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1.  Development of a rabbit corneal equivalent using an acellular corneal matrix of a porcine substrate 
Molecular Vision  2008;14:2180-2189.
Purpose
The tissue equivalent that mimics the structure and function of normal tissue is a major bioengineering challenge. Tissue engineered replacement of diseased or damaged tissue has become a reality for some types of tissue such as skin and cartilage. The tissue engineered corneal epithelium, stroma, and endothelium scaffold are promising concepts in overcoming the current limitations of a cornea replacement with an allograft.
Methods
The acellular corneal matrix from porcine (ACMP) was examined as a potential corneal cell sheet frame. The physical and mechanical properties of strength, expansion, transparency, and water content of the ACMP were measured. The major antigens of the cell components were completely removed with series of extraction methods, the major antigens of the cell components were identified by hematoxylin and eosin (HE), immunofluorescence staining, and scanning electron microscopy. The structural properties were investigated by HE stain and scanning electron microscopy. The three types of rabbit corneal cells were cultured in vitro, and characteristics were investigated by colony formation efficiency (CFE), BrdU staining, immunofluorescence staining, and western blot assay of keratin 3 (K3), vimentin, and aquaporin A. The biocompatibility of the ACMP was investigated for one month using rabbit corneal stroma and three types of cultured corneal cells both in vivo and in vitro. The three types of cultured rabbit corneal cells were seeded onto ACMP of each side at a cell density of 5.0×103 cells/mm2.
Results
The optical and mechanical properties of the ACMP were similar to the normal porcine cornea. The collagen fiber interconnected to the network, formed regular collagen bundles of the ACMP, and was parallel to the corneal surface. The ACMP was transferred to the rabbit cornea stroma, which showed an intact epithelium and keratocytes in the implant region. There were no inflamed cells or new vessel invasion one month after transplantation. The three types of cultured rabbit corneal cells were positive for K3, vimentin, and aquaporin A. CFE and BrdU (5-bromo-2′-deoxyuridine) staining showed no statistical difference. The cultured rabbit corneal limbal epithelial cells, keratocyte cells, and endothelial cells formed a confluent cell sheet on the ACMP, which consisted of one to two cell layers. Immunofluorescence and scanning electron microscopy examination showed that the cells steadily adhered to the surface of the ACMP and maintained their conformation and special molecule expression such as K3, vimentin, and aquaporin A. Rabbit corneal epithelium-ACMP, keratocytes-ACMP, and endothelium-ACMP scaffold was built in vitro.
Conclusions
The rabbit corneal scaffold was made by the ACMP as a frame with three types of allogeneic rabbit corneal cells. This is a new concept in treating injured corneas.
PMCID: PMC2592998  PMID: 19052652
2.  Pirfenidone-loaded liposomes for lung targeting: preparation and in vitro/in vivo evaluation 
Background
The purpose of this study was to develop novel pirfenidone (PFD)-loaded liposomes for targeting to the lung.
Methods
The liposomes were prepared by the film hydration method, and their in vitro/vivo characteristics were evaluated.
Results
The PFD liposomes appeared visually as green to yellowish suspensions and were spherical in shape. The particle size was 582.3±21.6 nm and the entrapment efficiency was relatively high (87.2%±5.7%). The liposomes showed typical sustained and prolonged drug-release behavior in vitro and fitted well with the Weibull distribution equation. The relatively slower time taken to reach a minimal plasma PFD concentration in vivo suggests that PFD liposomes have a sustained-release profile, which is consistent with the results of the in vitro release study. The PFD liposomes showed the largest area under the curve for the lung. The high distribution of PFD achieved in the lungs using this liposomal formulation may be explained by physical entrapment of the liposomes in the vascular network of the lung. Histopathological results indicated that liposomal PFD could alleviate pathological injury in lung tissue.
Conclusion
This liposomal formulation can enable sustained release of PFD and increase targeting to the lung.
doi:10.2147/DDDT.S84046
PMCID: PMC4500626  PMID: 26185416
pirfenidone; liposomes; lung targeting; in vivo; histopathological
3.  Exosomes serve as tumour markers for personalized diagnostics owing to their important role in cancer metastasis 
Journal of Extracellular Vesicles  2015;4:10.3402/jev.v4.27522.
Exosomes, membrane vesicles of 40–100 nm in diameter, are derived from endosomes in various cells. The bioactive molecules specifically packed into exosomes can be horizontally transferred into recipient cells changing their biological properties, by which tumour cells continuously modify their surrounding microenvironment and distant target cells favouring cancer metastasis. It has been suspected for a long time that exosomes participate in the whole process of tumour metastasis. Although there is much unknown and many controversies in the role of cancer exosome, the major contribution of tumour-associated exosomes to different steps of cancer metastasis are demonstrated in this review. Mainly because these exosomes are easily accessible and capable of representing their parental cells, exosomes draw much attention as a promising biomarker for tumour screening, diagnosis and prognosis. Currently, researchers have found numerous biomarkers in exosomes with great potential to be utilized in personalized medicine. In this article, we summarize the roles of biomarkers, which are validated by clinical samples. Even though many conundrums remain, such as exosome extraction, large multicentre validation of biomarkers and data interpretation, exosomes are certain to be used in clinical practice in the near future as the field rapidly expands.
doi:10.3402/jev.v4.27522
PMCID: PMC4475684  PMID: 26095380
extracellular vesicle; metastasis; malignant neoplasms; microenvironment; personalized diagnostics; cancer biomarkers
4.  Draft Genome Sequence of Gluconobacter oxydans NL71, a Strain That Efficiently Biocatalyzes Xylose to Xylonic Acid at a High Concentration 
Genome Announcements  2015;3(3):e00615-15.
