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1.  Model-driven intracellular redox status modulation for increasing isobutanol production in Escherichia coli 
Background
Few strains have been found to produce isobutanol naturally. For building a high performance isobutanol-producing strain, rebalancing redox status of the cell was very crucial through systematic investigation of redox cofactors metabolism. Then, the metabolic model provided a powerful tool for the rational modulation of the redox status.
Results
Firstly, a starting isobutanol-producing E. coli strain LA02 was engineered with only 2.7 g/L isobutanol produced. Then, the genome-scale metabolic modeling was specially carried out for the redox cofactor metabolism of the strain LA02 by combining flux balance analysis and minimization of metabolic adjustment, and the GAPD reaction catalyzed by the glyceraldehyde-3-phosphate dehydrogenase was predicted as the key target for redox status improvement. Under guidance of the metabolic model prediction, a gapN-encoding NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase pathway was constructed and then fine-tuned using five constitutive promoters. The best strain LA09 was obtained with the strongest promoter BBa_J23100. The NADPH/NADP + ratios of strain LA09 reached 0.67 at exponential phase and 0.64 at stationary phase. The redox modulations resulted in the decrease production of ethanol and lactate by 17.5 and 51.7% to 1.32 and 6.08 g/L, respectively. Therefore, the isobutanol titer was increased by 221% to 8.68 g/L.
Conclusions
This research has achieved rational redox status improvement of isobutanol-producing strain under guidance of the prediction and modeling of the genome-scale metabolic model of isobutanol-producing E. coli strain with the aid of synthetic promoters. Therefore, the production of isobutanol was dramatically increased by 2.21-fold from 2.7 to 8.68 g/L. Moreover, the developed model-driven method special for redox cofactor metabolism was of very helpful to the redox status modulation of other bio-products.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0291-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13068-015-0291-2
PMCID: PMC4522091
Isobutanol; Redox balance; Genome-scale metabolic model; Synthetic promoters; Glyceraldehyde-3-phosphate dehydrogenase
2.  TMPRSS4 facilitates epithelial-mesenchymal transition of hepatocellular carcinoma and is a predictive marker for poor prognosis of patients after curative resection 
Scientific Reports  2015;5:12366.
TMPRSS4 (Transmembrane protease serine 4) is up-regulated in a broad spectrum of cancers. However, little is known about the biological effects of TMPRSS4 on hepatocellular carcinoma (HCC) and the related mechanisms. In the present study, we found that overexpression of TMPRSS4 significantly promoted the invasion, migration, adhesion and metastasis of HCC. Further more, TMPRSS4 induced EMT of HCC, which was mediated via snail and slug as a result of Raf/MEK/ERK1/2 activation, and inhibition of ERK1/2 activation by its inhibitor was associated with reduced cell invasion and reversion of EMT. In addition, we demonstrated that TMPRSS4 remarkably suppressed the expression of RECK, an inhibitor of angiogenesis, and drastically induced tumor angiogenesis and growth. More important, in clinical HCC specimens, TMPRSS4 expression was significantly correlated with tumor staging and was inversely correlated with E-cadherin and RECKS expression. Expression of TMPRSS4 is significantly associated with HCC progression and is an independent prognostic factor for postoperative worse survival and recurrence. In conclusion, TMPRSS4 functions as a positive regulator of Raf/MEK/ERK1/2 pathway and promotes HCC progression by inducing EMT and angiogenesis. The increase of TMPRSS4 expression may be a key event for HCC progression and may be regarded as a potential prognostic marker for HCC.
doi:10.1038/srep12366
PMCID: PMC4507176  PMID: 26190376
3.  Nighttime Systolic Blood-Pressure Load Is Correlated with Target-Organ Damage Independent of Ambulatory Blood-Pressure Level in Patients with Non-Diabetic Chronic Kidney Disease 
PLoS ONE  2015;10(7):e0131546.
Background
The impacts of blood pressure (BP) load on target-organ damage in patients with chronic kidney disease (CKD) are largely unclear. We examined whether BP load is correlated with target-organ damage (TOD) in Chinese CKD patients independent of BP level.
Methods
We recruited 1219 CKD patients admitted to our hospital division in this cross-sectional study. The TOD were measured by estimated glomerular filtration rate (eGFR), proteinuria, left ventricular mass index (LVMI) and carotid intima-media thickness (cIMT) in this study. Univariate and multivariate linear analyses were used to evaluate the relationship between systolic blood pressure (SBP) load, diastolic blood pressure (DBP) load and these renal, cardiovascular parameters.
Results
In multivariable-adjusted models, BP load and ambulatory BP levels both independently correlated with LVMI, eGFR and proteinuria in all groups of CKD patients (p<0.05), 24-h SBP correlated with cIMT only in non-diabetic CKD patients without hypertension (p<0.05), while nighttime SBP load was associated with cIMT only in non-diabetic CKD patients (p<0.05). Furthermore, nighttime SBP load additionally increased coefficient of determination (R2) and correlated with LVMI, proteinuria in non-diabetic CKD patients without hypertension (R2 = 0.034, P<0.001 and R2 = 0.012, P = 0.006 respectively) and LVMI, cIMT, eGFR in non-diabetic CKD patients with hypertension (R2>0.008, P<0.05) in multivariable-adjusted model which already including the 24-h BP. BP load did not refine this correlation based on the 24-h BP level in diabetic CKD patients.
