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1.  Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells 
PLoS ONE  2015;10(7):e0132710.
Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.
doi:10.1371/journal.pone.0132710
PMCID: PMC4514595  PMID: 26207636
2.  Near-field collimation of light carrying orbital angular momentum with bull’s-eye-assisted plasmonic coaxial waveguides 
Scientific Reports  2015;5:12108.
The orbital angular momentum (OAM) of light, as an emerging hotspot in optics and photonics, introduces many degrees of freedom for applications ranging from optical communication and quantum processing to micromanipulation. To achieve a high degree of integration, optical circuits for OAM light are essential, which are, however, challenging in the optical regime owing to the lack of well-developed theory. Here we provide a scheme to guide and collimate the OAM beam at the micro- and nano-levels. The coaxial plasmonic slit was exploited as a naturally occurring waveguide for light carrying OAM. Concentric grooves etched on the output surface of the coaxial waveguide were utilized as a plasmonic metasurface to couple the OAM beam to free space with greatly increased beam directivity. Experimental results at λ = 532 nm validated the novel transportation and collimating effect of the OAM beam. Furthermore, dynamic tuning of the topological charges was demonstrated by using a liquid crystal spatial light modulator (SLM).
doi:10.1038/srep12108
PMCID: PMC4498324  PMID: 26159423
3.  Evaluation of hGM-CSF/hTNFα surface-modified prostate cancer therapeutic vaccine in the huPBL-SCID chimeric mouse model 
To validate its efficacy in the context of the human immune system, a novel therapeutic vaccine of hGM-CSF/hTNFα surface-modified PC-3 cells against human prostate cancer was evaluated in the human peripheral blood lymphocytes-severe combined immunodeficiency (huPBL-SCID) chimeric mouse model. The hGM-CSF or/and hTNFα modified vaccines inhibited prostate cancer growth effectively so as to prolong the mouse survival significantly. The splenocytes from the hGM-CSF/hTNFα vaccine-inoculated mice showed the strongest tumor-specific cytotoxicity against PC-3 cells and the highest production of IFNɤ. These features indicated that type 1 protective immune response was induced efficiently against human prostate cancer and further enhanced through synergetic adjuvant effects of hGM-CSF and hTNFα.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-015-0175-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13045-015-0175-8
PMCID: PMC4490636  PMID: 26108270
Cancer immunotherapy; Adjuvant; Synergetic effect; Human immune system; PC-3 prostate cancer cell
4.  Influence of nanotopography on periodontal ligament stem cell functions and cell sheet based periodontal regeneration 
Periodontal regeneration is an important part of regenerative medicine, with great clinical significance; however, the effects of nanotopography on the functions of periodontal ligament (PDL) stem cells (PDLSCs) and on PDLSC sheet based periodontal regeneration have never been explored. Titania nanotubes (NTs) layered on titanium (Ti) provide a good platform to study this. In the current study, the influence of NTs of different tube size on the functions of PDLSCs was observed. Afterward, an ectopic implantation model using a Ti/cell sheets/hydroxyapatite (HA) complex was applied to study the effect of the NTs on cell sheet based periodontal regeneration. The NTs were able to enhance the initial PDLSC adhesion and spread, as well as collagen secretion. With the Ti/cell sheets/HA complex model, it was demonstrated that the PDLSC sheets were capable of regenerating the PDL tissue, when combined with bone marrow mesenchymal stem cell (BMSC) sheets and HA, without the need for extra soluble chemical cues. Simultaneously, the NTs improved the periodontal regeneration result of the ectopically implanted Ti/cell sheets/HA complex, giving rise to functionally aligned collagen fiber bundles. Specifically, much denser collagen fibers, with abundant blood vessels as well as cementum-like tissue on the Ti surface, which well-resembled the structure of natural PDL, were observed in the NT5 and NT10 sample groups. Our study provides the first evidence that the nanotopographical cues obviously influence the functions of PDLSCs and improve the PDLSC sheet based periodontal regeneration size dependently, which provides new insight to the periodontal regeneration. The Ti/cell sheets/HA complex may constitute a good model to predict the effect of biomaterials on periodontal regeneration.
doi:10.2147/IJN.S83357
PMCID: PMC4484652  PMID: 26150714
titanium implant; titania nanotubes; periodontal ligament stem cells; periodontal regeneration; cell sheets
5.  Predicting Positive and Negative Relationships in Large Social Networks 
PLoS ONE  2015;10(6):e0129530.