Gluconobacter oxydans NL71, a selected strain in the crude lignocellulosic hydrolysate, catalyzed 600 g/liter xylose to 586.3 g/liter xylonic acid at 95.1% yield. The biocatalysis of xylose yielded three times higher than the best previous output, providing a possibility of the industrial scale utilization of lignocellulosic xylose. Due to its promising industrial applications, we sequenced the complete genome of strain G. oxydans NL71 to further our understanding of its overall metabolism.
doi:10.1128/genomeA.00615-15
PMCID: PMC4472894  PMID: 26089417
5.  Comparative Transcriptome Analysis of Cultivated and Wild Watermelon during Fruit Development 
PLoS ONE  2015;10(6):e0130267.
Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an important vegetable crop world-wide. Watermelon fruit quality is a complex trait determined by various factors such as sugar content, flesh color and flesh texture. Fruit quality and developmental process of cultivated and wild watermelon are highly different. To systematically understand the molecular basis of these differences, we compared transcriptome profiles of fruit tissues of cultivated watermelon 97103 and wild watermelon PI296341-FR. We identified 2,452, 826 and 322 differentially expressed genes in cultivated flesh, cultivated mesocarp and wild flesh, respectively, during fruit development. Gene ontology enrichment analysis of these genes indicated that biological processes and metabolic pathways related to fruit quality such as sweetness and flavor were significantly changed only in the flesh of 97103 during fruit development, while those related to abiotic stress response were changed mainly in the flesh of PI296341-FR. Our comparative transcriptome profiling analysis identified critical genes potentially involved in controlling fruit quality traits including α-galactosidase, invertase, UDP-galactose/glucose pyrophosphorylase and sugar transporter genes involved in the determination of fruit sugar content, phytoene synthase, β-carotene hydroxylase, 9-cis-epoxycarotenoid dioxygenase and carotenoid cleavage dioxygenase genes involved in carotenoid metabolism, and 4-coumarate:coenzyme A ligase, cellulose synthase, pectinesterase, pectinesterase inhibitor, polygalacturonase inhibitor and α-mannosidase genes involved in the regulation of flesh texture. In addition, we found that genes in the ethylene biosynthesis and signaling pathway including ACC oxidase, ethylene receptor and ethylene responsive factor showed highly ripening-associated expression patterns, indicating a possible role of ethylene in fruit development and ripening of watermelon, a non-climacteric fruit. Our analysis provides novel insights into watermelon fruit quality and ripening biology. Furthermore, the comparative expression profile data we developed provides a valuable resource to accelerate functional studies in watermelon and facilitate watermelon crop improvement.
doi:10.1371/journal.pone.0130267
PMCID: PMC4469606  PMID: 26079257
6.  Knockdown of SUMO-activating enzyme subunit 2 (SAE2) suppresses cancer malignancy and enhances chemotherapy sensitivity in small cell lung cancer 
Background
SUMO-activating enzyme subunit 2 (SAE2) is the sole E1-activating enzyme required for numerous important protein SUMOylation, abnormal of which is associated with carcinogenesis. SAE2 inactivation was recently reported to be a therapeutic strategy in cancers with Myc overexpression. However, the roles of SAE2 in small cell lung cancer (SCLC) are largely unknown.
Methods
Stably SAE2 knockdown in H446 cells were established with a lentiviral system. Cell viability, cell cycle, and apoptosis were analyzed using MTT assay and flow cytometric assay. Expression of SAE2 mRNA and protein were detected by qPCR, western blotting, and immunohistochemical staining. Cell invasion and migration assay were determined by transwell chamber assay. H446 cells with or without SAE2 knockdown, nude mice models were established to observe tumorigenesis.
Results
SAE2 was highly expressed in SCLC and significantly correlated with tumorigenesis in vivo. Cancer cells with RNAi-mediated reduction of SAE2 expression exhibited growth retardation and apoptosis increasing. Furthermore, down-regulation of SAE2 expression inhibited migration and invasion, simultaneously increased the sensitivity of H446 to etoposide and cisplatin.