Conclusion
Night-time SBP load was correlated with TOD in patients with non-diabetic chronic kidney disease independent of BP level.
doi:10.1371/journal.pone.0131546
PMCID: PMC4506060  PMID: 26186336
4.  The Novel Histone Deacetylase Inhibitor Thailandepsin A Inhibits Anaplastic Thyroid Cancer Growth 
The Journal of surgical research  2014;190(1):191-197.
Background
Anaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with anti-proliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC.
Methods
Human-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry.
Results
TDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly ADP ribose polymerase (PARP) were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently and the anti-apoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase following TDP-A treatment.
Conclusion
TDP-A induces a notable dose- and time-dependent anti-proliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations.
doi:10.1016/j.jss.2014.02.042
PMCID: PMC4063213  PMID: 24679699
thyroid cancer; anaplastic; HDAC inhibitor; thailandepsin A; TDP-A; growth inhibition
5.  Epstein - Barr virus Serology as a Potential Screening Marker for Nasopharyngeal Carcinoma among High-risk Individuals from Multiplex Families in Taiwan 
Background
Nasopharyngeal carcinoma (NPC) is an EBV associated cancer that is highly treatable when diagnosed early, with 5-year disease-free survival of ~90%. However, NPC is typically diagnosed at advanced stages, where disease-free survival is <50%. There is therefore a need for clinical tools to assist in early NPC detection, particularly in high-risk individuals.
Methods
We evaluated the ability of anti-EBV IgA antibodies to detect incident NPC among high-risk Taiwanese individuals. NPC cases (N=21) and age and sex-matched controls (N=84) were selected. Serum collected prior to NPC diagnosis was tested for ELISA-based IgA markers against the following EBV peptides: EBNA1, VCAp18, EAp138, Ead_p47, and VCAp18 + EBNA1 peptide mixture. The sensitivity, specificity, and screening program parameters were calculated.
Results
EBNA1 IgA had the best performance characteristics. At an optimized threshold value, EBNA1 IgA measured at baseline identified 80% of the high-risk individuals who developed NPC during follow-up (80% sensitivity). However, approximately 40% of high-risk individuals who did not develop NPC also tested positive (false positives). Application of EBNA1 IgA as a biomarker to detect incident NPC in a previously unscreened, high-risk population revealed that 164 individuals needed to be screened to detect 1 NPC and that 69 individuals tested positive per case detected.
Conclusions
EBNA1 IgA proved to be a sensitive biomarker for identifying incident NPC, but future work is warranted to develop more specific screening tools to decrease the number of false positives.
Impact
Results from this study could inform decisions regarding screening biomarkers and referral thresholds for future NPC early-detection program evaluations.
doi:10.1158/1055-9965.EPI-13-1262
PMCID: PMC4082438  PMID: 24769890
Epstein-Barr virus; EBV serology; familial NPC; cancer screening
6.  Resection of the main trunk of the superior mesenteric vein without reconstruction during surgery for giant pancreatic mucinous cystadenoma: A case report 
Pancreatic tumors, with peri-pancreatic main vascular invasion, especially the superior mesenteric vein (SMV) or the portal vein, are very common. In some cases, vascular resection and reconstruction are required for complete resection of pancreatic tumors. However, the optimum surgical method for venous management is controversial. Resection of the SMV without reconstruction during surgery for pancreatic tumors is rarely reported. Here we present the case of a 58-year-old woman with a giant pancreatic mucinous cystadenoma adhering to the SMV, who underwent an en bloc tumor resection, including the main trunk of the SMV and the spleen. No venous reconstruction was performed during surgery. No ischemic changes occurred in the bowel. The presence of several well-developed collateral vessels was shown by 3-dimensional computed tomography examination. The patient had an uneventful postoperative period and was discharged. This case indicated that the main trunk of the SMV can be resected without venous reconstruction if adequate collateralization has formed.
doi:10.3748/wjg.v21.i24.7604
PMCID: PMC4481460  PMID: 26140011
Superior mesenteric vein; Pancreatic mucinous cystadenoma; Collateral vessel; Main trunk; Reconstruction
7.  Genome-wide Association Study on Platinum-induced Hepatotoxicity in Non-Small Cell Lung Cancer Patients 
Scientific Reports  2015;5:11556.
