/pr300673x
PMCID: PMC3719870  PMID: 23078175
Hepatocellular carcinoma; liver cirrhosis; metabolic biomarker; cancer biomarker discovery; selected reaction monitoring; isotope dilution; mass spectrometry
10.  Endocardial Cells Form the Coronary Arteries by Angiogenesis through Myocardial-Endocardial VEGF Signaling 
Cell  2012;151(5):1083-1096.
Summary
The origins and developmental mechanisms of coronary arteries are incompletely understood. We showed here by fate mapping, clonal analysis and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.
doi:10.1016/j.cell.2012.10.023
PMCID: PMC3508471  PMID: 23178125
11.  Peritruncal Coronary Endothelial Cells Contribute to Proximal Coronary Artery Stems and Their Aortic Orifices in the Mouse Heart 
PLoS ONE  2013;8(11):e80857.
Avian embryo experiments proved an ingrowth model for the coronary artery connections with the aorta. However, whether a similar mechanism applies to the mammalian heart still remains unclear. Here we analyzed how the main coronary arteries and their orifices form during murine heart development. Apelin (Apln) is expressed in coronary vascular endothelial cells including peritruncal endothelial cells. By immunostaining, however, we did not find Apln expression in endothelial cells of the aorta during the period of coronary vessel development (E10.5 to E15.5). As a result of this unique expression difference, AplnCreERT2/+ genetically labels nascent coronary vessels forming on the heart, but not the aorta endothelium when pulse activated by tamoxifen injection at E10.5. This allowed us to define the temporal contribution of these distinct endothelial cell populations to formation of the murine coronary artery orifice. We found that the peritruncal endothelial cells were recruited to form the coronary artery orifices. These cells penetrate the wall of aorta and take up residence in the aortic sinus of valsalva. In conclusion, main coronary arteries and their orifices form through the recruitment and vascular remodeling of peritruncal endothelial cells in mammalian heart.
doi:10.1371/journal.pone.0080857
PMCID: PMC3836754  PMID: 24278332
12.  Paracrine Effects of Bone Marrow–Derived Endothelial Progenitor Cells: Cyclooxygenase-2/Prostacyclin Pathway in Pulmonary Arterial Hypertension 
PLoS ONE  2013;8(11):e79215.
Background
Endothelial dysfunction is the pathophysiological characteristic of pulmonary arterial hypertension (PAH). Some paracrine factors secreted by bone marrow–derived endothelial progenitor cells (BMEPCs) have the potential to strengthen endothelial integrity and function. This study investigated whether BMEPCs have the therapeutic potential to improve monocrotaline (MCT)-induced PAH via producing vasoprotective substances in a paracrine fashion.
Methods and Results
Bone marrow-derived mononuclear cells were cultured for 7 days to yield BMEPCs. 24 hours or 3 weeks after exposure to BMEPCs in vitro or in vivo, the vascular reactivity, cyclooxygenase-2 (COX-2) expression, prostacyclin (PGI2) and cAMP release in isolated pulmonary arteries were examined respectively. Treatment with BMEPCs could improve the relaxation of pulmonary arteries in MCT-induced PAH and BMEPCs were grafted into the pulmonary bed. The COX-2/prostacyclin synthase (PGIS) and its progenies PGI2/cAMP were found to be significantly increased in BMEPCs treated pulmonary arteries, and this action was reversed by a selective COX-2 inhibitor, NS398. Moreover, the same effect was also observed in conditioned medium obtained from BMEPCs culture.
Conclusions
Implantation of BMEPCs effectively ameliorates MCT-induced PAH. Factors secreted in a paracrine fashion from BMEPCs promote vasoprotection by increasing the release of PGI2 and level of cAMP.
doi:10.1371/journal.pone.0079215
PMCID: PMC3832480  PMID: 24260171
13.  A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress 
Journal of Experimental Botany  2013;65(1):143-158.
