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2.  Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion 
Journal of proteome research  2013;12(7):3353-3361.
We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ~12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successful quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.
PMCID: PMC3733379  PMID: 23763644
SRM; PRISM; targeted quantification; low-abundance protein; human serum; sensitivity; reproducibility
3.  miR-124 inhibits STAT3 signaling to enhance T cell-mediated immune clearance of glioma 
Cancer research  2013;73(13):3913-3926.
MicroRNAs (miRs) have been shown to modulate critical gene transcripts involved in tumorigenesis, but their role in tumor-mediated immune suppression is largely unknown. On the basis of miRNA gene expression in gliomas using tissue microarrays, in situ hybridization, and molecular modeling, miR-124 was identified as a lead candidate for modulating signal transducer and activator of transcription 3 (STAT3) signaling, a key pathway mediating immune suppression in the tumor microenvironment. miR-124 is absent in all grades and pathological types of gliomas. Upon up regulating miR-124 in glioma cancer stem cells (gCSCs), the STAT3 pathway was inhibited, and miR-124 reversed gCSC-mediated immune suppression of T-cell proliferation and induction of Foxp3+ regulatory T-cells (Tregs). Treatment of T-cells from immunosuppressed glioblastoma patients with miR-124 induced marked effector response including up regulation of IL-2, IFN-γ, and tumor necrosis factor (TNF)-α. Both systemic administration of miR-124 or adoptive miR-124-transfected T-cell transfers exerted potent anti-glioma therapeutic effects in clonotypic and genetically engineered murine models of glioblastoma and enhanced effector responses in the local tumor microenvironment. These therapeutic effects were ablated in both CD4+ and CD8+ depleted mice and nude mouse systems, indicating that the therapeutic effect of miR-124 depends on the presence of a T-cell-mediated antitumor immune response. Our findings highlight the potential application of miR-124 as a novel immunotherapeutic agent for neoplasms and serve as a model for identifying miRNAs that can be exploited as immune therapeutics.
PMCID: PMC3780786  PMID: 23636127
microRNAs; miR-124; cancer stem cells; glioblastoma multiforme; immune suppression; signal transducer and activator of transcription 3; regulatory T-cells
4.  Trends in Specific Immunotherapy for Allergic Rhinitis: A Survey of Chinese ENT Specialists 
Specific immunotherapy (SIT) is a suitable but uncommon treatment option for allergic rhinitis (AR) in China. The current understanding and attitude of Chinese ENT (ear, nose, and throat) specialists in regards to SIT is unclear. This study investigates current trends in the awareness and application status of SIT among Chinese ENT specialists.
We performed a nationwide, cross-sectional survey with a specially designed questionnaire given to 800 ENT specialists in China. A member of the trained research group conducted face-to-face interviews with each respondent.
Most of the respondents considered AR (96.0%) and allergic asthma (96.0%) the most suitable indications for SIT. Of all respondents, 77.0% recommended the application of SIT as early as possible; in addition, SIT was considered 'relatively controllable and safe' by most respondents (80.6%). The highest allergen-positive rate in AR was associated with house dust mite (47.7%) and obvious differences existed among geographical regions. Conventional subcutaneous immunotherapy was the most highly recommended treatment option (96.2%). 'The high cost of SIT' (86.6%) and 'lack of patient knowledge of SIT' (85.2%) were probably the main reasons for the lower clinical use of SIT in China.
Most cases showed that the opinions of Chinese ENT specialists appeared to be in agreement with recent SIT progress and international guidelines; however, many areas still need to enhance the standardization and use of SIT in China. Clinical guidelines for SIT require improvement; in addition, Chinese ENT specialists need continuing medical education on SIT.
PMCID: PMC4077955  PMID: 24991452
Allergic rhinitis; specific immunotherapy; data collection; Chinese otolaryngologists
5.  ‘RetinoGenetics’: a comprehensive mutation database for genes related to inherited retinal degeneration 
Inherited retinal degeneration (IRD), a leading cause of human blindness worldwide, is exceptionally heterogeneous with clinical heterogeneity and genetic variety. During the past decades, tremendous efforts have been made to explore the complex heterogeneity, and massive mutations have been identified in different genes underlying IRD with the significant advancement of sequencing technology. In this study, we developed a comprehensive database, ‘RetinoGenetics’, which contains informative knowledge about all known IRD-related genes and mutations for IRD. ‘RetinoGenetics’ currently contains 4270 mutations in 186 genes, with detailed information associated with 164 phenotypes from 934 publications and various types of functional annotations. Then extensive annotations were performed to each gene using various resources, including Gene Ontology, KEGG pathways, protein–protein interaction, mutational annotations and gene–disease network. Furthermore, by using the search functions, convenient browsing ways and intuitive graphical displays, ‘RetinoGenetics’ could serve as a valuable resource for unveiling the genetic basis of IRD. Taken together, ‘RetinoGenetics’ is an integrative, informative and updatable resource for IRD-related genetic predispositions.
