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1.  Alteration of Histone Acetylation Pattern during Long-Term Serum-Free Culture Conditions of Human Fetal Placental Mesenchymal Stem Cells 
PLoS ONE  2015;10(2):e0117068.
Increasing evidence suggests that the mesenchymal stem cells (MSCs) derived from placenta of fetal origin (fPMSCs) are superior to MSCs of other sources for cell therapy. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications, during which MSCs may undergo genetic and/or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic and epigenetic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical settings. To date, the genetic and epigenetic stability of fPMSCs after long-term in vitro expansion has not been fully investigated. In this report, alterations to histone acetylation and consequence on the expression pattern of fPMSCs following in vitro propagation under serum-free conditions were explored. The results show that fPMSCs maintain their MSC characteristics before they reached a senescent state. Furthermore, acetylation modification patterns were changed in fPMSCs along with gradually increased global histone deacetylase (HDAC) activity and expression of HDAC subtypes HDAC4, HDAC5 and HDAC6, as well as a down-regulated global histone H3/H4 acetylation during in vitro culturing. In line with the acetylation alterations, the expression of oncogenes Oct4, Sox2 and TERT were significantly decreased over the propagation period. Of note, the down-regulation of Oct4 was strongly associated with changes in acetylation. Intriguingly, telomere length in fPMSCs did not significantly change during the propagating process. These findings suggest that human fPMSCs may be a safe and reliable resource of MSCs and can be propagated under serum-free conditions with less risk of spontaneous malignancy, and warrants further validation in clinical settings.
doi:10.1371/journal.pone.0117068
PMCID: PMC4324636  PMID: 25671548
2.  A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum 
Journal of proteome research  2013;13(2):875-882.
Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase LC separations to fractionate and select target fractions for follow-on LC-SRM analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and LOQ values of ~130 pg/mL in serum and ~10 pg per 100 μg total protein mass in urine, respectively. A good correlation (R2 = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and ELISA. Based on an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between non-cancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.
doi:10.1021/pr400912c
PMCID: PMC3975687  PMID: 24251762
AGR2; PSA; prostate cancer; PRISM-SRM; human urine; human serum
3.  Cloning and analysis of expression patterns and transcriptional regulation of RghBNG in response to plant growth regulators and abiotic stresses in Rehmannia glutinosa 
SpringerPlus  2015;4:60.
RghBNG, a gene of unknown function, was cloned from Rehmannia glutinosa by reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA of RghBNG was 548 bp with a282-bp open reading frame. It encoded a polypeptide of 93 amino acids with a predicted molecular weight of 10.5 kDa and a theoretical isoelectric point of 9.25. Bioinformatics analysis indicated that RghBNG had no homology to any known plant genes, whereas the RghBNG polypeptide was highly similar to other plant proteins and possessed one conserved B12D protein family functional domain. Phylogenetic analysis revealed that RghBNG encoded for a dicot protein. RghBNG spatial and temporal expression patterns and responses to abiotic stresses and plant growth regulators were investigated by qRT-PCR. RghBNG transcripts were detected in roots, stems, leaves, petals, receptacles, stamens and pistils with the highest and lowest levels respectively observed in petals and leaves of mature plants. Additionally, RghBNG transcripts were detected at three developmental stages of roots, stems and leaves; the highest levels were observed in roots at seedling stage; Transcript levels changed to varying degrees in different tissues and stages; We also studied the effects of abiotic stress and plant growth regulators in roots and leaves. RghBNG expression was significantly increased (p < 0.01) by chromium, gibberellic acid and NaCl, with the highest levels induced by chromium stress; In contrast, 6-benzyladenine reduced expression. These results strongly suggest that RghBNG is involved in R. glutinosa growth, development and response to plant growth regulators and abiotic stresses.
doi:10.1186/s40064-015-0830-0
PMCID: PMC4320158
Rehmannia glutinosa; RghBNG gene; Cloning and expression; qRT-PCR; Abiotic stress; Plant growth regulator
4.  Aryl Hydrocarbon Receptors in Osteoclast Lineage Cells Are a Negative Regulator of Bone Mass 
PLoS ONE  2015;10(1):e0117112.