A novel tomato chloroplast-targeted DnaJ protein, LeCDJ1 was found to contribute to the maintenance of photosystem II under chilling stress and this maintenance effect was, at least partially, independent of D1 protein synthesis
DnaJ proteins act as essential molecular chaperones in protein homeostasis and protein complex stabilization under stress conditions. The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1), whose expression was upregulated by treatment at 4 and 42 °C, and with high light, NaCl, polyethylene glycol, and H2O2, were investigated here using sense and antisense transgenic tomatoes. The sense plants exhibited not only higher chlorophyll content, fresh weight and net photosynthetic rate, but also lower accumulation of reactive oxygen species and membrane damage under chilling stress. Moreover, the maximal photochemistry efficiency of photosystem II (PSII) (F v/F m) and D1 protein content were higher in the sense plants and lower in the antisense plants, and the photoinhibitory quenching was lower in the sense plants and higher in the antisense plants, suggesting that the inhibition of PSII was less severe in the sense plants and more severe in the antisense plants compared with the wild type. Furthermore, the PSII protein complexes were also more stable in the sense plants. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher F v/F m, D1 protein content and PSII stability than the SM-untreated antisense plants. This finding suggested that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, chloroplast heat-shock protein 70 was identified as the partner of LeCDJ1. These results indicate that LeCDJ1 has essential functions in maintaining PSII under chilling stress.
doi:10.1093/jxb/ert357
PMCID: PMC3883286  PMID: 24227338
Chilling stress; D1 protein; DnaJ protein; Hsp70; photosystem II; tomato.
14.  Genetic Cre-loxP assessment of epicardial cell fate using Wt1-driven Cre Alleles 
Circulation research  2012;111(11):e276-e280.
Rationale
Wt1-Cre based tools are important reagents for studying epicardial cell fate and gene function.
Objective
To better describe the properties of Wt1-Cre based tools to enhance their use in Cre-loxP based experiments.
Methods and Results
In contrast to recently reported results, we show that constitutive Wt1GFPCre in combination with certain Cre-activated reporters can be used to trace (pro)epicardial cell fate. Wt1CreERT2 can be efficiently induced by tamoxifen administration. We show substantial labeling of coronary endothelial cells when induction is performed at late but not early stages of heart development.
Conclusions
Wt1-based Cre alleles are useful tools for genetic lineage tracing of epicardial cells and mesothelium of other organs. Use of these tools with proper understanding of their properties and limitations enables genetic labeling of epicardial cells and their derivatives.
doi:10.1161/CIRCRESAHA.112.275784
PMCID: PMC3532881  PMID: 23139287
lineage tracing; Wt1; epicardium
15.  Hyperglycemia-Induced Inhibition of DJ-1 Expression Compromised the Effectiveness of Ischemic Postconditioning Cardioprotection in Rats 
Ischemia postconditioning (IpostC) is an effective way to alleviate ischemia and reperfusion injury; however, the protective effects seem to be impaired in candidates with diabetes mellitus. To gain deep insight into this phenomenon, we explored the role of DJ-1, a novel oncogene, that may exhibit powerful antioxidant capacity in postconditioning cardioprotection in a rat model of myocardial ischemia reperfusion injury. Compared with normal group, cardiac DJ-1 was downregulated in diabetes. Larger postischemic infarct size as well as exaggeration of oxidative stress was observed, while IpostC reversed the above changes in normal but not in diabetic rats. DJ-1 was increased after ischemia and postconditioning contributed to a further elevation; however, no alteration of DJ-1 was documented in all subgroups of diabetic rats. Alteration of the cardioprotective PI3K/Akt signaling proteins may be responsible for the ineffectiveness of postconditioning in diabetes. There is a positive correlation relationship between p-Akt and DJ-1 but a negative correlation between infarct size and DJ-1, which may partially explain the interaction of DJ-1 and IpostC cardioprotection. Our result indicates a beneficial role of DJ-1 in myocardial ischemia reperfusion. Downregulation of cardiac DJ-1 may be responsible for the compromised diabetic heart responsiveness to IpostC cardioprotection.