Database URL:
PMCID: PMC4060621  PMID: 24939193
6.  The 2011 Survey on Hypertensive Disorders of Pregnancy (HDP) in China: Prevalence, Risk Factors, Complications, Pregnancy and Perinatal Outcomes 
PLoS ONE  2014;9(6):e100180.
Hypertensive disorders of pregnancy (HDP) are a group of medical complications in pregnancy and also a risk factor for severe pregnancy outcomes, but it lacks a large-scale epidemiological investigation in recent years. This survey represents a multicenter cross-sectional retrospective study to estimate the prevalence and analyze the risk factors for HDP among the pregnant women who had referred for delivery between January 1st 2011 and December 31st 2011 in China Mainland. A total of 112,386 pregnant women were investigated from 38 secondary and tertiary specialized or general hospitals randomly selected across the country, of which 5,869 had HDP, accounting for 5.22% of all pregnancies. There were significant differences in the prevalence of HDP between geographical regions, in which the North China showed the highest (7.44%) and Central China showed the lowest (1.23%). Of six subtypes of HDP, severe preeclampsia accounted for 39.96%, gestational hypertension for 31.40%, mild preeclampsia for 15.13%, chronic hypertension in pregnancy for 6.00%, preeclampsia superimposed on chronic hypertension for 3.68% and eclampsia for 0.89%. A number of risk factors for HDP were identified, including twin pregnancy, age of >35 years, overweight and obesity, primipara, history of hypertension as well as family history of hypertension and diabetes. The prevalence of pre-term birth, placental abruption and postpartum hemorrhage were significantly higher in women with HDP than those without HDP. The possible risk factors confirmed in this study may be useful for the development of early diagnosis and appropriate treatment of HDP.
PMCID: PMC4061123  PMID: 24937406
7.  New index to predict esophageal variceal bleeding in cirrhotic patients 
AIM: To develop a safe, simple, noninvasive and affordable system to predict esophageal variceal bleeding (EVB) in decompensated cirrhosis patients.
METHODS: Four hundred and eighty-six patients with decompensated cirrhosis (238 males and 248 females), with a mean age of 63.1 ± 11.2 years, were admitted to Changshu Affiliated Hospital of Suzhou University between May 2008 and March 2011. Patients enrolled in this study underwent ultrasound-Doppler (US-Doppler) to assess left gastric vein (LGV) blood flow velocity (LGVV) and blood flow direction (LGVBFD), and were evaluated by the Model For End-Stage Liver Disease (MELD) scoring system. All patients received follow-up evaluations every three months. The resulting data were entered into a database after each time point collection.
RESULTS: Four hundred and sixteen patients completed follow-up evaluations for an average of 31.6 mo (range: 12 to 47 mo). Fifty-one (12.3%) patients experienced EVB. The change in the MELD score over three months (ΔMELD), LGVV and LGVBFD were independently associated with EVB occurrence. MELD-US-Doppler Index (MUI), a new index, was developed and calculated using the following logistic regression equation: MUI = Logit (P) = 1.667 (ΔMELD) + 2.096 (LGVV) - 3.245 (LGVBFD) - 1.697. The area under the receiver operating characteristic curve for prediction of EVB occurrence was significantly higher for the MUI [0.858 (95%CI: 0.774-0.920)] than for ΔMELD [0.734 (95%CI: 0.636-0.817); P < 0.05], LGVV [0.679 (95%CI: 0.578-0.769); P < 0.05] or LGVBFD [0.726 (95%CI: 0.627-0.810); P < 0.05] alone. When the MUI was set at 46, the index had high diagnostic accuracy (85.8%), with high specificity (80%) and sensitivity (87.27%).
CONCLUSION: The MUI, a noninvasive and affordable index, can predict EVB occurrence in decompensated cirrhotic patients and serve as an alternative when conventional endoscopic screening is declined.
PMCID: PMC4051942  PMID: 24944493
Portal hypertension; Ultrasound-Doppler; Esophageal variceal bleeding; Decompensated cirrhosis; Endoscopy
8.  Utility of diffusion-weighted imaging to assess hepatocellular carcinoma viability following transarterial chemoembolization 
Oncology Letters  2014;8(2):831-836.
