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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.  KEAP1 Is a Redox Sensitive Target That Arbitrates the Opposing Radiosensitive Effects of Parthenolide in Normal and Cancer Cells 
Cancer research  2013;73(14):4406-4417.
Elevated oxidative stress is observed more frequently in cancer cells than in normal cells. It is therefore expected that additional exposure to a low level of reactive oxygen species (ROS) will push cancer cells toward death, whereas normal cells might maintain redox homeostasis through adaptive antioxidant responses. We previously demonstrated that parthenolide enhances ROS production in prostate cancer cells through activation of NADPH oxidase. The present study identifies KEAP1 as the downstream redox target that contributes to parthenolide’s radiosensitization effect in prostate cancer cells. In vivo, parthenolide increases radiosensitivity of mouse xenograft tumors but protects normal prostate and bladder tissues against radiation-induced injury. Mechanistically, parthenolide increases the level of cellular ROS and causes oxidation of thioredoxin (TrX) in prostate cancer cells, leading to a TrX-dependent increase in a reduced state of KEAP1, which in turn leads to KEAP1-mediated PGAM5 and Bcl-xL (BCL2L1) degradation. In contrast, parthenolide increases oxidation of KEAP1 in normal prostate epithelial cells, leading to increased Nrf2 (NFE2L2) levels and subsequent Nrf2-dependent expression of antioxidant enzymes. These results reveal a novel redox-mediated modification of KEAP1 in controlling the differential effect of parthenolide on tumor and normal cell radiosensitivity. Further, they show it is possible to develop a tumor-specific radiosensitizing agent with radioprotective properties in normal cells.
doi:10.1158/0008-5472.CAN-12-4297
PMCID: PMC3715565  PMID: 23674500
Keap1; Nrf2; PGAM5; Bcl-xL; parthenolide; radiotherapy; prostate cancer; reactive oxygen species (ROS); redox modification; antioxidant proteins; mitochondrial function
3.  Central GLP-2 enhances hepatic insulin sensitivity via activating PI3K signaling in POMC neurons 
Cell metabolism  2013;18(1):86-98.
Glucagon-like peptides (GLP-1/2) are co-produced and highlighted as key modulators to improve glucose homeostasis and insulin sensitivity after bariatric surgery. However, it is unknown if CNS GLP-2 plays any physiological role in the control of glucose homeostasis and insulin sensitivity. We show that mice lacking GLP-2 receptor (GLP-2R) in POMC neurons display glucose intolerance and hepatic insulin resistance. GLP-2R activation in POMC neurons is required for GLP-2 to enhance insulin-mediated suppression of hepatic glucose production (HGP) and gluconeogenesis. GLP-2 directly modulates excitability of POMC neurons in GLP-2R- and PI3K-dependent manners. GLP-2 initiates GLP-2R-p85α interaction and facilitates PI3K-Akt-dependent FoxO1 nuclear exclusion in POMC neurons. Central GLP-2 suppresses basal HGP and enhances insulin sensitivity, which are abolished in POMC-p110α KO mice. Thus, CNS GLP-2 plays a key physiological role in the control of hepatic glucose production through activating PI3K-dependent modulation of membrane excitability and nuclear transcription of POMC neurons in the brain.
doi:10.1016/j.cmet.2013.06.014
PMCID: PMC3752162  PMID: 23823479
Glucagon-like peptides and receptors; Glucose homeostasis; Hypothalamus; Insulin sensitivity
4.  Pro-Inflammatory Stimuli Engage Brahma Related Gene 1 (Brg1) and Brahma (Brm) in Endothelial Injury 
Circulation research  2013;113(8):986-996.
Rationale
Endothelial dysfunction inflicted by inflammation is found in a host of cardiovascular pathologies. One hallmark event in this process is the aggregation and adhesion of leukocyte to the vessel wall mediated by the up-regulation of adhesion molecules (CAM) in endothelial cells at the transcriptional level. The epigenetic modulator(s) of CAM transactivation and its underlying pathophysiological relevance remain poorly defined.