doi:10.1155/2013/564902
PMCID: PMC3835206  PMID: 24303086
16.  Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide 
Cell Research  2013;23(11):1326-1329.
doi:10.1038/cr.2013.131
PMCID: PMC3817550  PMID: 24126715
17.  Efficacy of intensity-modulated radiotherapy combined with chemotherapy or surgery in locally advanced squamous cell carcinoma of the head-and-neck 
Objectives
Long-term locoregional control following intensity-modulated radiotherapy (IMRT) for locally advanced squamous cell carcinoma of the head-and-neck (SCCHN) remains challenging. This study aimed to assess the efficacy and toxicity of IMRT with and without chemotherapy or surgery in locally advanced SCCHN.
Materials and methods
Between January 2007 and January 2011, 61 patients with locally advanced SCCHN were treated with curative IMRT in the Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University; 28% underwent definitive IMRT and 72% postoperative IMRT, combined with simultaneous cisplatin-based chemotherapy in 58%. The mean doses of definitive and postoperative IMRT were 70.8 Gy (range, 66–74 Gy). Outcomes were analyzed using Kaplan–Meier curves. Acute and late toxicities were graded according to Radiation Therapy Oncology Group/European Organisation for Research and Treatment of Cancer radiation morbidity scoring criteria.
Results
At a median follow-up of 35 months, 3-year local recurrence-free survival (LRFS), regional recurrence-free survival (RRFS), distant metastasis-free survival (DMFS), disease-free survival (DFS), and overall survival (OS) were 83.8%, 86.1%, 82.4%, 53.2%, and 62%, respectively. Postoperative IMRT (n = 44, 72%) had significantly higher LRFS/OS/DMFS than definitive IMRT (n = 17, 28%; P < 0.05). IMRT combined with chemotherapy (n = 35, 58%) had significantly higher LRFS/OS/DMFS than IMRT alone (n = 26, 42%; P < 0.05). One year after radiotherapy, the incidence of xerostomia of grade 1, 2, or 3 was 13.1%, 19.7%, and 1.6%, respectively. No grade 4 acute or late toxicity was observed.
Conclusion
IMRT combined with surgery or chemotherapy achieved excellent long-term locoregional control and OS in locally advanced SCCHN, with acceptable early toxicity and late side-effects.
doi:10.2147/BTT.S48664
PMCID: PMC3804514  PMID: 24204121
SCCHN; IMRT; surgery; chemotherapy; prognosis analysis
18.  ¹H NMR-based metabolic profiling of human rectal cancer tissue 
Molecular Cancer  2013;12:121.
Background
Rectal cancer is one of the most prevalent tumor types. Understanding the metabolic profile of rectal cancer is important for developing therapeutic approaches and molecular diagnosis.
Methods
Here, we report a metabonomics profiling of tissue samples on a large cohort of human rectal cancer subjects (n = 127) and normal controls (n = 43) using 1H nuclear magnetic resonance (1H NMR) based metabonomics assay, which is a highly sensitive and non-destructive method for the biomarker identification in biological systems. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA) were applied to analyze the 1H-NMR profiling data to identify the distinguishing metabolites of rectal cancer.
Results
Excellent separation was obtained and distinguishing metabolites were observed among the different stages of rectal cancer tissues (stage I = 35; stage II = 37; stage III = 37 and stage IV = 18) and normal controls. A total of 38 differential metabolites were identified, 16 of which were closely correlated with the stage of rectal cancer. The up-regulation of 10 metabolites, including lactate, threonine, acetate, glutathione, uracil, succinate, serine, formate, lysine and tyrosine, were detected in the cancer tissues. On the other hand, 6 metabolites, including myo-inositol, taurine, phosphocreatine, creatine, betaine and dimethylglycine were decreased in cancer tissues. These modified metabolites revealed disturbance of energy, amino acids, ketone body and choline metabolism, which may be correlated with the progression of human rectal cancer.