Conclusions
SAE2 plays an important role in tumor growth, metastasis, and chemotherapy sensitivity of H446 and is a potential clinical biomarker and therapeutic target in SCLC with high c-Myc expression.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-015-0164-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13045-015-0164-y
PMCID: PMC4483218  PMID: 26063074
SUMO-activating enzyme subunit 2; Small cell lung cancer (SCLC); Chemotherapy sensitivity
7.  Manganese Superoxide Dismutase-Mediated Inside-Out Signaling in HaCaT Human Keratinocytes and SKH-1 Mouse Skin 
Antioxidants & Redox Signaling  2014;20(15):2347-2360.
Abstract
Aims: Inside-out signaling occurs when changes in organellar activity lead to alterations in cell signaling that culminate at the cell surface. Mitochondria are vital signaling platforms in cells that participate in radiation-induced inside-out signaling. However, the importance of the reactive oxygen species (ROS)-scavenging ability of mitochondria through manganese superoxide dismutase (MnSOD) is not established. Here, we used MnSOD heterozygous knockout and transgenic SKH-1 hairless, albino mice and MnSOD knockdown and overexpressing HaCaT human keratinocytes to study the effects of MnSOD on ultraviolet (UV) radiation-induced inside-out signaling. Results and Innovation: There is an inverse correlation between MnSOD expression and UV-induced activation of epidermal growth factor receptor (EGFR), as determined by phosphorylation at Tyr1068, both in vitro and in vivo, which correlates with increased ROS production (as measured by dihydroethidium fluorescence). EGFR activation is dependent on Nox4 expression and Src kinase activation, with Src activation upstream of Nox4 in regulation of EGFR activation. Enhanced EGFR activation in MnSOD knockdown cells is abrogated by treatment with the SOD mimetic MnTnBuOE-2-PyP5+. Conclusions: Our data demonstrate that the ROS-scavenging ability of mitochondria, through the expression of MnSOD, is important for UV-induced inside-out signaling. Decreased MnSOD expression enhances UV-induced activation of different oncogenic signaling pathways through an inside-out signaling-mediated mechanism. Inhibition of inside-out signaling by MnTnBuOE-2-PyP5+ mimics the effect of endogenous MnSOD, suggesting that pharmacological intervention by SOD mimetics could play an important role in the prevention of aberrant cell signaling, which may contribute to carcinogenesis and may prove valuable for the treatment or prevention of cancer in the future. Antioxid. Redox Signal. 20, 2347–2360.
doi:10.1089/ars.2013.5204
PMCID: PMC4005487  PMID: 24635018
8.  Promising response of anaplastic lymphoma kinase-positive large B-cell lymphoma to crizotinib salvage treatment: case report and review of literature 
Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (ALK + DLBCL) is a rare and poorly characterized subtype of lymphoma. Reports suggest that this type of tumor responds poorly to standard regimens for non-Hodgkin’s lymphoma, with rituximab playing no therapeutic role due to the absence of CD20 expression. In view of the expression of ALK in this disease, it is plausible that the ALK inhibitor crizotinib may be an effective treatment. We report a case of a 21-year-old male ALK + DLBCL patient. He initially received five cycles of CHOP-21 (vincristine, pirarubicin, cyclophosphamide and prednisone) and achieved a partial remission (PR) but soon deteriorated. He was subsequently treated with five courses of the salvage chemotherapy regimen ICE (ifosfamide, carboplatin and etoposide) and achieved PR again. He refused to accept an autologous stem-cell transplantation, after which the disease progressed rapidly. We administered two courses of an alternative salvage chemotherapy regimen containing GEMOX and dexamethasone with the addition of the ALK inhibitor crizotinib. His symptoms alleviated for a short time but soon worsened and the patient died of massive progressive disease.
PMCID: PMC4509179  PMID: 26221234
Anaplastic lymphoma kinase; non-hodgkin lymphoma; spleen; crizotinib
9.  Rutin ameliorates renal fibrosis and proteinuria in 5/6-nephrectomized rats by anti-oxidation and inhibiting activation of TGFβ1-smad signaling 
Objectives: Rutin, a polyphenolic flavonoid, was reported to have beneficial effect on drug induced nephropathy. The present study aimed to introduce 5/6 nephrectomized rat model to further evaluate its renal protective effect. Methods: Adult Wistar rats were induced to develop chronic renal failure through 5/6 nephrectomy (5/6 Nx). After that, animals were treated orally with saline, rutin at 15 and 45 mg/kg, and losartan (10 mg/kg) daily for 20 weeks; sham-operated animals were also involved as control. After treatment for 8 and 20 weeks, blood and urine samples were collected for biochemical examination; all the kidney remnants were collected for histological examination. The protein levels of TGF-β1, smad2 and phosphorylated-smad2 (p-smad2) in kidney were measured. Immunohistochemistry was used to analyze the expression of TGF-β1, fibronectin and collagen IV in kidney tissues. Results: Results suggested that rutin could reduce the proteinurea, blood urine nitrogen and blood creatinine in 5/6 Nx animals significantly, as well as oxidation stress in the kidney. By histological examination, rutin administration alleviated glomerular sclerosis scores and tubulointerstitial injuries in a dose-dependent manner (P<0.01). Immunohistochemistry also suggested rutin could reduce the expression of TGF-β1, fibronectin and collagen IV in kidney tissues. By western blot, we found the rutin could reduce the TGF-β1, p-smad2 expression in the kidney tissues of rats. Conclusions: This study suggests that the rutin can improve renal function in 5/6 Nx rats effectively. Its effect may be due to its anti-oxidation and inhibiting TGFβ1-Smad signaling.