Objective
Our goal was to determine the involvement of Brg1 and Brm in CAM transactivation and its relevance in the pathogenesis of atherosclerosis.
Methods and Results
In the present study, we report that pro-inflammatory stimuli augmented the expression of Brg1 and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Over-expression of Brg1 and Brm promoted whereas knockdown of Brg1 and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Brg1 and Brm interacted with and were recruited to the CAM promoters by NF-κB/p65. Conversely, depletion of Brg1 and Brm disrupted the kinetics of p65 binding on CAM promoters and crippled CAM activation. Silencing of Brg1 and Brm also altered key epigenetic changes associated with CAM transactivation. Of intrigue, 17β-estradiol antagonized both the expression and activity of Brg1/Brm. Most importantly, endothelial-targeted elimination of Brg1/Brm conferred atheroprotective effects to Apoe−/− mice on a Western diet.
Conclusion
Therefore, our data suggest that Brg1 and Brm integrate various pro-inflammatory cues into CAM transactivation and endothelial malfunction and as such may serve as potential therapeutic targets in treating inflammation related cardiovascular diseases.
doi:10.1161/CIRCRESAHA.113.301296
PMCID: PMC4049295  PMID: 23963727
Endothelial dysfunction; inflammation; atherosclerosis; gene regulation; adhesion molecules
5.  The Proteasome Inhibitor, MG132, Attenuates Diabetic Nephropathy by Inhibiting SnoN Degradation In Vivo and In Vitro 
BioMed Research International  2014;2014:684765.
Transforming growth factor-β (TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-β signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF-β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-β were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF-β were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-β activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN.
doi:10.1155/2014/684765
PMCID: PMC4070544  PMID: 25003128
6.  The Role of Ubiquitination and Sumoylation in Diabetic Nephropathy 
BioMed Research International  2014;2014:160692.
Diabetic nephropathy (DN) is a common and characteristic microvascular complication of diabetes; the mechanisms that cause DN have not been clarified, and the epigenetic mechanism was promised in the pathology of DN. Furthermore, ubiquitination and small ubiquitin-like modifier (SUMO) were involved in the progression of DN. MG132, as a ubiquitin proteasome, could improve renal injury by regulating several signaling pathways, such as NF-κB, TGF-β, Nrf2-oxidative stress, and MAPK. In this review, we summarize how ubiquitination and sumoylation may contribute to the pathology of DN, which may be a potential treatment strategy of DN.
doi:10.1155/2014/160692
PMCID: PMC4065738  PMID: 24991536
7.  High Glucose Induces Sumoylation of Smad4 via SUMO2/3 in Mesangial Cells 
BioMed Research International  2014;2014:782625.
Recent studies have shown that sumoylation is a posttranslational modification involved in regulation of the transforming growth factor-β (TGF-β) signaling pathway, which plays a critical role in renal fibrosis in diabetic nephropathy (DN). However, the role of sumoylation in the regulation of TGF-β signaling in DN is still unclear. In the present study, we investigated the expression of SUMO (SUMO1 and SUMO2/3) and Smad4 and the interaction between SUMO and Smad4 in cultured rat mesangial cells induced by high glucose. We found that SUMO1 and SUMO2/3 expression was significantly increased in the high glucose groups compared to the normal group (P < 0.05). Smad4 and fibronectin (FN) levels were also increased in the high glucose groups in a dose-dependent manner. Coimmunoprecipitation and confocal laser scanning revealed that Smad4 interacted and colocalized with SUMO2/3, but not with SUMO1 in mesangial cells. Sumoylation (SUMO2/3) of Smad4 under high glucose condition was strongly enhanced compared to normal control (P < 0.05). These results suggest that high glucose may activate TGF-β/Smad signaling through sumoylation of Samd4 by SUMO2/3 in mesangial cells.
doi:10.1155/2014/782625
PMCID: PMC4058256  PMID: 24971350
8.  Escherichia coli O157:H7 Super-Shedder and Non-Shedder Feedlot Steers Harbour Distinct Fecal Bacterial Communities 
PLoS ONE  2014;9(5):e98115.
Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed “super-shedders”. A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a “normal” microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.
doi:10.1371/journal.pone.0098115
PMCID: PMC4032279  PMID: 24858731
9.  Whole brain radiotherapy plus simultaneous in-field boost with image guided intensity-modulated radiotherapy for brain metastases of non-small cell lung cancer 
Background
Whole brain radiotherapy (WBRT) plus sequential focal radiation boost is a commonly used therapeutic strategy for patients with brain metastases. However, recent reports on WBRT plus simultaneous in-field boost (SIB) also showed promising outcomes. The objective of present study is to retrospectively evaluate the efficacy and toxicities of WBRT plus SIB with image guided intensity-modulated radiotherapy (IG-IMRT) for inoperable brain metastases of NSCLC.
Methods
Twenty-nine NSCLC patients with 87 inoperable brain metastases were included in this retrospective study. All patients received WBRT at a dose of 40 Gy/20 f, and SIB boost with IG-IMRT at a dose of 20 Gy/5 f concurrent with WBRT in the fourth week. Prior to each fraction of IG-IMRT boost, on-line positioning verification and correction were used to ensure that the set-up errors were within 2 mm by cone beam computed tomography in all patients.
Results
The one-year intracranial control rate, local brain failure rate, and distant brain failure rate were 62.9%, 13.8%, and 19.2%, respectively. The two-year intracranial control rate, local brain failure rate, and distant brain failure rate were 42.5%, 30.9%, and 36.4%, respectively. Both median intracranial progression-free survival and median survival were 10 months. Six-month, one-year, and two-year survival rates were 65.5%, 41.4%, and 13.8%, corresponding to 62.1%, 41.4%, and 10.3% of intracranial progression-free survival rates. Patients with Score Index for Radiosurgery in Brain Metastases (SIR) >5, number of intracranial lesions <3, and history of EGFR-TKI treatment had better survival. Three lesions (3.45%) demonstrated radiation necrosis after radiotherapy. Grades 2 and 3 cognitive impairment with grade 2 radiation leukoencephalopathy were observed in 4 (13.8%) and 4 (13.8%) patients. No dosimetric parameters were found to be associated with these late toxicities. Patients received EGFR-TKI treatment had higher incidence of grades 2–3 cognitive impairment with grade 2 leukoencephalopathy.
Conclusions
WBRT plus SIB with IG-IMRT is a tolerable and effective treatment for NSCLC patients with inoperable brain metastases. However, the results of present study need to be examined by the prospective investigations.
doi:10.1186/1748-717X-9-117
PMCID: PMC4035738  PMID: 24884773
Whole brain radiotherapy; Simultaneous in-field boost; Brain metastases; Non-small cell lung cancer
10.  Expert consensus on acute exacerbation of chronic obstructive pulmonary disease in the People’s Republic of China 
Chronic obstructive pulmonary disease (COPD) is a common disease that severely threatens human health. Acute exacerbation of COPD (AECOPD) is a major cause of disease progression and death, and causes huge medical expenditures. This consensus statement represents a description of clinical features of AECOPD in the People’s Republic of China and a set of recommendations. It is intended to provide clinical guidelines for community physicians, pulmonologists and other health care providers for the prevention, diagnosis, and treatment of AECOPD.
doi:10.2147/COPD.S58454
PMCID: PMC4008287  PMID: 24812503
COPD; AECOPD; recommendations; guidelines
11.  Expression of RABEX-5 and its clinical significance in prostate cancer 
Background
While recent research has shown that expression of RABEX-5 in breast cancer and colorectal cancer has a crucial impact on tumor development, there is little information regarding RABEX-5 expression in prostate cancer. This study investigated the expression of RABEX-5 in prostate cancer by real time quantitative polymerase chain reaction and evaluated its association with clinicopathological variables, including prostate cancer patient prognosis.