Aryl hydrocarbon receptors (AhRs) play a critical role in various pathological and physiological processes. Although recent research has identified AhRs as a key contributor to bone metabolism following studies in systemic AhR knockout (KO) or transgenic mice, the cellular and molecular mechanism(s) in this process remain unclear. In this study, we explored the function of AhR in bone metabolism using AhRRANKΔOc/ΔOc (RANKCre/+;AhRflox/flox) mice. We observed enhanced bone mass together with decreased resorption in both male and female 12 and 24-week-old AhRRANKΔOc/ΔOc mice. Control mice treated with 3-methylcholanthrene (3MC), an AhR agonist, exhibited decreased bone mass and increased bone resorption, whereas AhRCtskΔOc/ΔOc (CtskCre/+;AhRflox/flox) mice injected with 3MC appeared to have a normal bone phenotype. In vitro, bone marrow-derived macrophages (BMDMs) from AhRRANKΔOc/ΔOc mice exhibited impaired osteoclastogenesis and repressed differentiation with downregulated expression of B lymphocyte-induced maturation protein 1 (Blimp1), and cytochrome P450 genes Cyp1b1 and Cyp1a2. Collectively, our results not only demonstrated that AhR in osteoclast lineage cells is a physiologically relevant regulator of bone resorption, but also highlighted the need for further studies on the skeletal actions of AhR inhibitors in osteoclast lineage cells commonly associated with bone diseases, especially diseases linked to environmental pollutants known to induce bone loss.
doi:10.1371/journal.pone.0117112
PMCID: PMC4304837  PMID: 25615839
5.  Proteomic approaches for site-specific O-GlcNAcylation analysis 
Bioanalysis  2014;6(19):2571-2580.
O -GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago and its significance has been implicated in several disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically challenging to characterize comprehensively and quantitatively because of its low abundance, low stoichiometry and extremely labile nature under conventional collision-induced dissociation tandem MS conditions. Herein, we review the recent advances addressing these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, including specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification and quantitative analysis of O-GlcNAcylation.
doi:10.4155/bio.14.239
PMCID: PMC4275047  PMID: 25411699
6.  Protective effects of acacetin isolated from Ziziphora clinopodioides Lam. (Xintahua) on neonatal rat cardiomyocytes 
Chinese Medicine  2014;9(1):28.
Background
The total flavonoids from ethanol extract of the aerial part of Ziziphora clinopodioides Lam. (Lamlaceae) (Xintahua) showed protective activities against rat acute myocardial ischemia in rats. This study aims to isolate acacetin, a flavonoid, from the aerial part of Z. clinopodioides, to develop an HPLC method for its detection, and to evaluate its protective effects on neonatal rat cardiomyocytes.
Methods
Sephadex LH-20 silicagel and pillar layer chromatography silica gel were applied for the isolation and purification of acacetin and its structure was elucidated on the basis of 1H and 13C NMR spectroscopy. The content of acacetin in Z. clinopodioides collected from three different origins was determined by HPLC. The neonatal rat cardiomyocytes were isolated and cultured in vitro to establish a hypoxia/reoxygenation injury model. The viability of cardiomyocytes was measured by the MTT method. Changes of malondialdehyde (MDA) content in the medium were also determined.
Results
The acacetin content in various batches of Z. clinopodioides ranged from 45.50 to 47.41 μg/g. Acacetin of 25, 10, 5 μg/mL significantly decreased the MDA content in a model of hypoxia/reoxygenation injury (P < 0.001, P < 0.001 and P = 0.033, respectively).
Conclusions
Acacetin protects neonatal cardiomyocytes from the damage induced by hypoxia/reoxygenation stress through reduction of lipid peroxidation and enhancement of the antioxidant activity.
doi:10.1186/s13020-014-0028-3
PMCID: PMC4272544  PMID: 25558275
Acacetin; Ziziphora clinopodioides; Neonatal rat cardiomyocytes; Hypoxia/reoxygenation; HPLC
7.  eQTL and receptor pharmacology implicate Arg1 and the GABA-A receptor as therapeutic targets in neuroblastoma 
Cell reports  2014;9(3):1034-1046.