PMCID: PMC4503034  PMID: 26191162
Rutin; 5/6 nephrectomy; TGF-β1; chronic renal failure
10.  Rectal gastrointestinal stromal tumor as an incidental finding in a patient with rectal polyps 
A patient who was diagnosed as rectal polyps in the local hospital went to our hospital for surgical treatment. Abdominal CT demonstrated a large irregular extra-luminal tumor of at least 5 cm cross-section on the ventral side of the lower rectal wall. Intraoperatively, a large irregular extra-luminal tumor (about 5×4.5×4 cm) was found. Anterior resection with end colostomy and rectal stump (Hartmann’s procedure) was performed. Postoperative histological examination showed simultaneous development of rectal GIST and polyps.
PMCID: PMC4503193  PMID: 26191322
Rectal polyps; GISTs; simultaneous development; KIT mutation
11.  Balanced ratio of plasma to packed red blood cells improves outcomes in massive transfusion: A large multicenter study 
Resuscitation with the early administration of plasma can improve the survival of patients undergoing surgery or trauma patients who require massive transfusion. To ascertain the optimal ratio of fresh frozen plasma (FFP) to packed red blood cells (pRBCs) in massive transfusions, the records of 1,048 patients who received a massive transfusion at 20 hospitals were retrospectively reviewed. The patients were stratified into three groups according to the ratio of FFP to pRBCs. These were the low (<1:2.3), middle (1:2.3–0.75) and high (≥1:0.75) ratio groups. For 24-h treatment, the middle FFP:pRBC ratio led to a lower mortality rate (9.31%) compared with that in the low (11.83%) and high (11.44%) ratio groups (P=0.477). For 72-h treatment, the middle FFP:pRBC ratio also lead to the lowest mortality rate (7.25%), which was significantly lower than the ratios in the low (10.39%) and high (13.65%) ratio groups (P=0.007). The length of hospital stay, ICU stay, and FFP:pRBC ratio in 72 h were found to be significant associated with mortality. The optimal ratio of FFP to pRBCs of 1:2.3–0.75 in 72 h can improve the survival of patients undergoing massive transfusions.
doi:10.3892/etm.2015.2461
PMCID: PMC4486907  PMID: 26170909
fresh frozen plasma; packed red blood cells; surgical transfusion
12.  Tim-4 Inhibits NO Generation by Murine Macrophages 
PLoS ONE  2015;10(4):e0124771.
Objective
T cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation.
Methods
Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot.
Results
Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages.
Conclusion
These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.
doi:10.1371/journal.pone.0124771
PMCID: PMC4408120  PMID: 25905790
13.  Synergistic effects of high dietary calcium and exogenous parathyroid hormone in promoting osteoblastic bone formation in mice 
The British Journal of Nutrition  2015;113(6):909-922.
In the present study, we investigated whether high dietary Ca and exogenous parathyroid hormone 1–34 fragments (PTH 1–34) have synergistic effects on bone formation in adult mice, and explored the related mechanisms. Adult male mice were fed a normal diet, a high-Ca diet, a PTH-treated diet, or a high-Ca diet combined with subcutaneously injected PTH 1–34 (80 μg/kg per d) for 4 weeks. Bone mineral density, trabecular bone volume, osteoblast number, alkaline phosphatase (ALP)- and type I collagen-positive areas, and the expression levels of osteoblastic bone formation-related genes and proteins were increased significantly in mice fed the high-Ca diet, the PTH-treated diet, and, even more dramatically, the high-Ca diet combined with PTH. Osteoclast number and surface and the ratio of receptor activator for nuclear factor-κB ligand (RANKL):osteoprotegerin (OPG) were decreased in the high-Ca diet treatment group, increased in the PTH treatment group, but not in the combined treatment group. Furthermore, third-passage osteoblasts were treated with high Ca (5 mm), PTH 1–34 (10− 8 m) or high Ca combined with PTH 1–34. Osteoblast viability and ALP activity were increased in either the high Ca-treated or PTH-treated cultures and, even more dramatically, in the cultures treated with high Ca plus PTH, with consistent up-regulation of the expression levels of osteoblast proliferation and differentiation-related genes and proteins. These results indicate that dietary Ca and PTH play synergistic roles in promoting osteoblastic bone formation by stimulating osteoblast proliferation and differentiation.
doi:10.1017/S0007114514004309
PMCID: PMC4392706  PMID: 25744000
Parathyroid hormone; High-calcium diet; Osteoblasts; Osteoclasts; Synergistic effects; Bone formation
14.  Identification and characterization of rye genes not expressed in allohexaploid triticale 
BMC Genomics  2015;16(1):281.