Methods
A total of 180 patients with primary prostate cancer treated by radical prostatectomy were enrolled. Real time quantitative polymerase chain reaction was utilized to investigate mRNA expression level of RABEX-5 in 180 paired prostate cancer/adjacent non-cancerous tissues. RABEX-5 mRNA expression was divided into high expression group and low expression group and correlations between RABEX-5 mRNA and clinicopathological factors were then evaluated. Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the association between RABEX-5 mRNA expression and prognosis of patients with prostate cancer.
Results
Our study showed that RABEX-5 mRNA was significantly upregulated in prostate cancer tissues. The data indicated that high expression of RABEX-5 mRNA was significantly associated with lymph node metastasis (P = 0.001), clinical stage (P = 0.004), biochemical recurrence (P = 0.009), preoperative prostate-specific antigen (P < 0.001), and Gleason score (P < 0.001). High RABEX-5 mRNA expression was a significant predictor of poor biochemical recurrence free survival and overall survival both in univariate and multivariate analysis.
Conclusion
This is to our knowledge the first report investigating tumor RABEX-5 mRNA expression level in prostate cancer. We have shown that high RABEX-5 mRNA expression is a strong predictor of poor prognosis in prostate cancer patients treated by radical prostatectomy, and multivariate analysis confirmed RABEX-5 mRNA as an independent prognostic factor.
doi:10.1186/1756-9966-33-31
PMCID: PMC3996945  PMID: 24716822
12.  Microcantilever-Based Label-Free Characterization of Temperature-Dependent Biomolecular Affinity Binding 
This paper presents label-free characterization of temperature-dependent biomolecular affinity binding on solid surfaces using a microcantilever-based device. The device consists of a Parylene cantilever one side of which is coated with a gold film and functionalized with molecules as an affinity receptor to a target analyte. The cantilever is located in a poly(dimethylsiloxane) (PDMS) microfluidic chamber that is integrated with a transparent indium tin oxide (ITO) resistive temperature sensor on the underlying substrate. The ITO sensor allows for real-time measurements of the chamber temperature, as well as unobstructed optical access for reflection-based optical detection of the cantilever deflection. To test the temperature-dependent binding between the target and receptor, the temperature of the chamber is maintained at a constant setpoint, while a solution of unlabeled analyte molecules is continuously infused through the chamber. The measured cantilever deflection is used to determine the target-receptor binding characteristics. We demonstrate label-free characterization of temperature-dependent binding kinetics of the platelet-derived growth factor (PDGF) protein with an aptamer receptor. Affinity binding properties including the association and dissociation rate constants as well as equilibrium dissociation constant are obtained, and shown to exhibit significant dependencies on temperature.
doi:10.1016/j.snb.2012.02.045
PMCID: PMC3979549  PMID: 24723743
aptamer; affinity binding; microcantilever; label-free detection; temperature dependence
13.  Clinical Analysis of Thyroid Carcinoma Showing Thymus-Like Differentiation: Report of 8 Cases 
International Surgery  2013;98(2):95-100.
Thyroid carcinoma showing thymus-like differentiation (CASTLE) is a kind of rare neoplasm of the thyroid gland. Because thyroid CASTLE is rare and difficult to diagnose, its clinicopathologic features have not been well defined, and no universally accepted treatment recommendation is available. We analyzed retrospectively the clinicopathologic data of 8 patients with thyroid CASTLE who underwent surgery and radiotherapy at the Shengjing Hospital of China Medical University between December 2008 and June 2012. All patients accepted radical surgery. All patients accepted postoperative radiotherapy, except one 79-year-old patient. There was no evidence of recurrence or metastasis during the follow-up period. The pattern of immunohistochemical staining was similar to that of thymic carcinoma. Six of 8 CASTLE cases expressed CD5. All 8 CASTLE patients were negatively expressed in thyroglobulin, thyroid transcription factor 1, and calcitonin. Patients with thyroid CASTLE have good outcomes after radical resection and postoperative radiotherapy. Positive CD5 immunoreactivity can contribute to diagnosis of this disease.
doi:10.9738/INTSURG-D-12-00034.1
PMCID: PMC3723174  PMID: 23701142
CASTLE; Thyroid neoplasms; CD5; Thymus
14.  FUsed in Sarcoma Is a Novel Regulator of Manganese Superoxide Dismutase Gene Transcription 
Antioxidants & Redox Signaling  2014;20(10):1550-1566.