SUMMARY
The development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma, and that benzodiazepines in clinical use may have potential for neuroblastoma therapy.
doi:10.1016/j.celrep.2014.09.046
PMCID: PMC4251494  PMID: 25437558
8.  β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells 
Over-activation of SUMOylation is correlated with poor prognosis in multiple myeloma (MM), with the mechanism unclear. Wnt signaling is one of the aberrantly regulated pathways related to cancer tumorigenesis and progression. Whether SUMOylation is involved in regulating the activity of Wnt/β-catenin pathway, however, has not been reported in MM. Here we found that the TOPflash reporter activity and the expression of Wnt/β-catenin target genes can be down-regulated after interference with SUMOylation through SUMO-1 small interfering RNA (siRNA). SUMOylation inhibition down-regulated β-catenin at protein level via promotion of ubiquitin-proteasomal mediated degradation. Furthermore, over-expression of β-catenin rescued Wnt/β-catenin pathway activity and partially prevented increased apoptosis and growth inhibition induced by SUMOylation inhibition, indicating that β-catenin was responsible for the observed effect on Wnt/β-catenin pathway. To gain a clearer view, we exploited the inter-protein interactions of β-catenin and SUMO-1 in myeloma cell lines. Immunoprecipitation and immunofluorescence assay proved that β-catenin is subjected to SUMOylation in vivo, which may, at least partially explain the impact of SUMOylation inhibition on β-catenin. The association of SUMO-1 and β-catenin was confirmed in myeloma patient samples. Taken together, our data proved that SUMOylation inhibition down-regulates Wnt/β-catenin pathway by promoting the ubiquitin-proteasomal mediated degradation of β-catenin. SUMOylation of β-catenin is part of the mechanisms involved in the dysregulated proliferation of myeloma cells.
PMCID: PMC4300696  PMID: 25628940
Multiple myeloma; SUMO-1; SUMOylation; Wnt/β-catenin
9.  Immune therapeutic targeting of glioma cancer stem cells 
Targeted oncology  2010;5(3):217-227.
Glioblastoma multiforme (GBM) is a lethal cancer that responds poorly to radiotherapy and chemotherapy. Glioma cancer stem cells (gCSCs) have been shown to recapitulate the characteristic features of GBM and to mediate chemotherapy and radiation resistance. Immunotherapeutic targeting of this cell population holds therapeutic promise but must be considered in the context of the immunosuppressive properties mediated by the gCSC. Recent findings have indicated that this goal will be challenging because the gCSC can suppress both the innate and adaptive immune systems by a variety of gCSC-secreted products and cell-membrane interactions. In this review article, we will attempt to reconcile the disparate research findings regarding the potential of immune targeting of the gCSC and propose several novel solutions.
doi:10.1007/s11523-010-0151-8
PMCID: PMC4262967  PMID: 20737294
Glioma cancer stem cells; STAT3; glioblastoma multiforme; immunotherapy
12.  Do we really need closed-suction drainage in total hip arthroplasty? A meta-analysis 
International Orthopaedics  2013;37(11):2109-2118.
Purpose
The clinical use of closed-suction drainage, which aims to reduce postoperative wound haematomas and infection, is common. This study was performed to determine whether closed-suction drainage is safe and effective in promoting wound healing and reducing blood loss and other complications compared with no-drainage in total hip arthroplasty.
Methods
The literature search was based on PubMed, the Cochrane Library, MEDLINE, and EMBASE. The data were evaluated using the generic evaluation tool designed by the Cochrane Bone, Joint and Muscle Trauma Group, and then analysed using RevMan 5.0. Twenty randomised controlled trials involving 3,186 patients were included in our analysis.
Results
The results of our meta-analysis indicate that closed-suction drainage reduces the requirement for dressing reinforcement, but increases the rate of homologous blood transfusion. No significant difference was observed in the incidence of infection, blood loss, changes in haemoglobin and haematocrit, functional assessment, or other complications when the drainage group was compared with the no-drainage group.