In a social network, users hold and express positive and negative attitudes (e.g. support/opposition) towards other users. Those attitudes exhibit some kind of binary relationships among the users, which play an important role in social network analysis. However, some of those binary relationships are likely to be latent as the scale of social network increases. The essence of predicting latent binary relationships have recently began to draw researchers' attention. In this paper, we propose a machine learning algorithm for predicting positive and negative relationships in social networks inspired by structural balance theory and social status theory. More specifically, we show that when two users in the network have fewer common neighbors, the prediction accuracy of the relationship between them deteriorates. Accordingly, in the training phase, we propose a segment-based training framework to divide the training data into two subsets according to the number of common neighbors between users, and build a prediction model for each subset based on support vector machine (SVM). Moreover, to deal with large-scale social network data, we employ a sampling strategy that selects small amount of training data while maintaining high accuracy of prediction. We compare our algorithm with traditional algorithms and adaptive boosting of them. Experimental results of typical data sets show that our algorithm can deal with large social networks and consistently outperforms other methods.
doi:10.1371/journal.pone.0129530
PMCID: PMC4468140  PMID: 26075404
6.  Flaxseed Oil Containing α-Linolenic Acid Ester of Plant Sterol Improved Atherosclerosis in ApoE Deficient Mice 
Plant sterols (PS) have potential preventive function in atherosclerosis due to their cholesterol-lowering ability. Dietary α-linolenic acid in flaxseed oil is associated with a reduction in cardiovascular events through its hypolipidemic and anti-inflammation properties. This study was designed to evaluate the effects of flaxseed oil containing α-linolenic acid ester of PS (ALA-PS) on atherosclerosis and investigate the underlying mechanisms. C57BL/6 mice were administered a regular diet and apoE knockout (apoE-KO) mice were given a high fat diet alone or supplemented with 5% flaxseed oil with or without 3.3% ALA-PS for 18 weeks. Results demonstrated that flaxseed oil containing ALA-PS was synergistically interaction in ameliorating atherosclerosis as well as optimizing overall lipid levels, inhibiting inflammation and reducing oxidative stress. These data were associated with the modification effects on expression levels of genes involved in lipid metabolism (PPARα, HMGCR, and SREBPs), inflammation (IL-6, TNF, MCP-1, and VCAM-1), and oxidative stress (NADPH oxidase).
doi:10.1155/2015/958217
PMCID: PMC4477243  PMID: 26180602
7.  Early B-cell Factor 1 Regulates Adipocyte Morphology and Lipolysis in White Adipose Tissue 
Cell metabolism  2014;19(6):981-992.
Summary
White adipose tissue (WAT) morphology characterized by hypertrophy (i.e. fewer but larger adipocytes) associates with increased adipose inflammation, lipolysis, insulin resistance and risk of diabetes. However, the causal relationships and the mechanisms controlling WAT morphology are unclear. Herein, we identified EBF1 as an adipocyte-expressed transcription factor with decreased expression/activity in WAT hypertrophy. In human adipocytes, the regulatory targets of EBF1 were enriched for genes controlling lipolysis and adipocyte morphology/differentiation and in both humans and murine models, reduced EBF1 levels associated with increased lipolysis and adipose hypertrophy. Although EBF1 did not affect adipose inflammation, TNFα reduced EBF1 gene expression. High fat diet-intervention in Ebf1+/− mice resulted in more pronounced WAT hypertrophy and attenuated insulin sensitivity compared with wild-type littermate controls. We conclude that EBF1 is an important regulator of adipose morphology and fat cell lipolysis and may constitute a link between WAT inflammation, altered lipid metabolism, adipose hypertrophy and insulin resistance.
doi:10.1016/j.cmet.2014.03.032
PMCID: PMC4109056  PMID: 24856929
8.  Modified sequential therapy vs quadruple therapy as initial therapy in patients with Helicobacter infection 
AIM: To evaluate the efficacy and safety of modified sequential therapy and to compare modified sequential therapy with standard quadruple therapy for Helicobacter pylori (H. pylori) eradication.
METHODS: In total, 200 consecutive patients who were diagnosed with H. pylori-infected chronic gastritis by electronic endoscopy and rapid urease testing from December 2012 to October 2013 were enrolled in this study. The patients had not previously received H. pylori eradication treatment, and were randomized into two groups. The patients in Group A (n = 101) were treated with ilaprazole + bismuth potassium citrate + amoxicillin and clavulanate potassium + levofloxacin, and the patients in Group B (n = 99) were administered a modified sequential therapy composed of ilaprazole at 5 mg bid and amoxicillin and clavulanate potassium at 914 mg for the first five days followed by ilaprazole at 5 mg bid, furazolidone at 100 mg bid and levofloxacin at 500 mg qid for the next five days. Four to six weeks after the end of treatment, a 14C-urea breath test was performed for all the subjects to confirm the eradication of H. pylori. The intention-to-treat and per-protocol eradication rates were determined.