Background
One of the most important evolutionary processes in plants is polyploidization. The combination of two or more genomes in one organism often initially leads to changes in gene expression and extensive genomic reorganization, compared to the parental species. Hexaploid triticale (x Triticosecale) is a synthetic hybrid crop species generated by crosses between T. turgidum and Secale cereale. Because triticale is a recent synthetic polyploid it is an important model for studying genome evolution following polyploidization. Molecular studies have demonstrated that genomic sequence changes, consisting of sequence elimination or loss of expression of genes from the rye genome, are common in triticale. High-throughput DNA sequencing allows a large number of genes to be surveyed, and transcripts from the different homeologous copies of the genes that have high sequence similarity can be better distinguished than hybridization methods previously employed.
Results
The expression levels of 23,503 rye cDNA reference contigs were analyzed in 454-cDNA libraries obtained from anther, root and stem from both triticale and rye, as well as in five 454-cDNA data sets created from triticale seedling shoot, ovary, stigma, pollen and seed tissues to identify the classes of rye genes silenced or absent in the recent synthetic hexaploid triticale. Comparisons between diploid rye and hexaploid triticale detected 112 rye cDNA contigs (~0.5%) that were totally undetected by expression analysis in all triticale tissues, although their expression was relatively high in rye tissues. Non-expressed rye genes were found to be strikingly less similar to their closest BLASTN matches in the wheat genome or in the other Triticum genomes than a test set of 200 random rye genes. Genes that were not detected in the RNA-seq data were further characterized by testing for their presence in the triticale genome by PCR using genomic DNA as a template.
Conclusion
Genes with low similarity between rye sequences and their closest matches in the Triticum genome have a higher probability to be repressed or absent in the allopolyploid genome.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1480-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1480-x
PMCID: PMC4396786  PMID: 25886913
Allopolyploidization; Gene repression; Gene deletion; Gene silencing; Triticale; High-throughput DNA sequencing; Tissue-specific expression
15.  Mapsnp: An R Package to Plot a Genomic Map for Single Nucleotide Polymorphisms 
PLoS ONE  2015;10(4):e0123609.
Single-nucleotide polymorphism (SNP) is one of the most common sources of genetic variations of the genome. Currently, SNPs are a main target for most genetic association studies. Visualizing genomic coordinates of SNPs, including their physical location relative to their host gene, and the structure of the relevant transcripts, may provide intuitive supplements to the understanding of their functions. Nevertheless, to date, no such easy-to-use programming tools exist. Therefore, we developed an R package, “mapsnp”, to plot genomic map for a panel of SNPs within a genome region of interest, including the relative chromosome location and the transcripts in the region. mapsnp is a simple and flexible software package which can be used to visualize a genomic map for SNPs, integrating a chromosome ideogram, genomic coordinates, SNP locations and SNP labels.
doi:10.1371/journal.pone.0123609
PMCID: PMC4390227  PMID: 25853637
16.  Meta-Analysis of the Association between Vitamin D and Autoimmune Thyroid Disease 
Nutrients  2015;7(4):2485-2498.
Although emerging evidence suggests that low levels of vitamin D may contribute to the development of autoimmune disease, the relationship between vitamin D reduction and autoimmune thyroid disease (AITD), which includes Graves’ disease (GD) and Hashimoto thyroiditis (HT), is still controversial. The aim was to evaluate the association between vitamin D levels and AITD through systematic literature review. We identified all studies that assessed the association between vitamin D and AITD from PubMed, Embase, CENTRAL, and China National Knowledge Infrastructure (CNKI) databases. We included studies that compared vitamin D levels between AITD cases and controls as well as those that measured the odds of vitamin D deficiency by AITD status. We combined the standardized mean differences (SMD) or the odds ratios (OR) in a random effects model. Twenty case-control studies provided data for a quantitative meta-analysis. Compared to controls, AITD patients had lower levels of 25(OH)D (SMD: −0.99, 95% CI: −1.31, −0.66) and were more likely to be deficient in 25(OH)D (OR 2.99, 95% CI: 1.88, 4.74). Furthermore, subgroup analyses result showed that GD and HT patients also had lower 25(OH)D levels and were more likely to have a 25(OH)D deficiency, suggesting that low levels of serum 25(OH)D was related to AITD.
doi:10.3390/nu7042485
PMCID: PMC4425156  PMID: 25854833
vitamin D; autoimmune thyroid disease; Graves’ disease; Hashimoto thyroiditis; meta-analysis
17.  EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells 
Oncotarget  2015;6(9):7244-7261.
Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.
PMCID: PMC4466682  PMID: 25779663
EGF; Arf6; ERK; Wnt5a; EMT
18.  Clinical significance of ALDH2 rs671 polymorphism in esophageal cancer: evidence from 31 case-control studies 
OncoTargets and therapy  2015;8:649-659.