The purpose of the present study was to evaluate whether diffusion-weighted imaging (DWI) can be used to assess hepatocellular carcinoma (HCC) viability following transarterial chemoembolization (TACE). A total of 41 consecutive patients were treated according to chemoembolization protocols. The follow-up was performed between six and eight weeks post-chemoembolization by multidetector computed tomography [or enhanced magnetic resonance imaging (MRI)] and DW-MRI on the same day. The presence of any residual tumor and the extent of tumor necrosis were evaluated according to the European Association for the Study of the Liver. The apparent diffusion coefficient (ADC) values of the entire area of the treated mass and the vital and necrotic tumor tissues were recorded. Correlation coefficients were also calculated to compare the percentage of necrosis with ADC values. The mean ADC values of the necrotic and vital tumor tissues were 2.22±0.31×10−3 mm2/sec and 1.42±0.25×10−3 mm2/sec, respectively (Mann-Whitney U test, P<0.001). The results from the receiver operating characteristic analysis showed that the threshold ADC value was 1.84×10−3 mm2/sec with 92.3% sensitivity and 100% specificity for identifying the necrotic tumor tissues. A significant linear regression correlation was identified between the ADC value of the entire area of the treated mass and the extent of tumor necrosis (r=0.58; P<0.001). In conclusion, DWI can be used to assess HCC viability following TACE.
PMCID: PMC4081130
hepatocellular carcinoma; chemoembolization; diffusion-weighted imaging; magnetic resonance imaging
9.  Determination of burn patient outcome by large-scale quantitative discovery proteomics 
Critical care medicine  2013;41(6):1421-1434.
Emerging proteomics techniques can be used to establish proteomic outcome signatures and to identify candidate biomarkers for survival following traumatic injury. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and multiplex cytokine analysis to profile the plasma proteome of survivors and non-survivors of massive burn injury to determine the proteomic survival signature following a major burn injury.
Proteomic discovery study.
Five burn hospitals across the U.S.
Thirty-two burn patients (16 non-survivors and 16 survivors), 19–89 years of age, were admitted within 96 h of injury to the participating hospitals with burns covering >20% of the total body surface area and required at least one surgical intervention.
Measurements and Main Results
We found differences in circulating levels of 43 proteins involved in the acute phase response, hepatic signaling, the complement cascade, inflammation, and insulin resistance. Thirty-two of the proteins identified were not previously known to play a role in the response to burn. IL-4, IL-8, GM-CSF, MCP-1, and β2-microglobulin correlated well with survival and may serve as clinical biomarkers.
These results demonstrate the utility of these techniques for establishing proteomic survival signatures and for use as a discovery tool to identify candidate biomarkers for survival. This is the first clinical application of a high-throughput, large-scale LC-MS-based quantitative plasma proteomic approach for biomarker discovery for the prediction of patient outcome following burn, trauma or critical illness.
PMCID: PMC3660437  PMID: 23507713
burn; inflammation; proteomic profiling; plasma proteins; LC-MS; biomarker
10.  Preoperative pelvic axial rotation: a possible predictor for postoperative coronal decompensation in thoracolumbar/lumbar adolescent idiopathic scoliosis 
European Spine Journal  2013;22(6):1264-1272.
The pelvis as the biomechanical foundation of spine, plays an important role in the balance of the stance and gait through the multi-link spinal-pelvic system. If the pelvic axial rotation (PAR) exists in adolescent idiopathic scoliosis (AIS) patients, it should theoretically have some effects on the body balance.
To explore the probable effects of preoperative PAR on the spinal balance in coronal plane in AIS patients with main thoracolumbar/lumbar (TL/L) curve after posterior spinal instrumentation.
Thirty-eight AIS patients (age: 15 ± 1.5 years) with main TL/L curve (51° ± 6.2°) were recruited retrospectively into this study. The mean follow-up period was 27 months (24–36 months). Standing full spine posteroanterior radiographs were taken preoperatively, 3 month and 1 year postoperatively, and at last follow-up. The convex/concave ratio (CV/CC ratio) of the anterior superior iliac spine laterally and the inferior ilium at the sacroiliac joint medially was measured on posteroanterior radiographs. According to the preoperative CV/CC ratios, the patients were divided into two groups: normal group (N-group: 0.95 ≤ CV/CC ≤ 1.05); and the asymmetrical group (A-group: CV/CC < 0.95, or >1.05).
In all the patients, the 3-month-postoperative CV/CC ratio (1.026 ± 0.087) was significantly different from the preoperative CV/CC ratio (0.969 ± 0.095, P < 0.001), indicating that the pelvis had rotated in the opposite direction of the corrective derotation load applied to the TL/L spine after surgery. No significant change was found in the CV/CC ratio from 3-month-postoperative to the last follow-up (1.013 ± 0.103, P > 0.05). There was no significant difference in the demographic, phenotypic, and treatment variables between the N- (n = 16) and A-groups (n = 22) (P > 0.05). However, more coronal decompensation occurred in the A-group after surgery (36.4 vs. 0.0 %, P = 0.013): two patients having trunk translation, three having lower instrumented vertebra (LIV) translation, and one having LIV tilt; meanwhile, one patient having both LIV translation and LIV tilt, and one having both trunk translation and LIV tilt.