Conclusions
Our results of the comparison between closed-suction drainage and no drainage in THA have indicated that the routine use of closed-suction drainage for elective total hip arthroplasty may be of more harm than benefit.
doi:10.1007/s00264-013-2053-8
PMCID: PMC3824906  PMID: 23982636
Closed-suction drainage; Total hip arthroplasty; Blood loss; Transfusion; Meta-analysis
13.  12-hydroxyeicosatetraenoic acid is associated with variability in aspirin-induced platelet inhibition 
Background
Aspirin is one of the most widely used non-steroidal anti-inflammatory drugs (NSAIDs). It is also a commonly used anti-platelet drug, which inhibits the formation of the platelet activator, thromboxane A2 (TxA2) via inhibition of cyclooxygenase-1 (COX-1). However, the presence of a patient subset that fails to respond to aspirin despite reduced TxA2 concentrations suggests that the effect of aspirin might be more complex than exclusive COX-1 inhibition.
Methods
In this study we evaluated the impact of in vivo oral administration of a standard anti-platelet dose (75 mg) of aspirin in healthy volunteers on the acute impact of in vitro collagen-mediated platelet aggregation and generation of platelet-derived TxA2 and the 12-lipoxygenase (LOX) metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). The eicosanoids were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Results
Low-dose aspirin administration not only inhibited TxA2 generation but also decreased the production of 12-HETE. Furthermore, a significant correlation was observed between the levels of 12-HETE and collagen-induced platelet aggregation. Pre-treatment of platelets with the 12-LOX inhibitor, baicalein, prior to activation attenuated platelet aggregation.
Conclusions
These findings support a role for 12-HETE as a pro-aggregatory eicosanoid in platelet function and suggest a role for 12-HETE in variable sensitivity to aspirin. The study also highlights a potentially important mechanism by which aspirin impacts upon eicosanoid generation.
doi:10.1186/s12950-014-0033-4
PMCID: PMC4209229  PMID: 25349537
Aspirin; Platelet; 12-HETE; Thromboxane; Eicosanoids; Anti-inflammatory
14.  Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer 
Molecular oncology  2014;8(7):1169-1180.
Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing “signature” peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.
doi:10.1016/j.molonc.2014.02.004
PMCID: PMC4183720  PMID: 25266362
TMPRSS2-ERG gene fusion; ERG protein isoform; PRISM-SRM; Targeted quantification; Prostate cancer
15.  Characterization of PHB1 and Its Role in Mitochondrial Maturation and Yolk Platelet Degradation during Development of Artemia Embryos 
PLoS ONE  2014;9(10):e109152.
Background
To cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts) with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown.
Principal Finding
Our study investigated the role of prohibitin 1 (PHB1) in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein.
Conclusions/Significance
PHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.
doi:10.1371/journal.pone.0109152
PMCID: PMC4195616  PMID: 25310573
16.  Expediting SRM assay development for large-scale targeted proteomics experiments 
Journal of proteome research  2014;13(10):4479-4487.
Due to their high sensitivity and specificity, selected reaction monitoring (SRM) based targeted proteomics has become increasingly popular for biological and translational applications. Selection of optimal transitions and optimization of collision energy (CE) are important assay development steps for achieving sensitive detection and accurate quantification; however, these steps can be labor-intensive, especially, for large-scale applications. Herein, we explored several options for accelerating SRM assay development evaluated in the context of a relatively large set of 215 synthetic peptide targets. We first showed that HCD fragmentation is very similar to CID in triple quadrupole (QQQ) instrumentation, and by selection of the top six y fragment ions from HCD spectra, >86% of the top transitions optimized from direct infusion on QQQ instrument are covered. We also demonstrated that the CE calculated by existing prediction tools was less accurate for 3+ precursors, and a significant increase in intensity for transitions could be obtained using a new CE prediction equation constructed from the present experimental data. Overall, our study illustrated the feasibility of expediting the development of larger numbers of high-sensitivity SRM assays through automation of transition selection and accurate prediction of optimal CE to improve both SRM throughput and measurement quality.
doi:10.1021/pr500500d
PMCID: PMC4184450  PMID: 25145539
SRM; MRM; HCD; QQQ; transition selection; optimization; CE prediction; targeted quantification
17.  Long-gradient separations coupled with selected reaction monitoring for highly sensitive, large scale targeted protein quantification in a single analysis 
Analytical chemistry  2013;85(19):10.1021/ac402105s.