Abstract
Aims: FUsed in sarcoma (FUS) is a multifunctional DNA/RNA-binding protein that possesses diverse roles, such as RNA splicing, RNA transport, DNA repair, translation, and transcription. The network of enzymes and processes regulated by FUS is far from being fully described. In this study, we have focused on the mechanisms of FUS-regulated manganese superoxide dismutase (MnSOD) gene transcription. Results: Here we demonstrate that FUS is a component of the transcription complex that regulates the expression of MnSOD. Overexpression of FUS increased MnSOD expression in a dose-dependent manner and knockdown of FUS by siRNA led to the inhibition of MnSOD gene transcription. Reporter analyses, chromatin immunoprecipitation assay, electrophoretic mobility shift assay, affinity chromatography, and surface plasmon resonance analyses revealed the far upstream region of MnSOD promoter as an important target of FUS-mediated MnSOD transcription and confirmed that FUS binds to the MnSOD promoter and interacts with specificity protein 1 (Sp1). Importantly, overexpression of familial amyotropic lateral sclerosis (fALS)-linked R521G mutant FUS resulted in a significantly reduced level of MnSOD expression and activity, which is consistent with the decline in MnSOD activity observed in fibroblasts from fALS patients with the R521G mutation. R521G-mutant FUS abrogates MnSOD promoter-binding activity and interaction with Sp1. Innovation and Conclusion: This study identifies FUS as playing a critical role in MnSOD gene transcription and reveals a previously unrecognized relationship between MnSOD and mutant FUS in fALS. Antioxid. Redox Signal. 20, 1550–1566.
doi:10.1089/ars.2012.4984
PMCID: PMC3942683  PMID: 23834335
16.  Gastric foreign body granuloma caused by an embedded fishbone: A case report 
Fishbones are the most commonly ingested foreign bodies that cause gastrointestinal tract penetration. However, fishbones embedded in the gastrointestinal tract that lead to foreign body granulomas that mimic submucosal tumors are rare. Herein, we describe a 56-year-old woman who presented with a 20-day-history of upper abdominal pain. Endoscopy revealed an elevated lesion in the gastric antrum. An abdominal computed tomography scan showed a mass in the gastric antrum and a linear calcified lesion in the mass. An endoscopic ultrasonography examination revealed a 3.9 cm × 2.2 cm, irregular, hypoechoic mass with indistinct margins in the muscularis propria layer. The patient was initially diagnosed as having a submucosal tumor, and subsequent surgical resection showed that the lesion was a foreign body granuloma caused by an embedded fishbone. Our case indicated that the differential diagnosis of a foreign body granuloma should be considered in cases of elevated lesions in the gastrointestinal tract.