Background
Aldehyde dehydrogenase 2 (ALDH2), a critical enzyme for the detoxification of alcohol, is associated with many types of cancers. To verify the relationship of ALDH2 rs671 G>A polymorphism and esophageal cancer (EC), we performed a meta-analysis of a total of 31 published data including 8,510 patients and 16,197 controls.
Methods
The pooled odds ratio (OR) and the 95% confidence interval (CI) were calculated using a fixed or random-effects model. Heterogeneity (PH), publication bias, and sensitivity analysis were also determined.
Results
Although a protective effort was found in the rs671 homozygote comparison (AA/GG: OR=0.69; 95% CI=0.48–0.98), the heterozygote comparison was apparently associated with the risk of EC, particularly in the Chinese population (AG/GG: OR=1.39; 95% CI=1.03–1.87). Alcohol consumption remarkably increased this risk, especially in the AG genotype. Drinking men with the AG genotype appeared to show a higher risk (AG/GG: OR=4.39; 95% CI=1.24–6.55) than drinking women.
Conclusion
The present meta-analysis provided advanced information regarding the association of the ALDH2 A>G polymorphism and EC. Taken together, insights from this study suggested an enhanced effect on the development of EC through a genetic–environmental interaction.
doi:10.2147/OTT.S76526
PMCID: PMC4376259  PMID: 25848305
aldehyde dehydrogenase-2; single nucleotide polymorphism; SNP; esophageal cancer; EC; meta-analysis
19.  miR-188-5p inhibits tumour growth and metastasis in prostate cancer by repressing LAPTM4B expression 
Oncotarget  2015;6(8):6092-6104.
Elucidation of the molecular targets and pathways regulated by the tumour-suppressive miRNAs can shed light on the oncogenic and metastatic processes in prostate cancer (PCa). Using miRNA profiling analysis, we find that miR-188-5p was significantly down-regulated in metastatic PCa. Down-regulation of miR-188-5p is an independent prognostic factor for poor overall and biochemical recurrence-free survival. Restoration of miR-188-5p in PCa cells (PC-3 and LNCaP) significantly suppresses proliferation, migration and invasion in vitro and inhibits tumour growth and metastasis in vivo. We also find overexpression of miR-188-5p in PC-3 cells can significantly enhance the cells' chemosensitivity to adriamycin. LAPTM4B is subsequently identified as a direct target of miR-188-5p in PCa, and is found to be significantly over-expressed in PCa. Knockdown of LAPTM4B phenotypically copies miR-188-5p-induced phenotypes, whereas ectopic expression of LAPTM4B reverses the effects of miR-188-5p. We also find that restoration of miR-188-5p can inhibit the PI3K/AKT signaling pathway via the suppression of LAPTM4B. Taken together, this is the first report unveils that miR-188-5p acts as a tumour suppressor in PCa and may therefore serve as a useful therapeutic target for the development of new anticancer therapy.
PMCID: PMC4467424  PMID: 25714029
miRNA; metastasis; miR-188-5p; prostate cancer; LAPTM4B
20.  Hypoxia Precondition Promotes Adipose-Derived Mesenchymal Stem Cells Based Repair of Diabetic Erectile Dysfunction via Augmenting Angiogenesis and Neuroprotection 
PLoS ONE  2015;10(3):e0118951.
The aim of the present study was to examine whether hypoxia preconditioning could improve therapeutic effects of adipose derived mesenchymal stem cells (AMSCs) for diabetes induced erectile dysfunction (DED). AMSCs were pretreated with normoxia (20% O2, N-AMSCs) or sub-lethal hypoxia (1% O2, H-AMSCs). The hypoxia exposure up-regulated the expression of several angiogenesis and neuroprotection related cytokines in AMSCs, including vascular endothelial growth factor (VEGF) and its receptor FIK-1, angiotensin (Ang-1), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). DED rats were induced via intraperitoneal injection of streptozotocin (60 mg/kg) and were randomly divided into three groups—Saline group: intracavernous injection with phosphate buffer saline; N-AMSCs group: N-AMSCs injection; H-AMSCs group: H-AMSCs injection. Ten rats without any treatment were used as normal control. Four weeks after injection, the mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured. The contents of endothelial, smooth muscle, dorsal nerve in cavernoursal tissue were assessed. Compared with N-AMSCs and saline, intracavernosum injection of H-AMSCs significantly raised ICP and ICP/MAP (p<0.05). Immunofluorescent staining analysis demonstrated that improved erectile function by MSCs was significantly associated with increased expression of endothelial markers (CD31 and vWF) (p<0.01) and smooth muscle markers (α-SMA) (p<0.01). Meanwhile, the expression of nNOS was also significantly higher in rats receiving H-AMSCs injection than those receiving N-AMSCs or saline injection. The results suggested that hypoxic preconditioning of MSCs was an effective approach to enhance their therapeutic effect for DED, which may be due to their augmented angiogenesis and neuroprotection.