Long-gradient separations coupled to tandem MS were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional LC-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in LOQ for target proteins in human female serum. Based on at least one surrogate peptide per protein, an LOQ of 10 ng/mL was achieved for the two spiked proteins in non-depleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or sub-ng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and ELISA measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM potentially offers much higher multiplexing capacity than conventional LC-SRM due to an increase in average peak widths (~3-fold) for a 300-min gradient compared to a 45-min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.
doi:10.1021/ac402105s
PMCID: PMC3839867  PMID: 24004026
long-gradient; targeted quantification; low-abundance protein; human serum; sensitivity; reproducibility
18.  Genetic Variations in the Kir6.2 Subunit (KCNJ11) of Pancreatic ATP-Sensitive Potassium Channel Gene Are Associated with Insulin Response to Glucose Loading and Early Onset of Type 2 Diabetes in Childhood and Adolescence in Taiwan 
To investigate the role of E23K polymorphism of the KCNJ11 gene on early onset of type 2 diabetes in school-aged children/adolescents in Taiwan, we recruited 38 subjects with type 2 diabetes (ages 18.6 ± 6.6 years; body mass index percentiles 83.3 ± 15.4) and 69 normal controls (ages 17.3 ± 3.8 years; body mass index percentiles 56.7 ± 29.0) from a national surveillance for childhood/adolescent diabetes in Taiwan. We searched for the E23K polymorphism of the KCNJ11 gene. We found that type 2 diabetic subjects had higher carrier rate of E23K polymorphism of KCNJ11 gene than control subjects (P = 0.044). After adjusting for age, gender, body mass index percentiles, and fasting plasma insulin, the E23K polymorphism contributed to an increased risk for type 2 diabetes (P = 0.047). K23-allele-containing genotypes conferring increased plasma insulin level during OGTT in normal subjects. However, the diabetic subjects with the K23-allele-containing genotypes had lower fasting plasma insulin levels after adjustment of age and BMI percentiles. In conclusion, the E23K variant of the KCNJ11 gene conferred higher susceptibility to type 2 diabetes in children/adolescents. Furthermore, in normal glucose-tolerant children/adolescents, K23 allele carriers had a higher insulin response to oral glucose loading.
doi:10.1155/2014/983016
PMCID: PMC4189766  PMID: 25309595
19.  A synthetic Toll-like receptor 3 ligand mitigates profibrotic fibroblast responses by inducing autocrine interferon signaling 
Activation of Toll-like receptor-3 (TLR3) by exogenous microbial ligands or endogenous injury-associated ligands leads to production of type I interferon (IFN). Scleroderma patients with progressive skin fibrosis display an IFN-regulated gene signature, implicating TLR3 signaling in the disease. We now show that TLR3 expression was detected on foreskin, adult skin and lung fibroblasts, and TLR3 levels were significantly elevated in a subset of scleroderma skin biopsies. In explanted skin and lung fibroblasts, the synthetic TLR3 ligand polyinosinic: polycytidylic acid (Poly I:C), a double-stranded RNA analog, caused dose- and time-dependent stimulation of IFN-β production and generation of an IFN-response gene signature that was accompanied by substantial down-regulation of collagen and alpha-smooth muscle actin gene expression. Furthermore, Poly I:C abrogated transforming growth factor-β (TGF-β)-induced fibrotic responses and blocked canonical Smad signaling via up-regulation of inhibitory Smad7. Surprisingly, the inhibitory effects of Poly I:C in fibroblasts were independent of TLR3, and were mediated by the cytosolic receptors retinoic acid-inducible gene 1 (RIG1) and melanoma differentiation associated gene 5 (MDA5), and involved signaling via the IFN receptor. Taken together, these results demonstrate that induction of a fibroblast IFN response gene signature triggered by double-stranded RNA is associated with potent TLR3-independent anti-fibrotic effects. The characteristic IFN response gene signature seen in scleroderma lesions might therefore signify a tissue-autonomous protective attempt to restrict fibroblast activation during injury.
