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1.  Effect of Different Tumbling Marination Treatments on the Quality Characteristics of Prepared Pork Chops 
The effect of different tumbling marination treatments (control group, CG; conventional static marination, SM; vacuum continuous tumbling marination, CT; vacuum intermittent tumbling marination, IT) on the quality characteristics of prepared pork chops was investigated under simulated commercial conditions. The CT treatment increased (p<0.05) the pH value, b* value, product yield, tenderness, overall flavor, sensory juiciness and overall acceptability in comparison to other treatments for prepared boneless pork chops. The CT treatment decreased (p<0.05) cooking loss, shear force value, hardness, gumminess and chewiness compared with other treatments. In addition, CT treatment effectively improved springiness and sensory color more than other treatments. However, IT treatment achieved the numerically highest (p<0.05) L* and a* values. These results suggested that CT treatment obtained the best quality characteristics of prepared pork chops and should be adopted as the optimal commercial processing method for this prepared boneless pork chops.
doi:10.5713/ajas.14.0511
PMCID: PMC4283172  PMID: 25557823
Tumbling; Continuous; Intermittent; Quality Characteristic; Prepared Pork Chop
2.  Peripherally inserted central catheter thrombosis incidence and risk factors in cancer patients: a double-center prospective investigation 
Background
Peripherally inserted central catheters (PICCs) are widely used in chemotherapy, but the reported PICC thrombosis incidence varies greatly, and risks of PICC thrombosis are not well defined. This study was to investigate the incidence and risk factors of PICC-related upper extremity vein thrombosis in cancer patients.
Methods
This was a prospective study conducted in two tertiary referral hospitals from May 2010 to February 2013. Cancer patients who were subject to PICC placement were enrolled and checked by Doppler ultrasound weekly for at least 1 month. Univariable and multivariable logistic regression analyses were applied for identification of risk factors.
Results
Three hundred and eleven cancer patients were enrolled in the study. One hundred and sixty (51.4%) developed PICC thrombosis, of which 87 (54.4%) cases were symptomatic. The mean time interval from PICC insertion to thrombosis onset was 11.04±5.538 days. The univariable logistic regression analysis showed that complications (odds ratio [OR] 1.686, P=0.032), less activity (OR 1.476, P=0.006), obesity (OR 3.148, P=0.000), and chemotherapy history (OR 3.405, P=0.030) were associated with PICC thrombosis. Multivariate analysis showed that less activity (OR 9.583, P=0.000) and obesity (OR 3.466, P=0.014) were significantly associated with PICC thrombosis.
Conclusions
The incidence of PICC thrombosis is relatively high, and nearly half are asymptomatic. Less activity and obesity are risk factors of PICC-related thrombosis.
doi:10.2147/TCRM.S73379
PMCID: PMC4321638
PICC; complication; clinical study; catheter-related thrombosis; upper extremity vein thrombosis
3.  Induction of H2AX phosphorylation in tumor cells by gossypol acetic acid is mediated by phosphatidylinositol 3-kinase (PI3K) family 
Cancer Cell International  2014;14(1):141.
Background
H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including Ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. Our study shows that gossypol acetic acid (GAA) alone can induce γH2AX in Human mucoepidermoid carcinoma cell line (MEC-1) in vitro. Thus, we further examined the possible mechanisms of GAA to induce γH2AX in tumor cells.
Materials and methods
The PI3K inhibitors caffeine and wortmannin were used in an effort to identify the kinase(s) responsible for GAA -induced γH2AX in MEC-1 cells. DNA dependent protein kinase (DNA-PK) - proficient and –deficient cells, human glioma cell lines M059K and M059J, were also used to evaluate the kinases responsible for GAA induced H2AX phosphorylation. γH2AX expression was detected by immunofluorescent microscopy. Flow cytometry assay was used to assay γH2AX and cell cycle.
Results
GAA induced H2AX phosphorylation in a cell cycle-dependent manner and a significant G0/G1 phase arrest in MEC-1 cells was shown. Caffeine and wortmannin significantly inhibited GAA-induced H2AX phosphorylation in MEC-1 cells. GAA induced H2AX phosphorylation in M059K, but not in M059J. Taken together, these data suggested that GAA treatment alone could induce H2AX phosphorylation in a cell cycle dependent manner in MEC-1 and M059K, but not in M059J cells. A significant G0/G1 phase arrest was shown in MEC-1.
Conclusions
The member of PI3K family, DNA-PK, ATM and ATR are involved in the H2AX phosphorylation of MEC-1 cells.
doi:10.1186/s12935-014-0141-5
PMCID: PMC4272777  PMID: 25530717
Gossypol acetic acid; H2AX phosphorylation; DNA-PK; ATM; ATR
4.  Fabrication of Electrospun Poly(L-Lactide-co-ɛ-Caprolactone)/Collagen Nanoyarn Network as a Novel, Three-Dimensional, Macroporous, Aligned Scaffold for Tendon Tissue Engineering 
Tissue Engineering. Part C, Methods  2013;19(12):925-936.