doi:10.1371/journal.pone.0118951
PMCID: PMC4366267  PMID: 25790284
21.  Correlative association of interleukin-6 with intima media thickness: a meta-analysis 
Background: Involvement of inflammatory processes in the atherogenesis is now well-recognized. The present study examines the relationship between a proinflammatory cytokine, interleukin-6 (IL-6), and atherosclerosis by meta-analyzing the correlation coefficients between IL-6 and intima media thickness reported in the relevant studies. Method: Relevant research articles were searched in several electronic databases by using most relevant MeSH terms and keywords. For the meta-analyses, correlation coefficients of individual studies were first converted into Fisher’s z scores and then overall effect size was transformed into correlation coefficient. Between-study hete rogeneity was tested by I2 index and subgroup analyses were performed. Quality of the included studies was assessed and tests for publication bias were carried out. Results: Thirty seven studies were selected for the meta-analysis from which data of 14832 participants including healthy persons, persons at risk of CVD and patients of various diseases is used. The effect size (correlation coefficient) with 95% confidence interval (CI) between IL-6 and intima media thickness was 0.336 (0.327 to 0.345); P < 0.0001 in the overall meta-analysis, 0.446 (0.422 to 0.47); P < 0.0001 in patients suffering from any pathological condition; 0.478 (0.446 to 0.508); P < 0.0001 in patients suffering either from a CVD or a disease which poses risk of CVD; 0.327 (0.264 to 0.388); P < 0.0001 in patients suffering from a disease with no CVD risk; and 0.31 (0.291 to 0.327); P < 0.0001 in participants of community-based surveys. There was no significant publication bias.Conclusion: Intima media thickness is significantly correlated with IL-6 levels in the patients with CVD or a disease posing risk of CVD as well as in apparently healthy populations.
PMCID: PMC4443248  PMID: 26064414
Atherosclerosis; intima media thickness; inflammation; interleukin-6; cardiovascular
22.  Clinical, Virological and Immunological Features from Patients Infected with Re-Emergent Avian-Origin Human H7N9 Influenza Disease of Varying Severity in Guangdong Province 
PLoS ONE  2015;10(2):e0117846.
Background
The second wave of avian influenza H7N9 virus outbreak in humans spread to the Guangdong province of China by August of 2013 and this virus is now endemic in poultry in this region.
Methods
Five patients with H7N9 virus infection admitted to our hospital during August 2013 to February 2014 were intensively investigated. Viral load in the respiratory tract was determined by quantitative polymerase chain reaction (Q-PCR) and cytokine levels were measured by bead-based flow cytometery.
Results
Four patients survived and one died. Viral load in different clinical specimens was correlated with cytokine levels in plasma and broncho-alveolar fluid (BALF), therapeutic modalities used and clinical outcome. Intravenous zanamivir appeared to be better than peramivir as salvage therapy in patients who failed to respond to oseltamivir. Higher and more prolonged viral load was found in the sputum or endotracheal aspirates compared to throat swabs. Upregulation of proinflammatory cytokines IP-10, MCP-1, MIG, MIP-1α/β, IL-1β and IL-8 was found in the plasma and BALF samples. The levels of cytokines in the plasma and viral load were correlated with disease severity. Reactivation of herpes simplex virus type 1(HSV-1) was found in three out of five patients (60%).
Conclusion
Expectorated sputum or endotracheal aspirate specimens are preferable to throat swabs for detecting and monitoring H7N9 virus. Severity of the disease was correlated to the viral load in the respiratory tract as well as the extents of cytokinemia. Reactivation of HSV-1 may contribute to clinical outcome.
doi:10.1371/journal.pone.0117846
PMCID: PMC4344233  PMID: 25723593
23.  Wnt2 promotes non-small cell lung cancer progression by activating WNT/β-catenin pathway 
American Journal of Cancer Research  2015;5(3):1032-1046.
Background: Wnt2 is overexpressed and able to promote tumorigenesis in many types of cancer. However, its expression and role in lung cancer has not been well clarified yet. In this study, we aims to investigate the expression pattern, clinical significance and the underlying molecular mechanism of Wnt2 in non-small lung cancer (NSCLC). Methods: Immunohistochemical staining and ELISA assays were applied to detect Wnt2 level in tumor tissue and serum. EDU incorporation assays and colony formation assays were used to evaluate the growth-promoting effect of Wnt2 in vitro. Then we performed western blot and immunofluorescence assays to detect the activation of WNT signaling pathway. Finally mice engrafted with NSCLC tumor cells were used to assess the role of Wnt2 in vivo. Results: Immunohistochemical staining consisting of 264 NSCLC tumor tissues showed that a high level of Wnt2 was associated with a poor overall survival (OS) and relapse-free survival (RFS) of NSCLC patients (P = 0.002 and 0.0005, respectively). Multivariate analysis presented that Wnt2 level in tumor tissue was an independent prognostic factor (P = 0.049 for OS and P = 0.002 for RFS, respectively). Furthermore, ELISA assays for 181 individuals (116 NSCLC and 65 controls) revealed that serum Wnt2 levels in adenocarcinoma was significantly higher than that in healthy volunteers (P < 0.0001). In vitro H460 cell line stably overexpressing Wnt2 showed enhanced growth activity than the control cells whereas knockdown of Wnt2 by siRNA in H1299 cells resulted in decreased growth activity. Additionally, Wnt2 level in tumor tissues was significantly associated with Ki-67 level (rs: 0.316; P < 0.0001). Immunofluorescence and Western blot assays detected the translocation of β-catenin from cytoplasm into nucleus, which indicated that Wnt2 probably promotes proliferation by activating WNT/β-catenin pathway. In vivo H460 cells expressing exogenous Wnt2 showed increased growth-promoting effect in Balb/c nude mice than control cells. Conclusions: The present study for the first time suggested that Wnt2 was both a prognostic and a diagnostic biomarker for NSCLC. Tumor-derived Wnt2 can promote growth activity of NSCLC cells through activating WNT/β-catenin signaling pathway.