The present study confirmed the existence of PAR in AIS patients, and indicated that the pelvis would experience an active rebalancing in the transverse plane within 3 months after spinal correction, and since then, its position would remain stable. Moreover, TL/L-AIS patients with preoperative asymmetrical PAR probably had greater risk of coronal decompensation postoperatively.
PMCID: PMC3676562  PMID: 23392555
Idiopathic scoliosis; Pelvis; Rotation; Transverse plane; Decompensation
11.  Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications 
Nature protocols  2013;9(1):64-75.
Reversible modifications of cysteine thiols play a significant role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation, and S-glutathionylation, have been recognized for their significance in various physiological and pathological processes. Here we describe a procedure for the enrichment of peptides containing reversible cysteine modifications. Starting with tissue or cell lysate samples, all of the unmodified free thiols are blocked using N-ethylmaleimide (NEM). This is followed by the selective reduction of those cysteines bearing the reversible modification(s) of interest. The reduction is achieved by using different reducing reagents that react specifically with each type of cysteine modification (e.g., ascorbate for S-nitrosylation). This protocol serves as a general approach for enrichment of thiol-containing proteins or peptides derived from reversibly modified proteins. The approach utilizes a commercially available thiol-affinity resin (Thiopropyl Sepharose 6B) to directly capture free thiol-containing proteins through a disulfide exchange reaction followed by on-resin protein digestion and multiplexed isobaric labeling to facilitate LC–MS/MS based quantitative site-specific analysis of cysteine-based reversible modifications. The overall approach requires a simpler workflow with increased specificity compared to the commonly used biotinylation-based assays. The procedure for selective enrichment and analyses of S-nitrosylation and the level of total reversible cysteine modifications (or total oxidation) is presented to demonstrate the utility of this general strategy. The entire protocol requires approximately 3 days for sample processing with an additional day for LC-MS/MS and data analysis.
PMCID: PMC4038159  PMID: 24336471
Mass spectrometry; post-translational modification; cysteine modification; redox modification; cysteine derivatisation; on-resin digestion; on-resin reaction; thiol enrichment; S-nitrosylation; reversibly oxidized cysteines; S-glutathionylation; S-acylation; iTRAQ; isobaric tags for relative and absolute quantification; tandem mass tags; TMT; MASIC
12.  A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses 
Annals of the rheumatic diseases  2013;73(2):446-454.
Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis (SSc), and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) exerts potent fibrotic activities. The synthetic triterpenoid oleanane 2-cyano-3,12-dioxoolean-1,9-dien-28-oic (CDDO) is a PPAR-γ agonist with potential effects on TGF-β signaling and dermal fibrosis.
To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma.
Material and methods
The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of type I constitutively active TGF-β receptor was evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the anti-fibrotic effects of CDDO, explanted skin fibroblasts cultured in 2-dimensional monolayers or in 3-dimensional full-thickness human skin equivelants were studied.
CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in 3-dimensional human skin equivalents. In 2-dimensional monolayer cultures, CDDO abrogated fibrogenic responses in explanted normal human skin fibroblasts. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated collagen synthesis. Remarkably, the anti-fibrotic effects of CDDO were independent of PPAR-γ.
The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signaling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.
PMCID: PMC4028127  PMID: 23515440
CDDO; triterpenoid; fibrosis; PPAR-γ; TGF-β; fibroblast; murine scleroderma
13.  Proteomic identification of tumor biomarkers associated with primary gallbladder cancer 
AIM: To identify potential biomarkers of primary gallbladder cancer (PGC).
METHODS: Fresh PGC, cholecystitis and normal gallbladder tissue specimens collected from 10 patients, respectively, were subjected to comparative proteomic analysis. The proteomic patterns of PGC were compared with those of cholecystitis and normal gallbladder tissues using two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were then identified using a MALDI-TOF mass spectrometer (MS) and database searches. To further validate these proteins, 20 samples of PGC tissues and normal tumor-adjacent tissues were collected for Western blot, quantitative real-time PCR, and immunohistochemical staining assay.
RESULTS: Seven differentially expressed protein spots were detected by 2-ED analysis by comparing the average maps of PGC, cholecystitis and normal gallbladder tissues. Six of the seven differentially expressed proteins were identified using MALDI-TOF MS, with three overexpressed and three underexpressed in PGC tissue. Protein levels of annexin A4 (ANXA4) were significantly elevated, and heat shock protein 90-beta (Hsp90β) and dynein cytoplasmic 1 heavy chain 1 (Dync1h1) were decreased in PGC tissues relative to the normal tumor-adjacent tissues as shown by Western blot analysis. However, levels of actin, aortic smooth muscle and gamma-actin were unchanged. In addition, the mRNA levels of all 5 proteins showed similar changes to those of the protein levels (P < 0.01). Further validation by immunohistochemical analysis showed the upregulated expression of ANXA4 and decreased expression of Hsp90β and Dync1h1 in the cytoplasm of PGC tissues relative to the normal tumor-adjacent tissues.