Tissue engineering techniques using novel scaffolding materials offer potential alternatives for managing tendon disorders. An ideal tendon tissue engineered scaffold should mimic the three-dimensional (3D) structure of the natural extracellular matrix (ECM) of the native tendon. Here, we propose a novel electrospun nanoyarn network that is morphologically and structurally similar to the ECM of native tendon tissues. The nanoyarn, random nanofiber, and aligned nanofiber scaffolds of a synthetic biodegradable polymer, poly(l-lactide-co-ɛ-caprolactone) [P(LLA-CL)], and natural collagen I complex were fabricated using electrospinning. These scaffolds were characterized in terms of fiber morphology, pore size, porosity, and chemical and mechanical properties for the purpose of culturing tendon cells (TCs) for tendon tissue engineering. The results indicated a fiber diameter of 632±81 nm for the random nanofiber scaffold, 643±97 nm for the aligned nanofiber scaffold, and 641±68 nm for the nanoyarn scaffold. The yarn in the nanoyarn scaffold was twisted by many nanofibers similar to the structure and inherent nanoscale organization of tendons, indicating an increase in the diameter of 9.51±3.62 μm. The nanoyarn scaffold also contained 3D aligned microstructures with large interconnected pores and high porosity. Fourier transform infrared analyses revealed the presence of collagen in the three scaffolds. The mechanical properties of the sample scaffolds indicated that the scaffolds had desirable mechanical properties for tissue regeneration. Further, the results revealed that TC proliferation and infiltration, and the expression of tendon-related ECM genes, were significantly enhanced on the nanoyarn scaffold compared with that on the random nanofiber and aligned nanofiber scaffolds. This study demonstrates that electrospun P(LLA-CL)/collagen nanoyarn is a novel, 3D, macroporous, aligned scaffold that has potential application in tendon tissue engineering.
doi:10.1089/ten.tec.2012.0328
PMCID: PMC3833310  PMID: 23557537
5.  Cell Surface Vimentin Is an Attachment Receptor for Enterovirus 71 
Journal of Virology  2014;88(10):5816-5833.
ABSTRACT
Enterovirus 71 (EV71) is a highly transmissible pathogenic agent that causes severe central nervous system diseases in infected infants and young children. Here, we reported that EV71 VP1 protein could bind to vimentin intermediate filaments expressed on the host cell surface. Soluble vimentin or an antibody against vimentin could inhibit the binding of EV71 to host cells. Accompanied with the reduction of vimentin expression on the cell surface, the binding of EV71 to cells was remarkably decreased. Further evidence showed that the N terminus of vimentin is responsible for the interaction between EV71 and vimentin. These results indicated that vimentin on the host cell surface may serve as an attachment site that mediated the initial binding and subsequently increased the infectivity of EV71.
IMPORTANCE This study delivers important findings on the roles of vimentin filaments in relation to EV71 infection and provides information that not only improves our understanding of EV71 pathogenesis but also presents us with potentially new strategies for the treatment of diseases caused by EV71 infections.
doi:10.1128/JVI.03826-13
PMCID: PMC4019121  PMID: 24623428
6.  MiR-451 is decreased in hypertrophic cardiomyopathy and regulates autophagy by targeting TSC1 
The molecular mechanisms that drive the development of cardiac hypertrophy in hypertrophic cardiomyopathy (HCM) remain elusive. Accumulated evidence suggests that microRNAs are essential regulators of cardiac remodelling. We have been suggested that microRNAs could play a role in the process of HCM. To uncover which microRNAs were changed in their expression, microRNA microarrays were performed on heart tissue from HCM patients (n = 7) and from healthy donors (n = 5). Among the 13 microRNAs that were differentially expressed in HCM, miR-451 was the most down-regulated. Ectopic overexpression of miR-451 in neonatal rat cardiomyocytes (NRCM) decreased the cell size, whereas knockdown of endogenous miR-451 increased the cell surface area. Luciferase reporter assay analyses demonstrated that tuberous sclerosis complex 1 (TSC1) was a direct target of miR-451. Overexpression of miR-451 in both HeLa cells and NRCM suppressed the expression of TSC1. Furthermore, TSC1 was significantly up-regulated in HCM myocardia, which correlated with the decreased levels of miR-451. As TSC1 is a known positive regulator of autophagy, we examined the role of miR-451 in the regulation of autophagy. Overexpression of miR-451 in vitro inhibited the formation of the autophagosome. Conversely, miR-451 knockdown accelerated autophagosome formation. Consistently, an increased number of autophagosomes was observed in HCM myocardia, accompanied by up-regulated autophagy markers, and the lipidated form of LC3 and Beclin-1. Taken together, our findings indicate that miR-451 regulates cardiac hypertrophy and cardiac autophagy by targeting TSC1. The down-regulation of miR-451 may contribute to the development of HCM and may be a potential therapeutic target for this disease.
doi:10.1111/jcmm.12380
PMCID: PMC4224559  PMID: 25209900
hypertrophic cardiomyopathy; microRNA-451; tuberous sclerosis complex 1; autophagy; microRNAs microarray
7.  Uniaxial Cyclic Stretch Promotes Osteogenic Differentiation and Synthesis of BMP2 in the C3H10T1/2 Cells with BMP2 Gene Variant of rs2273073 (T/G) 
PLoS ONE  2014;9(9):e106598.
Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic bone formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of bone morphogenetic protein 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H10T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, 10% stretch). In the presence of mechanical stress, the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (P<0.05). In conclusion, these results suggested that BMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to bone transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.
doi:10.1371/journal.pone.0106598
PMCID: PMC4156358  PMID: 25191703
8.  Chemotherapy for transarterial chemoembolization in patients with unresectable hepatocellular carcinoma 
World Journal of Gastroenterology : WJG  2014;20(31):10960-10968.
AIM: To compare the efficacy of different chemotherapeutic agents during conventional transarterial chemoembolization (cTACE) in the treatment of unresectable hepatocellular carcinoma (HCC).
METHODS: A retrospective review was undertaken of patients with unresectable HCC undergoing cTACE from May 2003 to November 2011. A total of 107 patients were treated with at least one cTACE session. Irinotecan (CPT-11) was used as a chemotherapeutic agent in 24 patients, gemcitabine (GEM) in 24 and doxorubicin in 59.
RESULTS: The time to progression and overall survival rates were significantly superior in patients treated with CPT-11 compared with the GEM or doxorubicin treated groups (11.4, 8.2, 9.5 mo, P = 0.02 and 21.7, 12.7, 14.5 mo, P = 0.004, respectively). Subgroup analysis showed that for intermediate-stage HCC, CPT-11 resulted in a significantly longer time to progression and overall survival compared with the GEM or doxorubicin treated groups (P = 0.022; P = 0.003, respectively). There were no significant differences in adverse events among the three groups (P > 0.05).
CONCLUSION: For patients treated with cTACE, the chemotherapeutic agent CPT-11 was significantly associated with improved overall survival and delayed tumor progression compared with GEM or doxorubicin. There were no significant differences in clinical adverse events between the three agents. CPT-11 thus appears to be a promising agent when combined with cTACE for the treatment of HCC.
doi:10.3748/wjg.v20.i31.10960
PMCID: PMC4138477  PMID: 25152600
Irinotecan; Gemcitabine; Transarterial chemoembolization; Hepatocellular carcinoma; Overall survival
9.  Advanced abdominal pregnancy: an increasingly challenging clinical concern for obstetricians 
Advanced abdominal pregnancy is rare. The low incidence, high misdiagnosis rate, and lack of specific clinical signs and symptoms explain the fact that there are no standard diagnostic and treatment options available for advanced abdominal pregnancy. We managed a case of abdominal pregnancy in a woman who was pregnant for the first time. This case was further complicated by a concurrent singleton intrauterine pregnancy; the twin pregnancy was not detected until 20 weeks of pregnancy. The case was confirmed at 26 weeks gestational age using MRI to be an abdominal combined with intrauterine pregnancy. The pregnancy was terminated by cesarean section at 33 + 5 weeks gestation. We collected the relevant data of the case while reviewing the advanced abdominal pregnancy-related English literature in the Pubmed, Proquest, and OVID databases. We compared and analyzed the pregnancy history, gestational age when the diagnosis was confirmed, the placental colonization position, the course of treatment and surgical processes, related concurrency rate, post-operative drug treatment programs, and follow-up results with the expectation to provide guidance for other physicians who might encounter similar cases.
PMCID: PMC4203159  PMID: 25337188
Advanced abdominal pregnancy; obstetricians; clinical
10.  Female Sex Is Associated with Worse Prognosis in Patients with Hypertrophic Cardiomyopathy in China 
PLoS ONE  2014;9(7):e102969.
Background
Sex plays an important role in the clinical expression and prognosis of various cardiovascular diseases. This study was designed to observe the effects of sex on hypertrophic cardiomyopathy (HCM).
Methods and Results
A total of 621 unrelated patients with HCM without heart failure (460 males) were enrolled from 1999 to 2011. Compared to male patients, at baseline female patients were older at diagnosis (49.6±17.2 years vs. 46.7±14.4 years, P = 0.033), and had greater frequency of left ventricular outflow tract obstruction (72/161, 44.7% vs. 149/460, 32.4%, P = 0.005). During the average four year follow-up period (range 2–7 years), survival analysis showed that the incidences of mortality from all causes, cardiovascular death and progression to chronic heart failure were greater in women than in men (P = 0.031, 0.040 and 0.012, respectively). After adjustment for multiple factors that may confound survival and cardiac function, female sex remained an independent risk factor for all-cause mortality, cardiovascular death, and chronic heart failure [hazard ratio (HR) 2.19, 95% confidence interval (CI) 1.21–3.95, P = 0.010; HR 2.19, 95% CI 1.17–4.09, P = 0.014; HR 1.73, 95% CI 1.12–2.69, P = 0.014, respectively] in HCM patients. Subgroup analysis revealed that female sex as a risk factor was identified only in patients younger than 50 years old (P = 0.011, 0.011 and 0.009, respectively), but not for those 50 years or older.