RESULTS: A total of 190 of the 200 patients completed the study. All 200 patients were included in the intention-to-treat analysis, whereas 190 patients were included in the per-protocol analysis. In the intention-to-treat analysis, the rates of H. pylori eradication in Groups A and B were 85.15% (86/101) and 81.82% (81/99), respectively. In the per-protocol analysis, the H. pylori eradication rates in Groups A and B were 88.66% (86/97) and 87.09% (81/93), respectively. No significant difference was observed (χ2 = 0.109, P = 0.741) in the eradication rate between Groups A and B. The rates of adverse effects observed in the groups were similar at 6.19% (6/97) for Group A and 7.53% (7/93) for Group B (P > 0.05). No mortality or major morbidities were observed in any of the patients. Symptomatic improvements in the presentation of stomachache, acid regurgitation, and burning sensation were not significantly different between the two groups.
CONCLUSION: Ilaprazole-based 10-d standard quadruple therapy does not offer an incremental benefit over modified sequential therapy for the treatment of H. pylori infection, as both treatment regimens appear to be effective, safe, and well-tolerated as initial treatment options.
doi:10.3748/wjg.v21.i20.6310
PMCID: PMC4445109  PMID: 26034367
Helicobacter pylori; Chronic gastritis; Sequential therapy; Quadruple therapy; Initial therapy; Ilaprazole
9.  Polypeptide from Chlamys farreri restores endoplasmic reticulum (ER) redox homeostasis, suppresses ER stress, and inhibits ER stress-induced apoptosis in ultraviolet B-irradiated HaCaT cells 
Objective: To investigate the effects of polypeptide from Chlamys farreri (PCF) on ultraviolet B (UVB)-induced apoptosis in human keratinocyte HaCaT cells. Methods: In HaCaT cells at 4 h or 18 h after UVB irradiation, the cell viability was measured by MTT assay. Cellular apoptosis was detected with annexin V-FITC/PI staining by flow cytometry. The expression levels of PDI, Ero-1α, GRP78, and CHOP were assessed by Western blot analysis. Mitochondrial membrane potential (MMP) was measured by fluorescent probe JC-1. Caspase activities were detected with fluorogenic substrates. Results: PCF alleviated cell viability loss and inhibited apoptosis in HaCaT cells after UVB irradiation. Moreover, PCF increased the expression levels of PDI and Ero-1α, which were related with the ER redox homeostasis. Furthermore, PCF treatment inhibited the expression of GRP78 at 4 h after UVB irradiation, and suppressed CHOP expression at 18 h post-irradiation, indicating that PCF could inhibit UVB-evoked ER stress in the early stage post-irradiation, and suppress the ER stress-induced apoptosis in the late stage. In addition, PCF alleviated UVB-induced MMP loss, and inhibited the activation of caspase-9/-3, in HaCaT cells after UVB irradiation. On the other hand, MMP loss and caspase-9/-3 activation could be partly blocked by the ER stress inhibitor 4-PBA. Conclusions: PCF inhibits UVB-induced apoptosis through restoring ER redox homeostasis, suppressing ER stress, and inhibiting ER stress-induced mitochondrial apoptosis in HaCaT cells. These findings provide evidence for the mechanism underlying UVB-induced skin damages, and support the promising role of PCF in treatment of the diseases.
PMCID: PMC4494147  PMID: 26175857
HaCaT cells; UVB irradiation; endoplasmic reticulum (ER); mitochondria; apoptosis
10.  Antioxidant activities and phenolic compounds of date plum persimmon (Diospyros lotus L.) fruits 
In the present study, phenolic compounds are extracted from the date plum persimmon fruits using water, methanol and acetone as solvents. Antioxidant activities of the phenolic extracts are measured using four different tests, namely, DPPH, hydroxyl radical scavenging activities, chelating and reducing power assays. All the extracts show dose dependent DPPH radical scavenging activity, reducing and chelating powers and moreover, they are well correlated with the total phenolic and total flavonoid substances, suggesting direct contribution of phenolic compounds to these activities. In further, the extracts are identified and quantified by HPLC-ECD. Results show that gallic acid is the most abundant phenolic compound, with amounts ranging between 45.49and 287.47 μg/g dry sample. Myricetin is the dominant flavonoid in all extracts. Its level varied from 2.75 μg/g dry sample in acetone extract to 5.28 μg/g dry sample in water extract. On the basis of the results obtained, the date plum persimmon fruits phenolic extract is a potential source of natural antioxidants owing to its significant antioxidant activities.