Platinum-based chemotherapy has been shown to improve the survival of advanced non-small cell lung cancer (NSCLC) patients; the platinum-induced toxicity severely impedes the success of chemotherapy. Genetic variations, such as single nucleotide polymorphisms (SNPs), may contribute to patients’ responses to the platinum-based chemotherapy. To identify SNPs that modify the risk of hepatotoxicity in NSCLC patients receiving platinum-based chemotherapy, we performed a genome-wide association scan in 334 subjects followed by a replication study among 375 subjects. Consistent associations with platinum-induced hepatotoxicity risk was identified for SNP rs2838566 located at 21q22.3, as the minor A allele could significantly increase the risk of liver injury (OR = 3.78, 95%CI = 1.99–7.19, P = 4.90 × 10−5 for GWAS scan, OR = 1.89, 95%CI = 1.03–3.46, P = 0.039 for replication, and OR = 2.56, 95%CI = 1.65–3.95, P = 2.55 × 10−5 for pooled population). These results suggested that genetic variants at 21q22.3 may contribute to the susceptibility of platinum-induced hepatotoxicity in NSCLC patients.
doi:10.1038/srep11556
PMCID: PMC4477405  PMID: 26100964
8.  Impact of body mass index on complications following pancreatectomy: Ten-year experience at National Cancer Center in China 
AIM: To examine the impact of body mass index (BMI) on outcomes following pancreatic resection in the Chinese population.
METHODS: A retrospective cohort study using prospectively collected data was conducted at the Cancer Hospital of the Chinese Academy of Medical Sciences, China National Cancer Center. Individuals who underwent pancreatic resection between January 2004 and December 2013 were identified and included in the study. Persons were classified as having a normal weight if their BMI was < 24 kg/m2 and overweight/obese if their BMI was ≥ 24 kg/m2 as defined by the International Life Sciences Institute Focal Point in China. A χ2 test (for categorical variables) or a t test (for continuous variables) was used to examine the differences in patients’ characteristics between normal weight and overweight/obese groups. Multiple logistic regression models were used to assess the associations of postoperative complications, operative difficulty, length of hospital stay, and cost with BMI, adjusting for age, sex, and type of surgery procedures.
RESULTS: A total of 362 consecutive patients with data available for BMI calculation underwent pancreatic resection for benign or malignant disease from January 1, 2004 to December 31, 2013. Of the 362 patients, 156 were overweight or obese and 206 were of normal weight. One or more postoperative complications occurred in 35.4% of the patients following pancreatic resection. Among patients who were overweight or obese, 42.9% experienced one or more complications, significantly higher than normal weight (29.6%) individuals (P = 0.0086). Compared with individuals who had normal weight, those with a BMI ≥ 24.0 kg/m2 had higher delayed gastric emptying (19.9% vs 5.8%, P < 0.0001) and bile leak (7.7% vs 1.9%, P = 0.0068). There were no significant differences seen in pancreatic fistula, gastrointestinal hemorrhage, reoperation, readmission, or other complications. BMI did not show a significant association with intraoperative blood loss, operative time, length of hospital stay, or cost.
CONCLUSION: Higher BMI increases the risk for postoperative complications after pancreatectomy in the Chinese population. The findings require replication in future studies with larger sample sizes.
doi:10.3748/wjg.v21.i23.7218
PMCID: PMC4476883  PMID: 26109808
Body mass index; China; Pancreatectomy; Pancreatic cancer; Postoperative complications
9.  Thiol peroxidases ameliorate LRRK2 mutant-induced mitochondrial and dopaminergic neuronal degeneration in Drosophila 
Human Molecular Genetics  2014;23(12):3157-3165.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are common causes of familial Parkinson's disease (PD). LRRK2 has been shown to bind peroxiredoxin-3 (PRDX3), the most important scavenger of hydrogen peroxide in the mitochondria, in vitro. Here, we examined the interactions of LRRK2 and PRDX3 in Drosophila models by crossing transgenic LRRK2 and PRDX3 flies. As proof of principle experiments, we subsequently challenged LRRK2 and LRRK2/PRDX3 flies with a peroxidase mimic, Ebselen. We demonstrated that co-expression of PRDX3 with the LRRK2 kinase mutant G2019S in bigenic Drosophila ameliorated the G2019S mutant-induced reduction in peroxidase capacity, loss of dopaminergic neurons, shortened lifespan and mitochondrial defects of flight muscles in monogenic flies expressing the G2019S alone. Challenges with Ebselen recapitulated similar rescue of these phenotypic features in mutant-expressing Drosophila. The peroxidase mimic preserved neuronal and mitochondrial and neuronal integrity and improved mobility and survival in mutant-expressing Drosophila. Taken together, our study provides the first in vivo evidence to suggest that phosphoinhibition of endogenous peroxidases could be a mechanism in LRRK2-induced oxidant-mediated neurotoxicity. Our therapeutic experiments also highlight the potential of thiol peroxidases as neuroprotective agents in PD patients carrying LRRK2 mutations.
doi:10.1093/hmg/ddu026
PMCID: PMC4030771  PMID: 24459295
10.  miR-135b Promotes Cancer Progression by Targeting Transforming Growth Factor Beta Receptor II (TGFBR2) in Colorectal Cancer 
PLoS ONE  2015;10(6):e0130194.