CONCLUSION: Three proteins are identified as potential biomarkers of PGC using proteomic analysis. The functions of these proteins in the carcinogenesis of PGC remain to be studied.
PMCID: PMC4017066  PMID: 24833881
Primary gallbladder cancer; Proteomic analysis; Annexin A4; Dynein cytoplasmic 1 heavy chain 1; Heat shock protein 90-beta; Biomarker
14.  Fibrosis in systemic sclerosis: Emerging concepts and implications for targeted therapy 
Autoimmunity reviews  2010;10(5):267-275.
Systemic sclerosis (SSc) is a complex and incompletely understood disease associated with fibrosis in multiple organs. Recent findings identify transforming growth factor-ß (TGF-ß), Wnt ligands, toll-like receptor-mediated signaling, hypoxia, type I interferon, type 2 immune responses and mechanical stress as extracellular cues that modulate fibroblast function and differentiation, and as potential targets for therapy. Moreover, fibrillin-1 has a major role in storing and regulating the bioavailability of TGF-ß and other cytokines, and fibrillin-1 mutations are implicated in a congenital form of scleroderma called stiff skin syndrome. Fibrosis is due not only to the activation of tissue-resident fibroblasts and their transdifferentiation into myofibroblasts, but also the differentiation of bone marrow-derived fibrocytes, and transition of endothelial and epithelial cells, pericytes and adipocytes into activated mesenchymal cells. These responses are modulated by signaling mediators and microRNAs that amplify or inhibit TGF-ß and Wnt signaling. Gain-of-function and loss-of-function abnormalities of these mediators may account for the characteristic activated phenotype of SSc fibroblasts. The nuclear orphan receptor PPAR-γ plays a particularly important role in limiting the duration and intensity of fibroblast activation and differentiation, and impaired PPAR-γ expression or function in SSc may underlie the uncontrolled progression of fibrosis.
Identifying the perturbations in signaling pathways, mediators and differentiation programs that are responsible for SSc tissue damage allows their selective targeting. This in turn opens the door for therapies utilizing novel compounds, or drug repurposing by innovative uses of already-approved drugs. In view of the heterogeneous clinical presentation and unpredictable course of SSc, as well as its complex pathogenesis, only robust clinical trials incorporating the judicious application of biomarkers will be able to clarify the clinical utility of these innovative approaches.
PMCID: PMC3998379  PMID: 20863909
Fibrosis; Systemic sclerosis; Fibroblast; TGF-ß; Wnt; Collagen
15.  Formation of a Polarised Primitive Endoderm Layer in Embryoid Bodies Requires Fgfr/Erk Signalling 
PLoS ONE  2014;9(4):e95434.
The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.
PMCID: PMC3994041  PMID: 24752320
16.  Prognostic Significance of Cyclin D1 Expression in Colorectal Cancer: A Meta-Analysis of Observational Studies 
PLoS ONE  2014;9(4):e94508.
Cyclin D1 plays a vital role in cancer cell cycle progression and is overexpressed in many human cancers, including colorectal cancer (CRC). However, the prognostic value of cyclin D1 overexpression in colorectal cancer is conflicting and heterogeneous. We conducted a meta-analysis to more precisely evaluate its prognostic significance.
A comprehensive literature search for relevant studies published up to January 2014 was performed using PubMed, EMBASE, and ISI Web of Science. The pooled hazard ratio (HR) with 95% confidence intervals (CI) was used to estimate the effects.
22 studies with 4150 CRC patients were selected to evaluate the association between cyclin D1 and overall survival (OS), disease-free survival (DFS) and clinicopathological parameters. In a random-effects model, the results showed that cyclin D1 overexpression in CRC was significantly associated with both poor OS (HR = 0.73, 95% CI: 0.63–0.85, P<0.001) and DFS (HR = 0.60, 95% CI: 0.44–0.82, P = 0.001). Additionally, cyclin D1 overexpression was significantly associated with more relative older patients (≥60 years) (OR 0.62, 95% CI 0.44–0.89, P = 0.009), T3,4 tumor invasion (OR 0.70, 95% CI 0.57–0.85, P<0.001), N positive (OR 0.75, 95% CI 0.60–0.95, P = 0.016) and distant metastasis (OR 0.60, 95% CI 0.36–0.99, P = 0.047) of CRC.