doi:10.1007/s13197-011-0591-x
PMCID: PMC4008739  PMID: 24803703
Date plum persimmon; Antioxidant activities; Phenolic compounds
11.  Pubertal Bisphenol A Exposure Alters Murine Mammary Stem Cell (MaSC) Function Leading to Early Neoplasia in Regenerated Glands 
Perinatal exposure to bisphenol A (BPA) has been shown to cause aberrant mammary gland morphogenesis and mammary neoplastic transformation. Yet, the underlying mechanism is poorly understood. We tested the hypothesis that mammary glands exposed to BPA during a susceptible window may lead to its susceptibility to tumorigenesis through a stem-cell mediated mechanism. We exposed 21-day-old Balb/c mice to BPA by gavage (25 μg/kg/day) during puberty for 3 weeks, and a subset of animals were further challenged with one oral dose (30 mg/kg) of 7,12-dimethylbenz[a]anthracene (DMBA) at 2 months of age. Primary mammary cells were isolated at 6 weeks, and 2 and 4 months of age for MaSC quantification and function analysis. Pubertal exposure to the low-dose BPA increased lateral branches and hyperplasia in adult mammary glands and caused an acute increase of MaSC in 6-week-old glands and a delayed increase of luminal progenitors in 4-month-old adult gland. Most importantly, pubertal BPA exposure altered the function of MaSC from different age groups, causing early neoplastic lesions in their regenerated glands similar to those induced by DMBA exposure, which indicates that MaSCs are susceptible to BPA-induced transformation. Deep sequencing analysis on MaSC-enriched mammospheres identified a set of aberrantly expressed genes associated with early neoplastic lesions in human breast cancer patients. Thus, our study for the first time shows that pubertal BPA exposure altered MaSC gene expression and function such that they induced early neoplastic transformation.
doi:10.1158/1940-6207.CAPR-13-0260
PMCID: PMC3976434  PMID: 24520039
Bisphenol A; Breast Cancer; Mammary stem cell; Early neoplastic lesion; Risk assessment; RNAseq; Mammosphere
12.  Eruptive xanthomas presenting in tattoos 
doi:10.1503/cmaj.140383
PMCID: PMC4361110  PMID: 25623644
13.  Treatment With Lenalidomide Modulates T-Cell Immunophenotype and Cytokine Production in Patients With Chronic Lymphocytic Leukemia 
Cancer  2011;117(17):3999-4008.
BACKGROUND
Lenalidomide, an immunomodulatory agent, has activity in lymphoproliferative disorders. The authors, therefore, evaluated its effects on T-cell immunophenotype and cytokine production in patients with chronic lymphocytic leukemia (CLL).
METHODS
To study the immunomodulatory effects of lenalidomide in CLL, the authors recruited 24 patients with untreated CLL enrolled in a phase 2 clinical trial of lenalidomide and obtained peripheral blood specimens for immunologic studies consisting of enumeration of T cells and assessing their ability to synthesize cytokines after activation through T-cell receptor (TCR).
RESULTS
After 3 cycles of therapy, patients had a significant reduction in percentage (%) and absolute lymphocyte count (ALC) and an increase in percentage of T cells, percentage of activated CD8+ T cells producing IFN-γ, and percentage of regulatory T (TR) cells when compared with their respective levels before treatment. After 15 cycles of treatment, responder patients had significant reduction in percentage of lymphocytes and ALC, percentage of activated CD4+ T cells producing IL-2, IFN-γ, or TNF-α, and percentage of TR cells when compared with their perspective levels after 3 cycles of treatment. Furthermore, the numbers of activated CD4+ T cells producing IL-2, IFN-γ, or TNF-α, activated CD8+ T cells producing IFN-γ, and TR cells normalized to the range of healthy subjects.
CONCLUSIONS
Treatment with lenalidomide resulted in the normalization of functional T-cell subsets in responders, suggesting that lenalidomide may modulate cell-mediated immunity in patients with CLL.