Conclusion
Our results suggest that female sex is associated with worse survival and heart failure in HCM patients. Further studies are required to determine whether female hormones modify the clinical expression and prognosis of HCM.
doi:10.1371/journal.pone.0102969
PMCID: PMC4105411  PMID: 25047602
11.  Rare Variants in Genes Encoding MuRF1 and MuRF2 Are Modifiers of Hypertrophic Cardiomyopathy 
Modifier genes contribute to the diverse clinical manifestations of hypertrophic cardiomyopathy (HCM), but are still largely unknown. Muscle ring finger (MuRF) proteins are a class of muscle-specific ubiquitin E3-ligases that appear to modulate cardiac mass and function by regulating the ubiquitin-proteasome system. In this study we screened all the three members of the MuRF family, MuRF1, MuRF2 and MuRF3, in 594 unrelated HCM patients and 307 healthy controls by targeted resequencing. Identified rare variants were confirmed by capillary Sanger sequencing. The prevalence of rare variants in both MuRF1 and MuRF2 in HCM patients was higher than that in control subjects (MuRF1 13/594 (2.2%) vs. 1/307 (0.3%), p = 0.04; MuRF2 22/594 (3.7%) vs. 2/307 (0.7%); p = 0.007). Patients with rare variants in MuRF1 or MuRF2 were younger (p = 0.04) and had greater maximum left ventricular wall thickness (p = 0.006) than those without such variants. Mutations in genes encoding sarcomere proteins were present in 19 (55.9%) of the 34 HCM patients with rare variants in MuRF1 and MuRF2. These data strongly supported that rare variants in MuRF1 and MuRF2 are associated with higher penetrance and more severe clinical manifestations of HCM. The findings suggest that dysregulation of the ubiquitin-proteasome system contributes to the pathogenesis of HCM.
doi:10.3390/ijms15069302
PMCID: PMC4100095  PMID: 24865491
hypertrophic cardiomyopathy; muscle ring finger protein; rare variant; modifier
12.  Nanolayer encapsulation of insulin-chitosan complexes improves efficiency of oral insulin delivery 
Current oral insulin formulations reported in the literature are often associated with an unpredictable burst release of insulin in the intestine, which may increase the risk for problematic hypoglycemia. The aim of the study was to develop a solution based on a nanolayer encapsulation of insulin-chitosan complexes to afford sustained release after oral administration. Chitosan/heparin multilayer coatings were deposited onto insulin-chitosan microparticulate cores in the presence of poly(ethylene) glycol (PEG) in the precipitating and coating solutions. The addition of PEG improved insulin loading and minimized an undesirable loss of the protein resulting from redissolution. Nanolayer encapsulation and the formation of complexes enabled a superior loading capacity of insulin (>90%), as well as enhanced stability and 74% decreased solubility at acid pH in vitro, compared with nonencapsulated insulin. The capsulated insulin administered by oral gavage lowered fasting blood glucose levels by up to 50% in a sustained and dose-dependent manner and reduced postprandial glycemia in streptozotocin-induced diabetic mice without causing hypoglycemia. Nanolayer encapsulation reduced the possibility of rapid and erratic falls of blood glucose levels in animals. This technique represents a promising strategy to promote the intestinal absorption efficiency and release behavior of the hormone, potentially enabling an efficient and safe route for oral insulin delivery of insulin in diabetes management.
doi:10.2147/IJN.S59075
PMCID: PMC4014370  PMID: 24833901
oral insulin; diabetes mellitus; insulin-chitosan complexes; multilayer nanoencapsulation; polyethylene glycol; chitosan; heparin
13.  Tenascin-C Deficiency in Apo E−/− Mouse Increases Eotaxin Levels: Implications for Atherosclerosis 
Atherosclerosis  2013;227(2):267-274.
Aim
To investigate the potential role of inflammatory cytokines in apo E−/− mouse in response to deletion of Tenascin-C (TNC) gene.
Methods and results
We used antibody array and ELISA to compare the profile of circulating inflammatory cytokines in apo E−/− mice and apo E−/− TNC−/− double knockout mice. In addition, tissue culture studies were performed to investigate the activity of cells from each mouse genotype in vitro. Cytokine array analysis and subsequent ELISA showed that circulating eotaxin levels were selectively and markedly increased in response to TNC gene deletion in apo E−/− mice. In addition, considerable variation was noted in the circulating level of eotaxin among the control apo E−/− mouse group. Inbreeding of apo E−/− mice with high or low levels of plasma eotaxin showed that the level of eotaxin per se determines the extent of atherosclerosis in this mouse genotype. While endothelial cells from apo E−/− mice had low level of eotaxin expression, cells derived from apo E−/−TNC−/− mice expressed a high level of eotaxin. Transient transfection of eotaxin promoter-reporter constructs revealed that eotaxin expression is regulated at the transcriptional level by TNC. Histochemical analysis of aortic sections revealed the massive accumulation of mast cells in the adventitia of double KO mice lesions whereas no such accumulation was detected in the control group. Plasma from the apo E−/−TNC−/− mice markedly stimulated mast cell migration whereas plasma from the apo E−/− mice had no such effect.