The transforming growth factor beta (TGF-β) signaling pathway is a tumor-suppressor pathway that is commonly inactivated in colorectal cancer (CRC). The inactivation of TGFBR2 is the most common genetic event affecting the TGF-β signaling pathway. However, the mechanism by which cancer cells downregulate TGFBR2 is unclear. In this study, we found that the TGFBR2 protein levels were consistently upregulated in CRC tissues, whereas its mRNA levels varied in these tissues, suggesting that a post-transcriptional mechanism is involved in the regulation of TGFBR2. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we performed bioinformatic analyses to search for miRNAs that potentially target TGFBR2. We identified the specific targeting site of miR-135b in the 3’-untranslated region (3’-UTR) of TGFBR2. We further identified an inverse correlation between the levels of miR-135b and TGFBR2 protein, but not mRNA, in CRC tissue samples. By overexpressing or silencing miR-135b in CRC cells, we experimentally validated that miR-135b directly binds to the 3’-UTR of the TGFBR2 transcript and regulates TGFBR2 expression. Furthermore, the biological consequences of the targeting of TGFBR2 by miR-135b were examined using in vitro cell proliferation and apoptosis assays. We demonstrated that miR-135b exerted a tumor-promoting effect by inducing the proliferation and inhibiting the apoptosis of CRC cells via the negative regulation of TGFBR2 expression. Taken together, our findings provide the first evidence supporting the role of miR-135b as an oncogene in CRC via the inhibition of TGFBR2 translation.
doi:10.1371/journal.pone.0130194
PMCID: PMC4462589  PMID: 26061281
11.  Plasma Epidermal Growth Factor Decreased in the Early Stage of Parkinson’s Disease 
Aging and Disease  2015;6(3):168-173.
Epidermal growth factor (EGF) is a neurotrophic factor that plays an important role in Parkinson’s disease (PD). We measured plasma EGF level in PD, essential tremor (ET) and normal controls to investigate whether it changes in PD and whether it is associated with motor and non-motor symptoms of PD. 100 patients with PD, 40 patients with ET as disease control and 76 healthy persons were enrolled in the present study. Motor and non-motor symptoms were assessed by different scales. Plasma EGF levels of three groups were measured by enzyme-linked immunosorbent assay kit. Spearman test and linear logistics regression model were used to test the correlation of EGF with motor and non-motor symptoms of PD. Plasma EGF level was significantly decreased in early PD patients compared with normal control, but not in advanced PD patients. Interestingly, plasma EGF level was significantly increased in advanced PD and total PD patients compared with ET patients, but not in early PD patients. In addition, plasma EGF level was correlated with UPDRS-III scores in PD. Also plasma EGF level was correlated with UPDRS-III scores and NMS scores in early PD. Our results suggested that plasma EGF decreased in the early stage of PD and increased later on in the PD disease course. Also, plasma EGF level was increased significantly in PD compared with ET patients and correlated with motor and non-motor symptoms in early PD.
doi:10.14336/AD.2014.0925
PMCID: PMC4441398  PMID: 26029474
epidermal growth factor; Parkinson’s disease; essential tremor
12.  P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy 
Cell Research  2015;25(6):674-690.
Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.
doi:10.1038/cr.2015.61
PMCID: PMC4456628  PMID: 25998681
septic encephalopathy; leukocyte adhesion; P2RX7; ICAM-1; two-photon imaging; microglia; neurovascular injury
13.  Effects of alfalfa saponin extract on mRNA expression of Ldlr, LXRα, and FXR in BRL cells*  
We studied the effects of alfalfa saponin extract (ASE) on low density lipoprotein receptor (Ldlr), liver X receptor α (LXRα), and farnesoid X receptor (FXR) in normal and hyperlipidemic Buffalo rat liver (BRL) cells. Normal and hyperlipidemic BRL cells were divided into eight groups: normal, or normal cells treated with 50, 100, and 150 mg/L ASE, hyperlipidemic, or hyperlipidemic cells treated with 50, 100, and 150 mg/L ASE. After treatment for 24 h, Ldlr, LXRα, and FXR mRNA expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Data showed that mRNA expression of Ldlr in normal BRL cells was significantly up-regulated by ASE treatment and mRNA expressions of LXRα and FXR were significantly down-regulated both in normal and hyperlipidemic BRL cells after ASE treatment. Thus, ASE might ameliorate hepatic steatosis by regulating genes involved in cholesterol metabolism, including up-regulation of Ldlr as well as down-regulation of LXRα and FXR.
doi:10.1631/jzus.B1400343
PMCID: PMC4471599  PMID: 26055909
Alfalfa saponin extract; Hyperlipidemic BRL cells; Cholesterol metabolism; mRNA expression
14.  Generalized-stacking-fault energy and twin-boundary energy of hexagonal close-packed Au: A first-principles calculation 
Scientific Reports  2015;5:10213.