doi:10.3748/wjg.v20.i12.3388
PMCID: PMC3964412  PMID: 24696619
Gastric; Foreign body granuloma; Fishbone; Endoscopic ultrasonography; Computed tomography
17.  3,3′-Diindolylmethane inhibits the invasion and metastasis of nasopharyngeal carcinoma cells in vitro and in vivo by regulation of epithelial mesenchymal transition 
Nasopharyngeal carcinoma (NPC) is characterized by silent progression and atypical early symptoms. Early metastasis to the neck lymph nodes is common. However, conventional chemoradiotherapy is limited and unable to effectively control cervical lymph node metastasis of NPC. In addition, toxicities caused by chemoradiotherapy often induce damage to normal tissues and organs. Thus, the aim of this study was to investigate the ability of 3,3′-diindolylmethane (DIM) to inhibit the invasion and metastasis of NPC cells in vitro and in vivo. The migration and invasive abilities of the 5–8F human NPC cell line were detected using a Transwell assay. Lymph node metastasis in nude mice was observed following the implantation of xenograft tumors for 8 weeks. In addition, western blot analysis was used to detect the expression levels of epithelial mesenchymal transition (EMT)-associated key proteins in NPC cells treated with DIM in vitro and in vivo. The results demonstrated that DIM effectively inhibited the migration and invasion of NPC cells in vitro and the effect was concentration-dependent. In addition, DIM significantly delayed and reduced the occurrence of lymph node metastasis in the animal model. The expression levels of a number of key proteins associated with EMT were affected by DIM treatment. In the animal model, there were no signs of toxicity in the vital organs, including the heart, liver and kidney, of animals fed a diet containing DIM. Therefore, the results of the present study indicate that DIM affects the expression levels of a number of EMT-associated key proteins and induces the inhibition of invasion and metastasis of NPC cells in vitro and in vivo.
doi:10.3892/etm.2014.1649
PMCID: PMC4043566  PMID: 24926357
3,3′-diindolylmethane; nasopharyngeal carcinoma cell; invasion; metastasis; epithelial mesenchymal transition
18.  Microarray Profiling and Co-Expression Network Analysis of Circulating lncRNAs and mRNAs Associated with Major Depressive Disorder 
PLoS ONE  2014;9(3):e93388.
LncRNAs, which represent one of the most highly expressed classes of ncRNAs in the brain, are becoming increasingly interesting with regard to brain functions and disorders. However, changes in the expression of regulatory lncRNAs in Major Depressive Disorder (MDD) have not yet been reported. Using microarrays, we profiled the expression of 34834 lncRNAs and 39224 mRNAs in peripheral blood sampled from MDD patients as well as demographically-matched controls. Among these, we found that 2007 lncRNAs and 1667 mRNAs were differentially expressed, 17 of which were documented as depression-related gene in previous studies. Gene Ontology (GO) and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to fundamental metabolic processes and neurodevelopment diseases. To investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we also constructed co-expression networks composed of the lncRNAs and mRNAs, which shows significant correlated patterns of expression. In the MDD-derived network, there were a greater number of nodes and connections than that in the control-derived network. The lncRNAs located at chr10:874695-874794, chr10:75873456-75873642, and chr3:47048304-47048512 may be important factors regulating the expression of mRNAs as they have previously been reported associations with MDD. This study is the first to explore genome-wide lncRNA expression and co-expression with mRNA patterns in MDD using microarray technology. We identified circulating lncRNAs that are aberrantly expressed in MDD and the results suggest that lncRNAs may contribute to the molecular pathogenesis of MDD.
doi:10.1371/journal.pone.0093388
PMCID: PMC3968145  PMID: 24676134
19.  Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease 
Respiratory Research  2014;15(1):33.
Background
Stem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats.
Methods
The MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay.
Results
rAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions.
Conclusions
Vacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.