Conclusion
These observations support the emerging hypothesis that TNC expression controls eotaxin level in apo E−/− mice and that this chemokine plays a key role in the development of atherosclerosis.
doi:10.1016/j.atherosclerosis.2013.01.039
PMCID: PMC3621962  PMID: 23433402
Tenascin-C; Eotaxin; Atherosclerosis; Mast cells; Chemokines; Matricellular protein; Extracellular matrix
14.  rAAV/ABAD-DP-6His attenuates oxidative stress-induced injury of PC12 cells 
Neural Regeneration Research  2014;9(5):481-488.
Our previous studies have revealed that amyloid β (Aβ)-binding alcohol dehydrogenase (ABAD) decoy peptide antagonizes Aβ42-induced neurotoxicity. However, whether it improves oxidative stress injury remains unclear. In this study, a recombinant adenovirus constitutively secreting and expressing Aβ-ABAD decoy peptide (rAAV/ABAD-DP-6His) was successfully constructed. Our results showed that rAAV/ABAD-DP-6His increased superoxide dismutase activity in hydrogen peroxide-induced oxidative stress-mediated injury of PC12 cells. Moreover, rAAV/ABAD-DP-6His decreased malondialdehyde content, intracellular Ca2+ concentration, and the level of reactive oxygen species. rAAV/ABAD-DP-6His maintained the stability of the mitochondrial membrane potential. In addition, the ATP level remained constant, and apoptosis was reduced. Overall, the results indicate that rAAV/ABAD-DP-6His generates the fusion peptide, Aβ-ABAD decoy peptide, which effectively protects PC12 cells from oxidative stress injury induced by hydrogen peroxide, thus exerting neuroprotective effects.
doi:10.4103/1673-5374.130065
PMCID: PMC4153500  PMID: 25206842
nerve regeneration; neurodegenerative disease; gene therapy; Alzheimer's disease; amyloid beta peptide; amyloid beta binding alcohol dehydrogenase; adeno-associated virus; hydrogen peroxide; oxidative stress; mitochondrial dysfunction; NSFC grant; neural regeneration
15.  Dab2 attenuates brain injury in APP/PS1 mice via targeting transforming growth factor-beta/SMAD signaling 
Neural Regeneration Research  2014;9(1):41-50.
Transforming growth factor-beta (TGF-β) type II receptor (TβRII) levels are extremely low in the brain tissue of patients with Alzheimer's disease. This receptor inhibits TGF-β1/SMAD signaling and thereby aggravates amyolid-beta deposition and neuronal injury. Dab2, a specific adapter protein, protects TβRII from degradation and ensures the effective conduction of TGF-β1/SMAD signaling. In this study, we used an adenoviral vector to overexpress the Dab2 gene in the mouse hippocampus and investigated the regulatory effect of Dab2 protein on TGF-β1/SMAD signaling in a mouse model of Alzheimer's disease, and the potential neuroprotective effect. The results showed that the TβRII level was lower in APP/PS1 mouse hippocampus than in normal mouse hippocampus. After Dab2 expression, hippocampal TβRII and p-SMAD2/3 levels were significantly increased, while amyloid-beta deposition, microglia activation, tumor necrosis factor-α and interleulin-6 levels and neuronal loss were significantly attenuated in APP/PS1 mouse brain tissue. These results suggest that Dab2 can exhibit neuroprotective effects in Alzheimer's disease by regulating TGF-β1/SMAD signaling.
doi:10.4103/1673-5374.125328
PMCID: PMC4146324  PMID: 25206742
nerve regeneration; transforming growth factor-β1; Dab2; Alzheimer's disease; amyolid-beta; neuron; SMAD2; SMAD3; microglia; neural regeneration
16.  Genome-wide multi-omics profiling of colorectal cancer identifies immune determinants strongly associated with relapse 
Frontiers in Genetics  2013;4:236.
The use and benefit of adjuvant chemotherapy to treat stage II colorectal cancer (CRC) patients is not well understood since the majority of these patients are cured by surgery alone. Identification of biological markers of relapse is a critical challenge to effectively target treatments to the ~20% of patients destined to relapse. We have integrated molecular profiling results of several “omics” data types to determine the most reliable prognostic biomarkers for relapse in CRC using data from 40 stage I and II CRC patients. We identified 31 multi-omics features that highly correlate with relapse. The data types were integrated using multi-step analytical approach with consecutive elimination of redundant molecular features. For each data type a systems biology analysis was performed to identify pathways biological processes and disease categories most affected in relapse. The biomarkers detected in tumors urine and blood of patients indicated a strong association with immune processes including aberrant regulation of T-cell and B-cell activation that could lead to overall differences in lymphocyte recruitment for tumor infiltration and markers indicating likelihood of future relapse. The immune response was the biologically most coherent signature that emerged from our analyses among several other biological processes and corroborates other studies showing a strong immune response in patients less likely to relapse.
doi:10.3389/fgene.2013.00236
PMCID: PMC3834519  PMID: 24312117
colorectal cancer; relapse; variant analysis; integrative analysis; multi-omics; exome sequencing; systems biology; immune response
17.  High expression of astrocyte elevated gene-1 (AEG-1) is associated with progression of cervical intraepithelial neoplasia and unfavorable prognosis in cervical cancer 
Background
Astrocyte elevated gene-1(AEG-1) plays an important role in the development and progression of certain types of human cancers. However, the expression dynamics of AEG-1 in cervical cancer and its clinical/prognostic significance are unclear.