Although solid Au is usually most stable as a face-centered cubic (fcc) structure, pure hexagonal close-packed (hcp) Au has been successfully fabricated recently. However, the phase stability and mechanical property of this new material are unclear, which may restrict its further applications. Here we present the evidence that hcp → fcc phase transformation can proceed easily in Au by first-principles calculations. The extremely low generalized-stacking-fault (GSF) energy in the basal slip system implies a great tendency to form basal stacking faults, which opens the door to phase transformation from hcp to fcc. Moreover, the Au lattice extends slightly within the superficial layers due to the self-assembly of alkanethiolate species on hcp Au (0001) surface, which may also contribute to the hcp → fcc phase transformation. Compared with hcp Mg, the GSF energies for non-basal slip systems and the twin-boundary (TB) energies for and twins are larger in hcp Au, which indicates the more difficulty in generating non-basal stacking faults and twins. The findings provide new insights for understanding the nature of the hcp → fcc phase transformation and guide the experiments of fabricating and developing materials with new structures.
doi:10.1038/srep10213
PMCID: PMC4441118  PMID: 25998415
15.  miR-19b downregulates intestinal SOCS3 to reduce intestinal inflammation in Crohn’s disease 
Scientific Reports  2015;5:10397.
Although aberrant microRNA (miRNA) expression has frequently been observed in inflammatory bowel disease (IBD), its biological functions and targets remain largely unknown. Present study found that miR-19b was significantly downregulated in active Crohn’s disease (CD). Using bioinformatics analysis, suppressor of cytokine signalling 3 (SOCS3), a physiological regulator of innate and adaptive immunity that controls several immuno-inflammatory diseases, was predicted to be a potential target of miR-19b. An inverse correlation between miR-19b and SOCS3 protein levels, but not mRNA, was identified in active-CD intestinal tissue samples. By overexpressing or knocking down miR-19b in Caco2 cells and HT29 cells, it was experimentally validated that miR-19b is a direct regulator of SOCS3. Using a luciferase reporter assay, it was confirmed that miR-19b directly recognizes the 3’-untranslated region (3’-UTR) of SOCS3. Furthermore, overexpression of miR-19b decreased SOCS3 expression, leading to increased production of macrophage-inflammatory protein-3α (MIP-3α) in Caco2 cells. In contrast, knockdown of miR-19b increased SOCS3 and decreased MIP-3α. Finally, intracolonically delivered miR-19b decreased the severity of colitis induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS). Taken together, our findings suggest that miR-19b suppresses the inflammatory response by inhibiting SOCS3 to modulate chemokine production in intestinal epithelial cells (IECs) and thereby prevents the pathogenesis of CD.
doi:10.1038/srep10397
PMCID: PMC4441154  PMID: 25997679
16.  Pegylated liposomal doxorubicin (Lipo-Dox®) combined with cyclophosphamide and 5-fluorouracil is effective and safe as salvage chemotherapy in taxane-treated metastatic breast cancer: an open-label, multi-center, non-comparative phase II study 
BMC Cancer  2015;15:423.
Background
Anthracycline and taxane are classes of drugs that are frequently used in the adjuvant and palliative settings of metastatic breast cancer (MBC); however, treatment failure occurs in most cases. Limited data demonstrated favorable response in MBC after previous taxane-based treatment. The aim of this study was to evaluate the efficacy and safety of pegylated liposomal doxorubicin (Lipo-Dox®) used as part of a combination salvage therapy for patients with MBC whose tumors progressed during or after taxane-based treatment.
Methods
Patients with MBC who failed to respond to previous taxane-based treatments were recruited. Treatment with pegylated liposomal doxorubicin (40 mg/m2), cyclophosphamide (500 mg/m2), and 5-fluorouracil (500 mg/m2) was administered every 3 weeks. Tumor response to treatment was determined by using the Response Evaluation Criteria in Solid Tumor criteria version 1.0, and left ventricular ejection fraction was measured before and after treatment using echocardiography. Each patient was followed for 30 days after the last dose of study medication or until resolution/stabilization of any drug-related adverse event.
Results
Forty-five patients were recruited. As of December 2012, the median follow-up duration was 29.8 months, the overall response rate was 41.9 %, the median progression-free survival was 8.2 months, and the median overall survival was 36.6 months for all treated patients. Grade 3/4 neutropenia, leucopenia, and neutropenic fever were observed in 14 %, 9 %, and 1 % of the cycles, respectively. Other non-hematologic adverse effects were mild to moderate and were manageable. No decrease in left ventricular ejection function was noted.
Conclusion
This regimen of combined of pegylated liposomal doxorubicin, cyclophosphamide, and 5-fluorouracil exhibited a promising overall response rate, progression-free survival rate, and overall survival rate, with a safe cardiac toxicity profile and manageable adverse effects. This regimen could be considered as a treatment option for patients with MBC whose tumors progressed during or after taxane-based treatment.
doi:10.1186/s12885-015-1433-4
PMCID: PMC4440506  PMID: 25994543
Advanced breast cancer; Pegylated liposomal doxorubicin; Metastatic breast cancer; Taxane failure; Safety
17.  Serum MicroRNA Profiles Serve as Novel Biomarkers for the Diagnosis of Alzheimer's Disease 
Disease Markers  2015;2015:625659.