doi:10.4049/jimmunol.1300376
PMCID: PMC3924580  PMID: 23956427
Poly I:C; TLR; TGF-β; fibrosis; scleroderma; fibroblast; Smad7
20.  Association of Novel Polymorphisms in Lymphoid Enhancer Binding Factor 1 (LEF-1) Gene with Number of Teats in Different Breeds of Pig 
Lymphoid enhancer binding factor 1 (LEF-1) is a member of the T-cell specific factor (TCF) family, which plays a key role in the development of breast endothelial cells. Moreover, LEF-1 gene has been identified as a candidate gene for teat number trait. In the present study, we detected two novel mutations (NC_010450.3:g. 99514A>G, 119846C>T) by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism in exon 4 and intron 9 of LEF-1 in Guanzhong Black, Hanjiang Black, Bamei and Large White pigs. Furthermore, we analyzed the association between the genetic variations with teat number trait in these breeds. The 99514A>G mutation showed an extremely significant statistical relevance between different genotypes and teat number trait in Guanzhong (p<0.001) and Large White (p = 0.002), and significant relevance in Hanjiang (p = 0.017); the 119846C>T mutation suggested significant association in Guanzhong Black pigs (p = 0.042) and Large White pigs (p = 0.003). The individuals with “AG” or “GG” genotype displayed more teat numbers than those with “AA”; the individuals with “TC” or “CC” genotype showed more teat numbers than those with “TT”. Our findings suggested that the 99514A>G and 119846C>T mutations of LEF-1 affected porcine teat number trait and could be used in breeding strategies to accelerate porcine teat number trait improvement of indigenous pigs breeds through molecular marker assisted selection.
doi:10.5713/ajas.2013.13772
PMCID: PMC4150191  PMID: 25178368
Pig; LEF-1 Gene; Expression Profile; Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RLFP); Teat Number; Haplotype
21.  Abnormal Response of the Proliferation and Differentiation of Growth Plate Chondrocytes to Melatonin in Adolescent Idiopathic Scoliosis 
Abnormalities in the melatonin signaling pathway and the involvement of melatonin receptor MT2 have been reported in patients with adolescent idiopathic scoliosis (AIS). Whether these abnormalities were involved in the systemic abnormal skeletal growth in AIS during the peripubertal period remain unknown. In this cross-sectional case-control study, growth plate chondrocytes (GPCs) were cultured from twenty AIS and ten normal control subjects. Although the MT2 receptor was identified in GPCs from both AIS and controls, its mRNA expression was significantly lower in AIS patients than the controls. GPCs were cultured in the presence of either the vehicle or various concentrations of melatonin, with or without the selective MT2 melatonin receptor antagonist 4-P-PDOT (10 µM). Then the cell viability and the mRNA expression of collagen type X (COLX) and alkaline phosphatase (ALP) were assessed by MTT and qPCR, respectively. In the control GPCs, melatonin at the concentrations of 1, 100 nM and 10 µM significantly reduced the population of viable cells, and the mRNA level of COLX and ALP compared to the vehicle. Similar changes were not observed in the presence of 4-P-PDOT. Further, neither proliferation nor differentiation of GPCs from AIS patients was affected by the melatonin treatment. These findings support the presence of a functional abnormality of the melatonin signaling pathway in AIS GPCs, which might be associated with the abnormal endochondral ossification in AIS patients.
doi:10.3390/ijms150917100
PMCID: PMC4200781  PMID: 25257530
idiopathic scoliosis; melatonin; growth plate; chondrocytes; proliferation; differentiation; MT2 receptor; antagonist; dysfunction
22.  miR-30b, Down-Regulated in Gastric Cancer, Promotes Apoptosis and Suppresses Tumor Growth by Targeting Plasminogen Activator Inhibitor-1 
PLoS ONE  2014;9(8):e106049.
Background
Gastric cancer is one of the most common malignant diseases worldwide. Emerging evidence has shown that microRNAs (miRNAs) are associated with tumor development and progression. Our previous studies have revealed that H. pylori infection was able to induce the altered expression of miR-30b in gastric epithelial cells. However, little is known about the potential role of miR-30b in gastric cancer.