The meta-analysis results indicated that cyclin D1 is an unfavorable prognostic factor for CRC. Cyclin D1 overexpression might be associated with poor clinical outcome and some clinicopathological factors such as age, T category, N category and distant metastasis in CRC patients.
PMCID: PMC3984178  PMID: 24728073
17.  Placental mesenchymal stem cells of fetal and maternal origins demonstrate different therapeutic potentials 
Therapeutic potentials of mesenchymal stem cells (MSCs) from different sources have been evaluated in pre-clinical and clinical settings. Although MSCs from different sources share MSC-specific characteristics and functions, inconsistent or controversial results of pre-clinical and clinical applications of such cells are frequently reported. This may be partially due to the fact that MSCs isolated from different origins may differentially express some functions not typical for MSCs, and hence have different therapeutic potentials. The aim of this study is to investigate the differences in human placental MSCs (P-MSCs) of fetal and maternal origins in the aspects of clinical importance.
P-MSCs of fetal and maternal origins isolated from normal term placentas were characterized for their typical phenotype as well as their expression of receptors and growth factors of clinic interests. P-MSCs that preferentially express hepatocyte growth factor (HGF) and CD200 were evaluated for their therapeutic potentials in models of angiogenesis and allogeneic skin transplantation, in comparison with their HGF and CD200 negative partners.
Although all P-MSCs express typical MSC phenotype, fetal but not maternal P-MSCs express high levels of CD200 and HGF. Compared with HGF and CD200 negative P-MSCs, HGF and CD200 positive cells demonstrated significantly high potentials in promoting angiogenesis in vitro and increasing immunosuppressive function in vivo. These therapeutic potentials were at least in part due to their differences in HGF and CD200 expression, respectively.
We conclude that MSC origins may have significant impact on the therapeutic potentials of such cells, and should be taken into consideration in clinical applications.
PMCID: PMC4055134  PMID: 24721710
18.  Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry 
S-nitrosylation, the formation of S-nitrosothiol (SNO), is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. Although many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge because of the low abundance and labile nature of this modification. Herein we present further improvement and optimization of the recently reported resin-assisted cysteinyl peptide enrichment protocol for SNO identification and its application to mouse skeletal muscle to identify specific cysteine sites sensitive to S-nitrosylation by a quantitative reactivity profiling strategy. Our results indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione, an NO donor, at two different concentrations (i.e., 10 and 100 μM). The reactivity profiling experiments led to the identification of 488 SNO-modified sites from 197 proteins with specificity of ~95% at the unique peptide level, i.e., ~95% of enriched peptides contain cysteine residues as the originally SNO-modified sites. Among these sites, 281 from 145 proteins were considered more sensitive to S-nitrosylation based on the ratios of observed SNO levels between the two treatments. These SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are localized in mitochondria, contractile fiber, and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, these observed SNO-sensitive proteins are primarily involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism and insulin action.
PMCID: PMC3771501  PMID: 23277143
S-nitrosylation; Redox regulation; Chemical enrichment; Mouse muscle; Proteomics; LC–MS/MS; Free radicals
19.  A pan-BCL2 inhibitor renders bone marrow resident human leukemia stem cells sensitive to tyrosine kinase inhibition 
Cell stem cell  2013;12(3):316-328.
Leukemia stem cells (LSC) play a pivotal role in chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), in part, through alternative splicing of self-renewal and survival genes. To elucidate splice isoform regulators of human BC LSC maintenance, we performed whole transcriptome RNA sequencing; splice isoform-specific qRT-PCR, nanoproteomics, stromal co-culture and BC LSC xenotransplantation analyses. Cumulatively, these studies show that alternative splicing of multiple pro-survival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSC that are quiescent in the marrow niche and contribute to therapeutic resistance. Notably, a novel pan-BCL2 inhibitor, sabutoclax, renders marrow niche-resident BC LSC sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice isoform expression in BC LSC maintenance and suggest that combinatorial inhibition of pro-survival BCL2 family proteins and BCR-ABL may eliminate dormant LSC and obviate resistance.
PMCID: PMC3968867  PMID: 23333150
20.  Cortactin expression confers a more malignant phenotype to gastric cancer SGC-7901 cells 
AIM: To study the effects of cortactin on the tumor biology of SGC-7901 cells and identify the mechanism involved in the process.
METHODS: Cell lines in which cortactin was stably overexpressed or knocked down as well as the respective control cell lines were established by standard molecular methods. The effects of cortactin on the proliferation, migration and invasion capacity of SGC-7901 cells were assessed by the MTT assay, colony formation, flow cytometry, transwell migration and matrigel invasion. Nude mouse models were also used to assess the role of cortactin in the growth and metastasis of SGC-7901 cells in vivo. Western blotting analysis was performed to detect the expression of epidermal growth factor receptor (EGFR) and downstream molecules.