Alzheimer's disease (AD) is the most common type of dementia, and promptly diagnosis of AD is crucial for delaying the development of disease and improving patient quality of life. However, AD detection, particularly in the early stages, remains a substantial challenge due to the lack of specific biomarkers. The present study was undertaken to identify and validate the potential of circulating miRNAs as novel biomarkers for AD. Solexa sequencing was employed to screen the expression profile of serum miRNAs in AD and controls. RT-qPCR was used to confirm the altered miRNAs at the individual level. Moreover, candidate miRNAs were examined in the serum samples of patients with mild cognitive impairment (MCI) and vascular dementia (VD). The results showed that four miRNAs (miR-31, miR-93, miR-143, and miR-146a) were markedly decreased in AD patients' serum compared with controls. Receiver operating characteristic curve analysis demonstrated that this panel of four miRNAs could be used as potential biomarker for AD. Furthermore, miR-93, and miR-146a were significantly elevated in MCI compared with controls, and the panel of miR-31, miR-93 and miR-146a can be used to discriminate AD from VD. We established a panel of four serum miRNAs as a novel noninvasive biomarker for AD diagnosis.
doi:10.1155/2015/625659
PMCID: PMC4452867  PMID: 26078483
18.  Resveratrol Prevents Estrogen-DNA Adduct Formation and Neoplastic Transformation in MCF-10F Cells 
Exposure to estrogens is a risk factor for breast cancer. Specific estrogen metabolites may initiate breast cancer and other cancers. Genotoxicity may be caused by cytochrome P450 (CYP)–mediated oxidation of catechol estrogens to quinones that react with DNA to form depurinating estrogen-DNA adducts. CYP1B1 favors quinone formation by catalyzing estrogen 4-hydroxylation, whereas NAD(P)H quinone oxidoreductase 1 (NQO1) catalyzes the protective reduction of quinones to catechols. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1B1 expression through the aryl hydrocarbon receptor (AhR). Resveratrol has anticancer effects in diverse in vitro and in vivo systems and is an AhR antagonist that decreases CYP expression but induces NQO1 expression. The chemopreventive effect of resveratrol on breast cancer initiation was investigated in MCF-10F cells. Its effects on estrogen metabolism and formation of estrogen-DNA adducts were analyzed in culture medium by high-performance liquid chromatography, where as its effects on CYP1B1 and NQO1 were determined by immunoblotting and immunostaining. The antitransformation effects of resveratrol were also examined. TCDD induced expression of CYP1B1 and its redistribution in the nucleus and cytoplasm. Concomitant treatment with resveratrol dose-dependently suppressed TCDD-induced expression of CYP1B1, mainly in the cytoplasm. Resveratrol dose- and time-dependently induced expression of NQO1. NQO1 is mainly in the perinuclear membrane of control cells, but resveratrol induced NQO1 and its intracellular redistribution, which involves nuclear translocation of nuclear factor erythroid 2–related factor 2. Resveratrol decreased estrogen metabolism and blocked formation of DNA adducts in cells treated with TCDD and/or estradiol. Resveratrol also suppressed TCDD and/or estradiol-induced cell transformation. Thus, resveratrol can prevent breast cancer initiation by blocking multiple sites in the estrogen genotoxicity pathway.
doi:10.1158/1940-6207.CAPR-08-0037
PMCID: PMC4424907  PMID: 19138946
19.  Gut Microbial Dysbiosis May Predict Diarrhea and Fatigue in Patients Undergoing Pelvic Cancer Radiotherapy: A Pilot Study 
PLoS ONE  2015;10(5):e0126312.
Fatigue and diarrhea are the most frequent adverse effects of pelvic radiotherapy, while their etiologies are largely unknown. The aim of this study is to investigate the correlations between fatigue, diarrhea, and alterations in gut microbiota induced by pelvic radiotherapy. During the 5-week treatment of pelvic radiotherapy in 11 cancer patients, the general fatigue score significantly increased and was more prominent in the patients with diarrhea. The fatigue score was closely correlated with the decrease of serum citrulline (an indicator of the functional enterocyte mass) and the increases of systemic inflammatory proteins, including haptoglobin, orosomuoid, α1-antitrypsin and TNF-α. Serum level of lipopolysaccharide (LPS) was also elevated, especially in the patients with diarrhea indicating epithelial barrier breach and endotoxemia. Pyrosequencing analysis of 16S rRNA gene revealed that microbial diversity, richness, and the Firmicutes/Bacteroidetes ratio were significantly altered prior to radiotherapy in patients who later developed diarrhea. Pelvic radiotherapy induced further changes in fecal microbial ecology, some of which were specific to the patients with or without diarrhea. Our results indicate that gut microbial dysbiosis prior to radiation therapy may be exploited to predict development of diarrhea and to guide preventive treatment options. Radiation-induced dysbiosis may contribute to pelvic radiation disease, including mucositis, diarrhea, systemic inflammatory response, and pelvic radiotherapy-associated fatigue in cancer patients.
doi:10.1371/journal.pone.0126312
PMCID: PMC4425680  PMID: 25955845
20.  The lycopene β-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm 
BMC Plant Biology  2015;15:112.