Conclusion
Our findings firstly identify the distinguishing metabolites in different stages of rectal cancer tissues, indicating possibility of the attribution of metabolites disturbance to the progression of rectal cancer. The altered metabolites may be as potential biomarkers, which would provide a promising molecular diagnostic approach for clinical diagnosis of human rectal cancer. The role and underlying mechanism of metabolites in rectal cancer progression are worth being further investigated.
doi:10.1186/1476-4598-12-121
PMCID: PMC3819675  PMID: 24138801
19.  Research of the Methylation Status of miR-124a Gene Promoter among Rheumatoid Arthritis Patients 
Objective. To analyze the methylation status of miR-124a loci in synovial tissues of rheumatoid arthritis (RA) patients using methylation-specific polymerase chain reaction (MSP). Materials and Methods. DNA obtained from the frozen tissue of 7 RA samples, 6 osteoarthritis (OA) samples, and 3 healthy controls were undergoing bisulfite conversion and then analyzed for miR-124a promoter methylation using MSP assay. Results. miR-124-a1 and miR-124-a2 promoter methylation were both seen in 71.4% of RA samples compared to 16.7% of OA samples. miR-124-a3 promoter methylation was seen in 57.1% of RA samples and 0% of OA samples. All the three loci were unmethylated in 3 healthy controls. Conclusion. The methylation status of miR-124a seen in this study concurs with that reported in tumor cells, indicating epigenetic dysregulation constituents, a mechanism in the development of rheumatoid arthritis.
doi:10.1155/2013/524204
PMCID: PMC3810484  PMID: 24223605
20.  Developmental basis for filamin-A-associated myxomatous mitral valve disease 
Cardiovascular Research  2012;96(1):109-119.
Aims
We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development.
Methods and results
Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype.
Conclusions
These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.
doi:10.1093/cvr/cvs238
PMCID: PMC3444235  PMID: 22843703
Filamin-A; Serotonin; Myxomatous valve disease; Transglutaminase-2; Valve maturation
21.  Utilization of Metabolomics to Identify Serum Biomarkers for Hepatocellular Carcinoma in Patients with Liver Cirrhosis 
Analytica chimica acta  2012;743C:90-100.
Characterizing the metabolic changes pertaining to hepatocellular carcinoma (HCC) in patients with liver cirrhosis is believed to contribute towards early detection, treatment, and understanding of the molecular mechanisms of HCC. In this study, we compare metabolite levels in sera of 78 HCC cases with 184 cirrhotic controls by using ultra performance liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from patients with cirrhosis are selected by parametric and non-parametric statistical methods. Putative metabolite identifications for these ions are obtained through mass-based database search. Verification of the identities of selected metabolites is conducted by comparing their MS/MS fragmentation patterns and retention time with those from authentic compounds. Quantitation of these metabolites is performed in a subset of the serum samples (10 HCC and 10 cirrhosis) using isotope dilution by selected reaction monitoring (SRM) on triple quadrupole linear ion trap (QqQLIT) and triple quadrupole (QqQ) mass spectrometers. The results of this analysis confirm that metabolites involved in sphingolipid metabolism and phospholipid catabolism such as sphingosine-1-phosphate (S-1-P) and lysophosphatidylcholine (lysoPC 17:0) are up-regulated in sera of HCC vs. those with liver cirrhosis. Down-regulated metabolites include those involved in bile acid biosynthesis (specifically cholesterol metabolism) such as glycochenodeoxycholic acid 3-sulfate (3-sulfo-GCDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA), and taurochenodeoxycholate (TCDCA). These results provide useful insights into HCC biomarker discovery utilizing metabolomics as an efficient and cost-effective platform. Our work shows that metabolomic profiling is a promising tool to identify candidate metabolic biomarkers for early detection of HCC cases in high risk population of cirrhotic patients.