RESULTS: Cell lines in which cortactin was stably overexpressed or knocked down as well as control cell lines were successfully established and designated as LV5-cortactin-SGC, LV5-SGC, LV3-shRNA-SGC and LV3-SGC. Cortactin overexpression promoted SGC-7901 cell migration (340.7 ±12.6 vs 229.1 ± 23.2, P < 0.01) and invasion (71.6 ± 5.2 vs 48.4 ± 3.6, P < 0.01). Cortactin downregulation impaired SGC-7901 cell migration (136.2 ± 19.8 vs 225 ± 17) and invasion (29.2 ± 5.2 vs 49.6 ± 3.8, P < 0.01). The results from the MTT and colony formation assays results indicated increased LV5-cortactin-SGC cell proliferation and decreased LV3-shRNA-SGC cell proliferation compared to the control cells. Flow cytometry analysis demonstrated that cortactin overexpression promoted the proliferation index of SGC-7901 cells, and the results were reversed when cortactin was downregulated. Mouse tumor models confirmed that cortactin expression increased SGC-7901 cell proliferation and metastasis in vivo. Western blotting analysis revealed that cortactin elevated EGFR expression and activated the downstream molecules.
CONCLUSION: Cortactin expression promoted the migration, invasion and proliferation of SGC-7901 cells both in vivo and in vitro. The EGFR signaling pathway is mechanistically involved.
PMCID: PMC3964399  PMID: 24696610
Gastric cancer; Cortactin; Epidermal growth factor receptor; Invasion; Metastasis; Proliferation
21.  High and Low Doses of Ionizing Radiation Induce Different Secretome Profiles in a Human Skin Model 
PLoS ONE  2014;9(3):e92332.
It is postulated that secreted soluble factors are important contributors of bystander effect and adaptive responses observed in low dose ionizing radiation. Using multidimensional liquid chromatography-mass spectrometry based proteomics, we quantified the changes of skin tissue secretome – the proteins secreted from a full thickness, reconstituted 3-dimensional skin tissue model 48 hr after exposure to 3, 10 and 200 cGy of X-rays. Overall, 135 proteins showed statistical significant difference between the sham (0 cGy) and any of the irradiated groups (3, 10 or 200 cGy) on the basis of Dunnett adjusted t-test; among these, 97 proteins showed a trend of downregulation and 9 proteins showed a trend of upregulation with increasing radiation dose. In addition, there were 21 and 8 proteins observed to have irregular trends with the 10 cGy irradiated group either having the highest or the lowest level among all three radiated doses. Moreover, two proteins, carboxypeptidase E and ubiquitin carboxyl-terminal hydrolase isozyme L1 were sensitive to ionizing radiation, but relatively independent of radiation dose. Conversely, proteasome activator complex subunit 2 protein appeared to be sensitive to the dose of radiation, as rapid upregulation of this protein was observed when radiation doses were increased from 3, to 10 or 200 cGy. These results suggest that different mechanisms of action exist at the secretome level for low and high doses of ionizing radiation.
PMCID: PMC3958549  PMID: 24642900
22.  Identification and Characterization of a Ste20-Like Kinase in Artemia and Its Role in the Developmental Regulation and Resistance to Environmental Stress 
PLoS ONE  2014;9(3):e92234.
To adapt to extreme environments, the crustacean Artemia has evolved two alternative reproductive pathways. During ovoviviparous (direct) development, nauplius larvae are produced. In contrast, Artemia females release encysted diapause embryos (cysts) via the oviparous pathway. To date, the cellular mechanisms that regulate stress resistance of Artemia remain largely unknown. Ste20-like kinase (SLK) participates in multiple biological processes, including stress responses, apoptosis, and cell cycle progression.
Principal Finding
We isolated and characterized a member of the SLK superfamily termed ArSLK from Artemia parthenogenetica. The ArSLK gene is transcribed throughout both ovoviviparous and oviparous development; however, the protein is located mainly in the nuclei of stress-resistant diapause cysts, unlike the nauplii and nauplius-destined embryos where it is cytoplasmic. Interestingly, exposure of nauplii to heat shock, acidic pH, and UV irradiation induced the translocation of ArSLK from cytoplasm to nucleus. This translocation was reversed following stress removal. Moreover, under physiologically-stressful conditions, the nauplius larvae produced by adults after gene knockdown of endogenous ArSLK by RNAi, lost the ability of free-swimming much earlier than those of control larvae from females injected with GFP dsRNA.
Taken together, this study demonstrated that trafficking of ArSLK between the cytoplasm and the nucleus participates in regulating the stress resistance of Artemia. Our findings may provide significant insight into the functions of members of the SLK superfamily.