Background
Lycopene β-cyclase (LCYB) is a key enzyme catalyzing the biosynthesis of β-carotene, the main source of provitamin A. However, there is no documented research about this key cyclase gene’s function and relationship with β-carotene content in wheat. Therefore, the objectives of this study were to clone TaLCYB and characterize its function and relationship with β-carotene biosynthesis in wheat grains. We also aimed to obtain more information about the endogenous carotenoid biosynthetic pathway and thus provide experimental support for carotenoid metabolic engineering in wheat.
Results
In the present study, a lycopene β-cyclase gene, designated TaLCYB, was cloned from the hexaploid wheat cultivar Chinese Spring. The cyclization activity of the encoded protein was demonstrated by heterologous complementation analysis. The TaLCYB gene was expressed differentially in different tissues of wheat. Although TaLCYB had a higher expression level in the later stages of grain development, the β-carotene content still showed a decreasing tendency. The expression of TaLCYB in leaves was dramatically induced by strong light and the β-carotene content variation corresponded with changes of TaLCYB expression. A post-transcriptional gene silencing strategy was used to down-regulate the expression of TaLCYB in transgenic wheat, resulting in a decrease in the content of β-carotene and lutein, accompanied by the accumulation of lycopene to partly compensate for the total carotenoid content. In addition, changes in TaLCYB expression also affected the expression of several endogenous carotenogenic genes to varying degrees.
Conclusion
Our results suggest that TaLCYB is a genuine lycopene cyclase gene and plays a crucial role in β-carotene biosynthesis in wheat. Our attempt to silence it not only contributes to elucidating the mechanism of carotenoid accumulation in wheat but may also help in breeding wheat varieties with high provitamin A content through RNA interference (RNAi) to block specific carotenogenic genes in the wheat endosperm.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0514-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-015-0514-5
PMCID: PMC4433027  PMID: 25943989
Lycopene; Lycopene β-cyclase; β-carotene; Provitamin A; RNA interference; Wheat
21.  Endothelial dysfunction and enhanced contractility in microvessels from ovariectomised rats: roles of oxidative stress and perivascular adipose tissue 
Hypertension  2014;63(5):1063-1069.
Ovarian hormone loss increases reactive oxidative species (ROS), endothelial dysfunction and cardiovascular disease. Since perivascular adipose tissue (PVAT) regulates endothelial function, we hypothesized that ROS in PVAT mediate adverse microvascular effects of ovarian hormone deficiency. Rats were ovariectomized (OVX) or sham-operated (SHAM) and given vehicle or tempol for 6 weeks. Mesenteric resistance arterioles from OVX compared to SHAM rats had dysfunctional responses to acetylcholine (ACh) including: decreased ACh induced endothelium-dependent relaxation; (50±6 vs 72±2%) and endothelium-dependent relaxation factor (EDRF; 17±4 vs 37±2%) and increased endothelium-dependent contracting factor (EDCF; 27±5 vs 9±3%). OVX rat mesenteric arterioles had increased contractions to the thromboxane/prostanoid receptor (TP-R) agonist U-46,619 (58±3 vs 40±5%) and increased of ROS (tempo-9-AC fluorescence) with U-46,619 (0.65±0.17 vs 0.14±0.06 units) or ACh (0.49±0.09 vs 0.09±0.05 units) and increased p22phox protein expression (0.89±0.05 vs 0.18±0.04 units) while nitric oxide (NO) activity (DAF-FM fluorescence) with ACh was reduced (0.39±0.1 vs 0.70±0.10 units). No differences were found in endothelium-dependent hyperpolarizing factor (EDHF) or contractile responses to phenylephrine (PE). PVAT enhanced ACh induced relaxation, EDRF and NO only in SHAM rats. Tempol prevented OVX-induced endothelial dysfunction and restored the enhancing effects of PVAT on ACh induced relaxation, EDRF and NO in OVX rat vessels but both tempol and PVAT were required to normalize the enhanced U-46,619 contractions after OVX. In conclusion, ovariectomy redirects endothelial responses from relaxation to contraction by reducing vascular NO, augmenting TP-R signaling and attenuating the vasodilatory effects of PVAT, all of which were dependent on ROS.
doi:10.1161/HYPERTENSIONAHA.113.02284
PMCID: PMC3984350  PMID: 24591333
reactive oxygen species; nitric oxide; endothelial dysfunction; ovariectomy; thromboxane prostanoid receptor
22.  A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy 
mAbs  2014;6(3):764-772.