doi:10.1186/1465-9921-15-33
PMCID: PMC3987841  PMID: 24661402
Chronic obstructive pulmonary disease; Mesenchymal stem cells; Alveolar epithelial type II cells; Niche
20.  Effects of Chaihu-Shugan-San and Shen-Ling-Bai-Zhu-San on p38 MAPK Pathway in Kupffer Cells of Nonalcoholic Steatohepatitis 
This study aimed to investigate the effects of Chaihu-Shugan-San (CSS), Shen-Ling-Bai-Zhu-San (SLBZS), and integrated recipe of the above two recipes on inflammatory markers and proteins involved in p38 MAPK pathway in Kupffer cells of NASH rats induced by high fat diet (HFD). Rats were administered at low or high dose of CSS, SLBZS, and integrated recipe except normal group and model group for 16 weeks. The levels of hepatic lipid, TNF-α, IL-1, and IL-6 in liver tissues were measured. Kupffer cells were isolated from livers to evaluate expressions of TLR4, p-p38 MAPK, and p38 MAPK by Western blotting. The results showed that the NASH model rats successfully reproduced typical pathogenetic and histopathological features. Levels of hepatic lipid and liver tissues inflammatory factors in high-dose SLBZS group and integrated recipe group were all lower than that of model group decreased observably. Expressions of TLR4, p-p38 MAPK, and p38 MAPK in Kupffer cells were decreased in all treatment groups, but there was no significant difference between treatment groups. The high-dose SLBZS group had the lowest expression levels of TLR4, and the most visible downtrend in the expression levels of p-p38 MAPK and p38 MAPK was found in the high-dose integrated recipe group. The ratio of p-p38 MAPK to total p38 MAPK protein was obviously increased in all treatment groups. Therefore, our study showed that the activation of p38 MAPK pathway in Kupffer cells might be related to the release of inflammatory factors such as TNF-α, IL-1, and IL-6 in NASH rats. High dose of SLBZS and integrated recipe might work as a significant anti-inflammatory effect in Kupffer cells of NASH rats induced by HFD through suppression of p38 MAPK pathway. It indicated that p38 MAPK pathway may be the possible effective target for the recipes.
doi:10.1155/2014/671013
PMCID: PMC3984846  PMID: 24795769
21.  Overexpression of LAPTM4B-35: A Novel Marker of Poor Prognosis of Prostate Cancer 
PLoS ONE  2014;9(3):e91069.
Background
Lysosome-associated protein transmembrane 4b-35 (LAPTM4B-35) is a member of the mammalian 4-tetratransmembrane spanning protein superfamily, which is overexpressed in several solid malignancies. However, the expression of LAPTM4B-35 and its role in the progression of prostate cancer (PCa) is unknown. The aim of the present study was to investigate the LAPTM4B-35 expression in PCa and its potential relevance to clinicopathological variables and prognosis.
Methods
Immunohistochemistry was used to determine the expression of LAPTM4B-35 protein in 180 PCa tissues in comparison with 180 normal benign prostatic hyperplasia (BPH) specimens. The correlation between the expression of the LAPTM4B-35 protein and the clinicopathologic characteristics of patients with PCa was analyzed.
Results
Statistical analysis showed that LAPTM4B-35 expression was significantly elevated in PCa compared with the BPH controls. High LAPTM4B-35 staining was present in 71.11% of all the cases with PCa. The overexpression of LAPTM4B-35 was significantly associated with the lymph node metastasis, seminal vesicle invasion, PCa stage, higher Gleason score, higher preoperative PSA, and biochemical recurrence (BCR). The Kaplan-Meier survival analysis showed that the high expression of LAPTM4B-35 was related to the poor overall survival and BCR-free survival of patients with PCa. Multivariate Cox analysis showed that LAPTM4B-35 was an independent prognostic factor for both overall survival and BCR-free survival of patients with PCa.
Conclusions
Overexpression of LAPTM4B-35 may be associated with tumor progression and poor prognosis in PCa and thus may serve as a new molecular marker to predict the prognosis of PCa patients.
doi:10.1371/journal.pone.0091069
PMCID: PMC3961215  PMID: 24651764
22.  Megakaryocytic Leukemia 1 (MKL1) Regulates Hypoxia Induced Pulmonary Hypertension in Rats 
PLoS ONE  2014;9(3):e83895.