Method
In present study, the methods of tissue microarrays (TMA) and immunohistochemistry (IHC) were utilized to investigate AEG-1 expression in cervical intraepithelial neoplasia (CIN) and cervical cancer. Receiver operating characteristic (ROC) curve analysis, χ2 test, Kaplan-Meier plots, and multivariate Cox regression analysis were used to analyze the data.
Results
The expression level of AEG-1 was increased from CIN I to CIN III. High expression of AEG-1 could be observed in 61.1% (55/90) of cervical cancer. Moreover, high expression of AEG-1 correlated with tumor size and lymph node metastasis (all P <0.05). More importantly, high expression of AEG-1 was closely associated with cervical cancer patient shortened survival time as evidenced by univariate and multivariate analysis (P <0.05).
Conclusions
Our data suggest for the first time that high expression of AEG-1 is associated significantly with progression of cervical cancer. AEG-1 overexpression, as examined by IHC, has the potential to be used as an immunomarker to predict prognosis of cervical cancer patients.
doi:10.1186/1477-7819-11-297
PMCID: PMC3866971  PMID: 24256614
AEG-1; Cervical cancer; Tissue microarrays; Prognosis
18.  Identification of Molecular Pathway Aberrations in Uterine Serous Carcinoma by Genome-wide Analyses 
Background
Uterine cancer is the fourth most common malignancy in women, and uterine serous carcinoma is the most aggressive subtype. However, the molecular pathogenesis of uterine serous carcinoma is largely unknown. We analyzed the genomes of uterine serous carcinoma samples to better understand the molecular genetic characteristics of this cancer.
Methods
Whole-exome sequencing was performed on 10 uterine serous carcinomas and the matched normal blood or tissue samples. Somatically acquired sequence mutations were further verified by Sanger sequencing. The most frequent molecular genetic changes were further validated by Sanger sequencing in 66 additional uterine serous carcinomas and in nine serous endometrial intraepithelial carcinomas (the preinvasive precursor of uterine serous carcinoma) that were isolated by laser capture microdissection. In addition, gene copy number was characterized by single-nucleotide polymorphism (SNP) arrays in 23 uterine serous carcinomas, including 10 that were subjected to whole-exome sequencing.
Results
We found frequent somatic mutations in TP53 (81.6%), PIK3CA (23.7%), FBXW7 (19.7%), and PPP2R1A (18.4%) among the 76 uterine serous carcinomas examined. All nine serous carcinomas that had an associated serous endometrial intraepithelial carcinoma had concordant PIK3CA, PPP2R1A, and TP53 mutation status between uterine serous carcinoma and the concurrent serous endometrial intraepithelial carcinoma component. DNA copy number analysis revealed frequent genomic amplification of the CCNE1 locus (which encodes cyclin E, a known substrate of FBXW7) and deletion of the FBXW7 locus. Among 23 uterine serous carcinomas that were subjected to SNP array analysis, seven tumors with FBXW7 mutations (four tumors with point mutations, three tumors with hemizygous deletions) did not have CCNE1 amplification, and 13 (57%) tumors had either a molecular genetic alteration in FBXW7 or CCNE1 amplification. Nearly half of these uterine serous carcinomas (48%) harbored PIK3CA mutation and/or PIK3CA amplification.
Conclusion
Molecular genetic aberrations involving the p53, cyclin E–FBXW7, and PI3K pathways represent major mechanisms in the development of uterine serous carcinoma.
doi:10.1093/jnci/djs345
PMCID: PMC3692380  PMID: 22923510
19.  miR-3928 activates ATR pathway by targeting Dicer 
RNA Biology  2012;9(10):1247-1254.
Alterations in microRNA (miRNA) expression have been observed in cells subjected to exogenous stresses, implying that miRNAs play an important role in cellular stress response; however, the underlying mechanism is still largely unknown. In the present study, we found that miR-3928 was implicated in cellular response to ionizing radiation. After exposed to X-rays, miR-3928 expression increased in 1.5 h and then decreased, meanwhile Dicer, a key component in the miRNA processing machinery, increased gradually. An oscillation was observed in the expression of both mature miR-3928 and Dicer mRNA from 2 h to 3.5 h in irradiated cells. Then, we verified that miR-3928 directly bound to the 3'-untranslated region of Dicer mRNA. Consequently, Dicer expression was suppressed and the maturation of other miRNAs including miR-185, miR-300, and miR-663, was inhibited. Overexpression of miR-3928 induced DNA damage, activated ATR, and phosphorylated Chk1 accompanied by G1 arrest. Taken together, these findings replenished ATR/Chk1 pathway by revealing a novel miRNA regulatory network in response to exogenous stress, in which miR-3928 plays an important role in regulating the expression of Dicer.
doi:10.4161/rna.21821
PMCID: PMC3583855  PMID: 22922797
DNA damage; Dicer; G1 arrest; ionizing radiation; microRNA
20.  Deletion of Tenascin-C gene Exacerbates Atherosclerosis and Induces Intraplaque Hemorrhage in Apo E Deficient Mice 
Aims
Tenascin-C (TNC), a matricellular protein, is upregulated in atherosclerotic plaques. We investigated whether the deletion of TNC gene affects the development of atherosclerosis in a murine model.