PMCID: PMC4449433  PMID: 26045984
Wnt2; WNT signaling pathway; non-small cell lung cancer; proliferation; tumor biomarker; β-catenin
24.  Concurrent paclitaxel-based chemo-radiotherapy for post-surgical microscopic residual tumor at the bronchial margin (R1 resection) in non-small-cell lung cancer 
BMC Cancer  2015;15:36.
Background
The microscopic residual tumor at the bronchial margin after radical surgery (R1 resection) affects prognosis negatively in non-small-cell lung cancer (NSCLC) patients. For patients with good performance status, a potential cure still exists. Here, we report the outcomes of concurrent paclitaxel-based chemo-radiotherapy (CRT) for NSCLC patients with microscopically positive bronchial margins or peribronchial infiltration.
Methods
A retrospective search in the clinical database was conducted in three hospitals. Patients were identified and evaluated if treated with radiotherapy combined with paclitaxel-based chemotherapy. The objects analyzed were local control time, progression-free survival (PFS), overall survival (OS), and treatment-related toxicity.
Results
Sixty-one patients with microscopic residual tumor at the bronchial stump following pulmonary lobectomy were identified. Forty-six patients who had received concurrent paclitaxel-based CRT were analyzed. The median follow-up was 40 months (range: 15.0–77.5 months). The 1-, 2- and 3-year survival rates were 97.8%, 60.9% and 36.9%, respectively. The local recurrences were recorded in 19.6% (9/46) patients. Median PFS and OS for the evaluated cohort were 23.0 [95% confidence interval (CI): 21.3–24.7] and 32.0 (95% CI: 23.7–40.3) months, respectively. The most common side effects were hematological toxicity (neutropenia, 93.5%; anemia, 89.1%; and thrombocytopenia, 89.1%) and no treatment-related deaths. Grade ≥2 acute radiation-induced pneumonitis and esophagitis were recorded in 43.5% (20/46) and 26.1% (12/46) patients, respectively. By univariate analysis, non-squamous cell lung cancer was associated with a significantly longer survival time (45.1 vs 26.4 months, p = 0.013).
Conclusions
For NSCLC patients with post-surgical microscopic residual tumor at the bronchial stump, concurrent paclitaxel-based chemo-radiotherapy achieved promising outcomes with accepted treatment-related toxicity.
doi:10.1186/s12885-015-1036-0
PMCID: PMC4324030  PMID: 25655307
25.  Prevention against diffuse spinal cord astrocytoma: can the Notch pathway be a novel treatment target? 
Neural Regeneration Research  2015;10(2):244-251.
This study was designed to investigate whether the Notch pathway is involved in the development of diffuse spinal cord astrocytomas. BALB/c nude mice received injections of CD133+ and CD133− cell suspensions prepared using human recurrent diffuse spinal cord astrocytoma tissue through administration into the right parietal lobe. After 7–11 weeks, magnetic resonance imaging was performed weekly. Xenografts were observed on the surfaces of the brains of mice receiving the CD133+ cell suspension, and Notch-immunopositive expression was observed in the xenografts. By contrast, no xenografts appeared in the identical position on the surfaces of the brains of mice receiving the CD133− cell suspension, and Notch-immunopositive expression was hardly detected either. Hematoxylin-eosin staining and immunohistochemical staining revealed xenografts on the convex surfaces of the brains of mice that underwent CD133+ astrocytoma transplantation. Some sporadic astroglioma cells showed pseudopodium-like structures, which extended into the cerebral white matter. However, it should be emphasized that the subcortex xenograft with Notch-immunopositive expression was found in the fourth mouse received injection of CD133− astrocytoma cells. However, these findings suggest that the Notch pathway plays an important role in the formation of astrocytomas, and can be considered a novel treatment target for diffuse spinal cord astrocytoma.
doi:10.4103/1673-5374.152378
PMCID: PMC4392672  PMID: 25883623
nerve regeneration; astrocytoma; mice; immunodeficiency (BALB/c) mice; Notch; nestin; glial fibrillary acidic protein; CD133; spinal cord; brain; MRI; neural regeneration

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