The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal.
doi:10.4161/mabs.28614
PMCID: PMC4011920  PMID: 24671001
interleukin 1; antibody; immunotherapy; drug discovery; animal models
23.  Gamma secretase inhibitor enhances sensitivity to doxorubicin in MDA-MB-231 cells 
Deregulated expression of molecular of the Notch signaling pathway is observed in malignant tumor. Notch signaling pathway is activated by a series of catalytic cleavage of the Notch receptor by gamma secretase. Gamma secretase inhibitor (GSI) have demonstrated clinical activity in patients with solid tumor. Triple negative breast cancer (TNBC) is related to poor prognosis and a high probability of lung and brain metastases. As first line therapy for TNBC, doxorubicin is partially effective in TNBC control. An understanding of the mechanisms for enhancing sensitivity to doxorubicin would be significant for future drug development. We hypothesized that a combination of cytotoxic chemotherapy doxorubicin to inhibit cell proliferation, together with GSI, would result in more effective outcome than either monotherapy alone. We treated MDA-MB-231 cell lines with doxorubicin and evaluated the monotherapy efficacy and in combination with GSI in both vitro and vivo. GSI-induced proliferation inhibition and apoptosis was achieved with an induction of PTEN and pro-apoptotic protein Bax expression and suppression of Notch-1, HES-1, CyclinD1 and anti-apoptotic protein Bcl-2. These results indicate that MDA-MB-231 cells are susceptible to a GSI, whether alone or in combination with doxorubicin, are correlated with changing of some surrogate marker. This study demonstrates practicability of combined use of GSI and doxorubicin, and together these results encourage new therapeutic method in triple negative breast cancer.
PMCID: PMC4503001  PMID: 26191129
Notch-1; doxorubicin; triple negative breast cancer
24.  Diagnostic Value of Circulating Chromogranin A for Neuroendocrine Tumors: A Systematic Review and Meta-Analysis 
PLoS ONE  2015;10(4):e0124884.
Background
In previous decades, chromogranin A (CgA) has been demonstrated to be the most promising biomarker for the diagnosis of neuroendocrine tumors (NETs), but its diagnostic value is still controversial. This meta-analysis aimed to estimate the potential diagnostic value of circulating CgA for NETs.
Methods
We collected relevant studies from several electronic databases as well as from reference lists. Diagnostic indices of CgA were pooled with random effects models. Pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and summary receiver operating characteristic (SROC) curves for the diagnosis of NETs were used to estimate the overall diagnostic efficiency.
Results
Through a search strategy, 13 studies met the inclusion criteria and were included. These studies contained 1260 patients with NETs and 967 healthy controls in the total sample. As a result, the overall sensitivity, specificity and diagnostic odds ratio (DOR) were 0.73 (95% CI: 0.71 to 0.76), 0.95 (95% CI: 0.93 to 0.96) and 56.29 (95% CI: 25.27 to 125.38), respectively, while the summary positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were 14.56 (95% CI: 6.62 to 32.02) and 0.26 (95% CI: 0.18 to 0.38), respectively. In addition, the area under the curve (AUC) of the circulating CgA in the diagnosis of NETs was 0.8962.
Conclusions
These data demonstrate that circulating CgA is an efficient biomarker for the diagnosis of NETs with high sensitivity and specificity, which indicates that it may be helpful for the clinical management of NETs. However, further studies are needed to clarify this issue.
doi:10.1371/journal.pone.0124884
PMCID: PMC4403810  PMID: 25894842
25.  The influence of SnoN gene silencing by siRNA on the cell proliferation and apoptosis of human pancreatic cancer cells 
Diagnostic Pathology  2015;10:30.
Background
The prognosis for pancreatic cancer (PC) is very poor. The SnoN gene may have a role in cell proliferation and apoptosis in human cancer. However, the influence of SnoN on cell proliferation and apoptosis in human PC cells remains unknown.
Methods
SnoN expression was assessed in SW1990 PC cell lines using real-time polymerase chain reaction (PCR). A luciferase reporter assay was used to confirm the target associations. The effect of SnoN on cell proliferation in vitro was confirmed using Cell Counting Kit-8. Apoptosis was confirmed using flow cytometry. Gene and protein expression were examined using real time PCR and Western blotting, respectively.
Results
SnoN siRNA significantly inhibited the growth of SW1990 cells by decreasing cell proliferation (P < 0.05) and increasing cell apoptosis (P < 0.05), compared with the blank group and the negative control group. The highest inhibition of cell proliferation appeared at 3 days post-transfection. Cell apoptosis more obvious at 48 h after transfection.
Conclusions
In summary, our results reveal that the RNAi-mediated downregulation of SnoN effectively inhibited the proliferation of PC cells. SnoN-siRNA also enhanced SW1990 PC cell apoptosis. These findings indicate that SnoN gene plays an important role in pancreatic cancer development, and might serve as a potential therapeutic target for pancreatic cancer. However, further in vivo studies are needed to clarify the influence of SnoN gene silencing by siRNA on pancreatic cancer therapy.
Virtual slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7609324661510147
doi:10.1186/s13000-015-0267-3
PMCID: PMC4407884  PMID: 25907906
SnoN gene; Proliferation; Apoptosis; Pancreatic cancer (PC)

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