Methods
TNC−/−/apo E−/− mice were generated and used for atherosclerosis studies. We compared these results to those observed in control groups of apo E−/− mice.
Results
The en face analysis of aortic area showed that the mean aortic lesion area of the double KO mice was significantly higher than control mice at different times after feeding of atherogenic diet; the accumulation of lesional macrophages and lipids were significantly higher, respectively. Analysis of cell adhesion molecules revealed that VCAM-1, but not ICAM-1, was upregulated 1 week after feeding of atherogenic diet in the double KO mouse as compared to apo E−/− mouse. Cell culture studies revealed that the expression of VCAM-1 in endothelial cells isolated from the double KO mouse is more sensitive to the TNFα stimulation than the cells isolated from apo E−/− mice. Cell adhesion studies showed that the adherence of RAW monocytic cells to the endothelial cells was significantly enhanced in the cultured endothelial cells from the TNC gene-deleted cells. Following the prolonged feeding of an atherogenic diet (28–30 weeks), the aortic and carotid atherosclerotic lesions frequently demonstrated large grossly visible areas of intraplaque hemorrhage in the double KO mice compared to control.
Conclusions
These data unveil a protective role for TNC in atherosclerosis and suggest that TNC signaling may have the potential to reduce atherosclerosis, in part by modulating VCAM-1 expression.
doi:10.1016/j.carpath.2011.12.005
PMCID: PMC3345312  PMID: 22300502
tenascin; atherosclerosis; plaque hemorrhage; extracellular matrix; VCAM-1
21.  The potential value of the neutral comet assay and γH2AX foci assay in assessing the radiosensitivity of carbon beam in human tumor cell lines 
Radiology and Oncology  2013;47(3):247-257.
Background
Carbon ions (12C6+) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The assessment of tumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of the neutral comet assay and γH2AX foci assay in assessing 12C6+ radiosensitivity of tumour cells.
Materials and methods
The doses of 12C6+ and X-rays used in the present study were 2 and 4 Gy. The survival fraction, DNA double-strand breaks (DSB) and repair kinetics of DSB were assayed with clonogenic survival, neutral comet assay and γH2AX foci assay in human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells at the time points of 0.5, 4, 16 and 24 h after 12C6+ and X-rays irradiation.
Results
The survival fraction for 12C6+ irradiation was much more inhibited than for X-rays (p < 0.05) in all three tumour cell lines tested. Substantial amounts of residual damage, assessed by the neutral comet assay, were present after irradiation (p < 0.05). The highest residual damage was observed at 0.5 or 4 h, both for 12C6+ and X-ray irradiation. However, the residual damage in HeLa and MEC-1 cells was higher for 12C6+ than X-rays (p < 0.05). The strongest induction of γH2AX foci was observed after 30 min, for all three tumour cell lines (p < 0.01). The franction of γH2AX foci persisted for at least 24 h after 12C6+ irradiation; in HeLa cells and MEC-1 was higher than after X-ray irradiation (p < 0.05). The correlation coefficients between the clonogenic survival, neutral comet assay and γH2AX foci assay were not statistically significant, except for some tumour cells at individual irradiation doses and types.
Conclusions
Our study demonstrated that the neutral comet assay and γ-H2AX foci assay could be used to assess the radiosensitivity of 12C6+ in human tumour cells.
doi:10.2478/raon-2013-0045
PMCID: PMC3794881  PMID: 24133390
human tumour cells; carbon ions; X-rays; radiation sensitivity; DNA double strand breaks; γH2AX
22.  Patch-Clamp Recordings from Lateral Line Neuromast Hair Cells of the Living Zebrafish 
Zebrafish are popular models for biological discovery. For investigators of the auditory and vestibular periphery, manipulations of hair cell and synaptic mechanisms have relied on inferences from extracellular recordings of physiological activity. We now provide data showing that hair cells and supporting cells of the lateral line can be directly patch-clamped, providing the first recordings of ionic channel activity, synaptic vesicle release, and gap junctional coupling in the neuromasts of living fish. Such capabilities will allow more detailed understanding of mechano-sensation of the zebrafish.
doi:10.1523/JNEUROSCI.4265-12.2013
PMCID: PMC3684625  PMID: 23407967
23.  Targeting Rac1 Signaling Inhibits Streptococcal M1 Protein-Induced CXC Chemokine Formation, Neutrophil Infiltration and Lung Injury 
PLoS ONE  2013;8(8):e71080.