doi:10.1016/j.aca.2012.07.013
PMCID: PMC3419576  PMID: 22882828
Metabolomics; biomarkers; liquid chromatography-mass spectrometry; hepatocellular carcinoma; selected reaction monitoring
22.  Upregulated MicroRNA-155 Expression in Peripheral Blood Mononuclear Cells and Fibroblast-Like Synoviocytes in Rheumatoid Arthritis 
Objective. This study was to screen for the miRNAs differently expressed in peripheral blood mononuclear cells (PBMC) of RA, to further identify the expression of miR-155 in RA PBMC and fibroblast-like synoviocytes (FLS), and to evaluate the function of miR-155 in RA-FLS. Methods. Microarray was used to screen for differentially expressed miRNAs in RA PBMC. miR-155 expression in PBMC and FLS of RA were identified by real-time PCR. Enforced overexpression and downexpression of miR-155 were used to investigate the function of miR-155 in RA-FLS. Expression of IKBKE which was previously identified as the actual target of miR-155 was examined by Western blot and real-time PCR in RA-FLS. Results. miR-155 levels were increased in both PBMC and FLS of RA and could be induced by TNF-α. Upregulation of miR-155 decreased MMP-3 levels and suppressed proliferation and invasion of RA-FLS. Inverse relationship between the expressions of miR-155 and the MMPs production-related protein IKBKE was found. Conclusion. An inflammatory milieu may alter miRNA expression profiles in rheumatoid arthritis. miR-155 is upregulated in RA-FLS, and it may be a protective factor against the inflammatory effect in part by attenuating expression of IKBKE.
doi:10.1155/2013/296139
PMCID: PMC3789322  PMID: 24151514
23.  A Small Molecule Inhibitor of Ubiquitin-Specific Protease-7 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Bortezomib Resistance 
Cancer cell  2012;22(3):345-358.
Summary
Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.
doi:10.1016/j.ccr.2012.08.007
PMCID: PMC3478134  PMID: 22975377
24.  Equal modulation of endothelial cell function by four distinct tissue-specific mesenchymal stem cells 
Angiogenesis  2012;15(3):443-455.
Mesenchymal stem cells (MSCs) can generate multiple end-stage mesenchymal cell types and constitute a promising population of cells for regenerative therapies. Additionally, there is increasing evidence supporting other trophic activities of MSCs, including the ability to enable formation of vasculature in vivo. Although MSCs were originally isolated from the bone marrow, the presence of these cells in the stromal vascular fraction of multiple adult tissues has been recently recognized. However, it is unknown whether the capacity to modulate vasculogenesis is ubiquitous to all MSCs regardless of their tissue of origin. Here, we demonstrated that tissue-resident MSCs isolated from four distinct tissues have equal capacity to modulate endothelial cell function, including formation of vascular networks in vivo. MSCs were isolated from four murine tissues, including bone marrow, white adipose tissue, skeletal muscle, and myocardium. In culture, all four MSC populations secreted a plethora of pro-angiogenic factors that unequivocally induced proliferation, migration, and tube formation of endothelial colony-forming cells (ECFCs). In vivo, co-implantation of MSCs with ECFCs into mice generated an extensive network of blood vessels with ECFCs specifically lining the lumens and MSCs occupying perivascular positions. Importantly, there were no differences among all four MSCs evaluated. Our studies suggest that the capacity to modulate the formation of vasculature is a ubiquitous property of all MSCs, irrespective of their original anatomical location. These results validate multiple tissues as potential sources of MSCs for future cell-based vascular therapies.
doi:10.1007/s10456-012-9272-2
PMCID: PMC3409933  PMID: 22527199
Mesenchymal stem cells; Endothelial cell; Vasculogenesis; Pericytes; Endothelial progenitor cells
25.  Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries 
Cell Research  2013;23(9):1075-1090.
Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.
doi:10.1038/cr.2013.83
PMCID: PMC3760626  PMID: 23797856
coronary artery; origin; development; subepicardial endothelial cell; angiogenesis

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