PMCID: PMC3956927  PMID: 24637947
23.  Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities 
Nature reviews. Rheumatology  2011;8(1):42-54.
Fibrosis in multiple organs is a prominent pathological finding and distinguishing hallmark of systemic sclerosis (SSc). Findings during the past 5 years have contributed to a more complete understanding of the complex cellular and molecular underpinning of fibrosis in SSc. Fibroblasts, the principal effector cells, are activated in the profibrotic cellular milieu by cytokines and growth factors, developmental pathways, endothelin 1 and thrombin. Innate immune signaling via Toll-like receptors, matrix-generated biomechanical stress signaling via integrins, hypoxia and oxidative stress seem to be implicated in perpetuating the process. Beyond chronic fibroblast activation, fibrosis represents a failure to terminate tissue repair, coupled with an expanded population of mesenchymal cells originating from bone marrow and transdifferentiation of epithelial cells, endothelial cells and pericytes. In addition, studies have identified intrinsic alterations in SSc fibroblasts resulting from epigenetic changes, as well as altered microRNA expression that might underlie the cell-autonomous, persistent activation phenotype of these cells. Precise characterization of the deregulated extracellular and intracellular signaling pathways, mediators and cellular differentiation programs that contribute to fibrosis in SSc will facilitate the development of selective, targeted therapeutic strategies. Effective antifibrotic therapy will ultimately involve novel compounds and repurposing of drugs that are already approved for other indications.
PMCID: PMC3954787  PMID: 22025123
24.  Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis 
PLoS Pathogens  2014;10(3):e1003994.
Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.
Author Summary
To date, studies on the regulation of physiology and virulence in Mycobacterium tuberculosis (Mtb) have focused on how transcriptional changes lead to adaptation. Interestingly, Mtb has numerous proteases that are essential for normal growth suggesting that protein turnover may also play an important regulatory role in the pathogen. We used novel methods to identify the set of proteins that are degraded by the essential Clp protease. The degradation of one protein, WhiB1, was required for normal growth confirming that inhibiting turnover of certain substrates can have a lethal effect. The understanding of essential pathways in Mtb will be important for the discovery of novel drugs to aid in the global fight against tuberculosis.
PMCID: PMC3946367  PMID: 24603869
25.  Endoscope-Guided Interstitial Intensity-Modulated Brachytherapy and Intracavitary Brachytherapy as Boost Radiation for Primary Early T Stage Nasopharyngeal Carcinoma 
PLoS ONE  2014;9(3):e90048.
Intracavitary brachytherapy (ICBT) is usually applied as boost radiotherapy for superficial residual of nasopharyngeal carcinoma (NPC) after primary extern-beam radiptherapy (ERT). Here, we evaluated the outcome of endoscope-guided interstitial intensity-modulated brachytherapy (IMBT) boost radiation for deep-seated residual NPC.
Methodology/Principal Findings
Two hundred and thirteen patients with residual NPC who were salvaged with brachytherapy boost radiation during 2005–2009 were analyzed retrospectively. Among these patients, 171 patients had superficial residual NPC (≤1 cm below the nasopharyngeal epithelium) were treated with ICBT boost radiation, and interstitial IMBT boost radiation was delivered to 42 patients with deep-seated residual NPC (>1 cm below the nasopharyngeal epithelium). We found that IMBT boost subgroup had a higher ratio of T2b (81.0% VS 34.5%, P<0.001) and stage II (90.5% VS 61.4%, P = 0.001) than that of ICBT boost subgroup. The dosage of external-beam radiotherapy in the nasopharyngeal (63.0±3.8 VS 62.6±4.3 Gray (Gy), P = 0.67) and regional lymph nodes (55.8±5.0 VS 57.5±5.7 Gy, P = 0.11) was comparable in both groups. For brachytherapy, IMBT subgroup had a lower boost radiation dosage than ICBT subgroup (11.0±2.9 VS 14.8±3.2 Gy, P<0.01). Though the IMBT group had deeper residual tumors and received lower boost radiation dosages, both subgroups had the similar 5-year actuarial overall survival rate (IMBT VS ICBT group: 96.8% VS 93.6%, P = 0.87), progression-free survival rate (92.4% VS 86.5%, P = 0.41) and distant metastasis-free survival rate (94.9% VS 92.7%, P = 0.64). Moreover, IMBT boost radiation subgroup had a similar local (97.4% VS 94.4%, P = 0.57) and regional (95.0% VS 97.2%, P = 0.34) control to ICBT subgroup. The acute and late toxicities rates were comparable between the both subgroups.
IMBT boost radiation may be a promising therapeutic selection for deep-seated residual NPC.
PMCID: PMC3940723  PMID: 24595299

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