Methods
We analyzed the expression of miR-30b in gastric cancer cell lines and human gastric cancer tissues. We examined the effect of miR-30b mimics on the apoptosis of gastric cancer cells in vitro by flow cytometry (FCM) and caspase-3/7 activity assays. Nude mouse xenograft model was used to determine whether miR-30b is involved in tumorigenesis of gastric cancer. The target of miR-30b was identified by bioinformatics analysis, luciferase assay and Western blot. Finally, we performed the correlation analysis between miR-30b and its target expression in gastric cancer.
Results
miR-30b was significantly down-regulated in gastric cancer cells and human gastric cancer tissues. Enforced expression of miR-30b promoted the apoptosis of gastric cancer cells in vitro, and miR-30b could significantly inhibit tumorigenicity of gastric cancer by increasing the apoptosis proportion of cancer cells in vivo. Moreover, plasminogen activator inhibitor-1 (PAI-1) was identified as the potential target of miR-30b, and miR-30b level was inversely correlated with PAI-1 expression in gastric cancer. In addition, silencing of PAI-1 was able to phenocopy the effect of miR-30b overexpression on apoptosis regulation of cancer cells, and overexpression of PAI-1 could suppressed the effect of promoting cell apoptosis by miR-30b, indicating PAI-1 is potentially involved in miR-30b-induced apoptosis on cancer cells.
Conclusion
miR-30b may function as a novel tumor suppressor gene in gastric cancer by targeting PAI-1 and regulating the apoptosis of cancer cells. miR-30b could serve as a potential biomarker and therapeutic target against gastric cancer.
doi:10.1371/journal.pone.0106049
PMCID: PMC4149503  PMID: 25170877
23.  A diffusion-based truncated projection artifact reduction method for iterative digital breast tomosynthesis reconstruction 
Physics in medicine and biology  2013;58(3):569-587.
Digital breast tomosynthesis (DBT) has strong promise to improve sensitivity for detecting breast cancer. DBT reconstruction estimates the breast tissue attenuation using projection views (PVs) acquired in a limited angular range. Because of the limited field of view (FOV) of the detector, the PVs may not completely cover the breast in the x-ray source motion direction at large projection angles. The voxels in the imaged volume cannot be updated when they are outside the FOV, thus causing a discontinuity in intensity across the FOV boundaries in the reconstructed slices, which we refer to as the truncated projection artifact (TPA). Most existing TPA reduction methods were developed for the filtered backprojection method in the context of computed tomography. In this study, we developed a new diffusion-based method to reduce TPAs during DBT reconstruction using the simultaneous algebraic reconstruction technique (SART). Our TPA reduction method compensates for the discontinuity in background intensity outside the FOV of the current PV after each PV updating in SART. The difference in voxel values across the FOV boundary is smoothly diffused to the region beyond the FOV of the current PV. Diffusion-based background intensity estimation is performed iteratively to avoid structured artifacts. The method is applicable to TPA in both the forward and backward directions of the PVs and for any number of iterations during reconstruction. The effectiveness of the new method was evaluated by comparing the visual quality of the reconstructed slices and the measured discontinuities across the TPA with and without artifact correction at various iterations. The results demonstrated that the diffusion-based intensity compensation method reduced the TPA while preserving the detailed tissue structures. The visibility of breast lesions obscured by the TPA was improved after artifact reduction.
doi:10.1088/0031-9155/58/3/569
PMCID: PMC4132954  PMID: 23318346
24.  Trauma-associated Human Neutrophil Alterations Revealed by Comparative Proteomics Profiling 
Proteomics. Clinical applications  2013;7(0):10.1002/prca.201200109.
PURPOSE
Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown.
EXPERIMENTAL DESIGN
We applied 2D-LC-MS/MS based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls.
RESULTS
A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways.
CONCLUSIONS AND CLINICAL RELEVANCE
Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs.
doi:10.1002/prca.201200109
PMCID: PMC3737403  PMID: 23589343
human neutrophil; LC-MS/MS; Proteomics; Trauma; Genomics
25.  Evaluation of selected binding domains for the analysis of ubiquitinated proteomes 
Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.
doi:10.1007/s13361-013-0619-8
PMCID: PMC3715598  PMID: 23649778
Ubiquitination; post-translation modification; affinity purification; proteomics; mass spectrometry

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