Infections with Streptococcus pyogenes exhibit a wide spectrum of infections ranging from mild pharyngitis to severe Streptococcal toxic shock syndrome (STSS). The M1 serotype of Streptococcus pyogenes is most commonly associated with STSS. In the present study, we hypothesized that Rac1 signaling might regulate M1 protein-induced lung injury. We studied the effect of a Rac1 inhibitor (NSC23766) on M1 protein-provoked pulmonary injury. Male C57BL/6 mice received NSC23766 prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Treatment with NSC23766 decreased M1 protein-induced neutrophil infiltration, edema formation and tissue injury in the lung. M1 protein challenge markedly enhanced Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Rac1 activity had no effect on M1 protein-induced expression of Mac-1 on neutrophils. However, Rac1 inhibition markedly decreased M1 protein-evoked formation of CXC chemokines in the lung. Moreover, NSC23766 completely inhibited M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. We conclude that these novel results suggest that Rac1 signaling is a significant regulator of neutrophil infiltration and CXC chemokine production in the lung. Thus, targeting Rac1 activity might be a potent strategy to attenuate streptococcal M1 protein-triggered acute lung damage.
doi:10.1371/journal.pone.0071080
PMCID: PMC3741375  PMID: 23951087
24.  Autosomal Recessive Transmission of MYBPC3 Mutation Results in Malignant Phenotype of Hypertrophic Cardiomyopathy 
PLoS ONE  2013;8(6):e67087.
Background
Hypertrophic cardiomyopathy (HCM) due to mutations in genes encoding sarcomere proteins is most commonly inherited as an autosomal dominant trait. Since nearly 50% of HCM cases occur in the absence of a family history, a recessive inheritance pattern may be involved.
Methods
A pedigree was identified with suspected autosomal recessive transmission of HCM. Twenty-six HCM-related genes were comprehensively screened for mutations in the proband with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and 376 healthy controls.
Results
A novel missense mutation (c.1469G>T, p.Gly490Val) in exon 17 of MYBPC3 was identified. Two siblings with HCM were homozygous for this mutation, whereas other family members were either heterozygous or wild type. Clinical evaluation showed that both homozygotes manifested a typical HCM presentation, but none of others, including 5 adult heterozygous mutation carriers up to 71 years of age, had any clinical evidence of HCM.
Conclusions
Our data identified a MYBPC3 mutation in HCM, which appeared autosomal recessively inherited in this family. The absence of a family history of clinical HCM may be due to not only a de novo mutation, but also recessive mutations that failed to produce a clinical phenotype in heterozygous family members. Therefore, consideration of recessive mutations leading to HCM is essential for risk stratification and genetic counseling.
doi:10.1371/journal.pone.0067087
PMCID: PMC3695947  PMID: 23840593
25.  Atorvastatin Enhance Efficacy of Mesenchymal Stem Cells Treatment for Swine Myocardial Infarction via Activation of Nitric Oxide Synthase 
PLoS ONE  2013;8(5):e65702.
Background
In a swine model of acute myocardial infarction (AMI), Statins can enhance the therapeutic efficacy of mesenchymal stem cell (MSCs) transplantation. However, the mechanisms remain unclear. This study aims at assessing whether atorvastatin (Ator) facilitates the effects of MSCs through activation of nitric oxide synthase (NOS), especially endothelial nitric oxide synthase (eNOS), which is known to protect against ischemic injury.
Methods and Results
42 miniswines were randomized into six groups (n = 7/group): Sham operation; AMI control; Ator only; MSC only, Ator+MSCs and Ator+MSCs+NG-nitrol-L-arginine (L-NNA), an inhibitor of NOS. In an open-heart surgery, swine coronary artery ligation and reperfusion model were established, and autologous bone-marrow MSCs were injected intramyocardium. Four weeks after transplantation, compared with the control group, Ator+MSCs animals exhibited decreased defect areas of both “perfusion” defined by Single-Photon Emission Computed Tomography (−6.2±1.8% vs. 2.0±5.1%, P = 0.0001) and “metabolism” defined by Positron Emission Tomography (−3.00±1.41% vs. 4.20±4.09%, P = 0.0004); Ejection fraction by Magnetic Resonance Imaging increased substantially (14.22±12.8% vs. 1.64±2.64%, P = 0.019). In addition, indices of inflammation, fibrosis, and apoptosis were reduced and survivals of MSCs or MSC-derived cells were increased in Ator+MSCs animals. In Ator or MSCs alone group, perfusion, metabolism, inflammation, fibrosis or apoptosis were reduced but there were no benefits in terms of heart function and cell survival. Furthermore, the above benefits of Ator+MSCs treatment could be partially blocked by L-NNA.
Conclusions
Atorvastatin facilitates survival of implanted MSCs, improves function and morphology of infarcted hearts, mediated by activation of eNOS and alleviated by NOS inhibitor. The data reveal the cellular and molecular mechanism for anti-AMI therapy with a combination of statin and stem cells.
doi:10.1371/journal.pone.0065702
PMCID: PMC3669282  PMID: 23741509

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