Hypoxia induced pulmonary hypertension (HPH) represents a complex pathology that involves active vascular remodeling, loss of vascular tone, enhanced pulmonary inflammation, and increased deposition of extracellular matrix proteins. Megakaryocytic leukemia 1 (MKL1) is a transcriptional regulator known to influence cellular response to stress signals in the vasculature. We report here that in response to chronic hypobaric hypoxia, MKL1 expression was up-regulated in the lungs in rats. Short hairpin RNA (shRNA) mediated depletion of MKL1 significantly ameliorated the elevation of pulmonary arterial pressure in vivo with a marked alleviation of vascular remodeling. MKL1 silencing also restored the expression of NO, a key vasoactive molecule necessary for the maintenance of vascular tone. In addition, hypoxia induced pulmonary inflammation was dampened in the absence of MKL1 as evidenced by normalized levels of pro-inflammatory cytokines and chemokines as well as reduced infiltration of pro-inflammatory immune cells in the lungs. Of note, MKL1 knockdown attenuated fibrogenesis in the lungs as indicated by picrosirius red staining. Finally, we demonstrate that MKL1 mediated transcriptional activation of type I collagen genes in smooth muscle cells under hypoxic conditions. In conclusion, we data highlight a previously unidentified role for MKL1 in the pathogenesis of HPH and as such lay down groundwork for future investigation and drug development.
doi:10.1371/journal.pone.0083895
PMCID: PMC3960100  PMID: 24647044
23.  Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN): an imaging demonstration 
OncoTargets and therapy  2014;7:441-446.
Background
Mesenchymal stem cells (MSCs) have been considered to hold great potential as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN) engineering on MSCs’ capacity for cancer cell-oriented migration.
Methods
MSCs were engineered with a PTEN-bearing plasmid and the expression was confirmed with Western blotting. A human glioma cell line (DBTRG) was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic system.
Results
The expression of transfected PTEN in MSCs was identified by immunoblotting analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring system, and a phagocytosis-like action of MSCs was also observed.
Conclusion
MSCs maintained their capacity for cancer cell-directed migration after they were engineered with anticancer genes. This study provides the first direct evidence of MSCs’ tropism post-anticancer gene engineering.
doi:10.2147/OTT.S59227
PMCID: PMC3962313  PMID: 24669193
gene therapy; mesenchymal stem cells; phosphatase and tensin homolog; cancer
24.  A young man with primary prostatic extra-gastrointestinal stromal tumor: a rare case report and review of the literature 
Mounting evidence demonstrates the presence of extragastrointestinal stromal tumor (EGIST) which originates from tissues outside the gastrointestinal (GI) tract and shares overlapping immunohistological features with gastrointestinal stromal tumor (GIST). GIST emanating from prostate is extremely rare. To our knowledge, there are only 3 definitely reported cases of primary prostatic EGIST. Herein, we report a case of prostatic EGIST in 31-year-old man with low urinary tract symptoms who was initially misdiagnosed as sarcoma of prostate. Imaging studies assist in determining the origin and location of EGIST. Immunohistochemical assessment (DOG-1, CD117, and CD34) helps in differentiating such lesion from other stromal tumors and in addressing an appropriate and optimal therapeutic strategy.
PMCID: PMC4014260  PMID: 24817976
Extragastrointestinal stromal tumor (EGIST); prostate; differential diagnosis; imatinib
25.  Severe anemia and melena caused by pyeloduodenal fistula due to renal stone-associated squamous cell carcinoma 
Pyeloduodenal fistula is a rare condition and its association with malignancy is even rarer. Herein we report the case of a 66-year-old man who was admitted to the hospital with a three-month history of intermittent melena and a more than 20-year history of right-side renal stones. Computed tomography showed a heterogeneous right renal mass with a staghorn stone that had invaded the duodenum and caused an internal fistula. An upper gastrointestinal series showed pyeloduodenal fistula. The patient underwent an exploratory operation and a biopsy was taken at the peripelvic region. Pathological examination verified the existence of squamous cell carcinoma. To our knowledge, this is the first case of pyeloduodenal fistula associated with renal stones, squamous cell carcinoma and upper urinary tract calculus presenting on melena. We report on the features of this rare entity but also review and summarize the etiology, diagnosis and treatment options that can be extrapolated from the existing literature.
PMCID: PMC3999026  PMID: 24772159
Calculus; Digestive tract; Pyeloduodenal fistula; Squamous cell carcinoma; Urinary tract

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