doi:10.1002/cncr.25983
PMCID: PMC4349201  PMID: 21858802
chronic lymphocytic leukemia; immunomodulatory agents; T cells; cytokines
14.  Combined histological and hematological assessment of iron-induced organ damage in a gerbil model of iron overload 
Background: Previous studies with gerbil models have suggested that excessive iron exposure causes cardiomyopathy and hepatic injury, but pathological analysis was not comprehensive, preventing a detailed understanding of how the metal induces this damage. Methods and results: Gerbils received single intraperitoneal injections of iron dextran (200 mg/kg) or saline and were then analyzed comprehensively for hematological and histological signs of organ damage. These tests included hematology parameters and determination of liver iron concentration, malondialdehyde levels and glutathione peroxidase activity; examination of heart and liver tissue stained with hematoxylin and eosin, Prussian-blue and Masson stain; and electron microscopy analysis of heart and liver ultrastructure. Iron-overloaded animals showed significantly different hematology parameters and significantly higher liver iron concentrations than saline-injected animals, as well as significantly higher malondialdehyde levels and significantly lower glutathione peroxidase activity. Histology analyses showed cellular damage, iron deposits, and both myocardial and liver fibrosis, while electron microscopy of heart and liver sections showed abundant iron deposition lysosomes, and disordered and swollen mitochondria. All these pathological changes increased with exposure time. Conclusions: This comprehensive assessment of iron overload in a gerbil model suggests that excessive iron deposition induces extensive cellular damage, particularly fibrosis in heart and liver. This damage may be the direct result of iron-mediated lipid peroxide damage and of iron deposition that cause compression of myocardial and liver cells, as well as vascular occlusion.
PMCID: PMC4399101  PMID: 25901205
Iron overload; lipid peroxidation; fibrosis; oxidative stress; ultrastructure
15.  Hispidulin prevents hypoxia-induced epithelial-mesenchymal transition in human colon carcinoma cells 
American Journal of Cancer Research  2015;5(3):1047-1061.
Epithelial-mesenchymal transition (EMT) is considered as the most important mechanism that underlies the initiation of cancer metastasis. Here we report that the naturally existing flavonoid, hispidulin is capable of preventing human colorectal cancer cells from hypoxia-induced EMT. The treatment of the cells with hispidulin reversed the EMT-related phenotype that has the morphological changes, down-regulation of E-cadherin, and hypoxia-induced cell migration and invasion. The effect was mediated at least in part by inhibiting the mRNA and protein expressions of HIF-1α via modulation of PTEN/PI3K/Akt pathway. In addition, we found that hispidulin-mediated prevention of the E-cadherin down-regulation and cell motility involved blockade of the hypoxia-induced up-regulation of Snail, Slug and Twist. Hispidulin was also effective in increasing expression of E-cadherin mRNA in HT29 colorectal cancer xenografts implanted in the nude mice. In summary, this study showed that hispidulin can prevent EMT induced by hypoxia, the environment that commonly exists in the center of a solid tumor. Given the low toxicity of hispidulin to the healthy tissues, our study suggests that hispidulin can serve as a safe therapeutic agent for suppressing cancer metastasis.
PMCID: PMC4449434  PMID: 26045985
Hispidulin; EMT; Hypoxia; PI3K/Akt; PTEN
16.  Evolutionary analysis of rubella viruses in mainland China during 2010–2012: endemic circulation of genotype 1E and introductions of genotype 2B 
Scientific Reports  2015;5:7999.
Rubella remains a significant burden in mainland China. In this report, 667 viruses collected in 24 of 31 provinces of mainland China during 2010–2012 were sequenced and analyzed, significantly extending previous reports on limited numbers of viruses collected before 2010. Only viruses of genotypes 1E and 2B were found. Genotype 1E viruses were found in all 24 provinces. Genotype 1E viruses were likely introduced into mainland China around 1997 and endemic transmission of primarily one lineage became established. Viruses reported here from 2010–2012 are largely in a single cluster within this lineage. Genotype 2B viruses were rarely detected in China prior to 2010. This report documents a previously undetected 2B lineage, which likely became endemic in eastern provinces of China between 2010 and 2012. Bayesian analyses were performed to estimate the evolutionary rates and dates of appearance of the genotype 1E and 2B viral linages in China. A skyline plot of viral population diversity did not provide evidence of reduction of diversity as a result of vaccination, but should be useful as a baseline for such reductions as vaccination programs for rubella become widespread in mainland China.
doi:10.1038/srep07999
PMCID: PMC4303870  PMID: 25613734
17.  The Emerging Nexus of Active DNA Demethylation and Mitochondrial Oxidative Metabolism in Post-Mitotic Neurons 
The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET) methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS) and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons.
doi:10.3390/ijms151222604
PMCID: PMC4284726  PMID: 25490140
active DNA demethylation; mitochondrial oxidative metabolism; TET (ten-eleven translocation) methylcytosine dioxygenases; post-mitotic neurons; neurodegeneration
18.  Construction and detection of the tissue-specific pINV-HPV16 E6/7 vector 
Oncology Letters  2014;9(2):857-862.
A tissue-specific promoter can control downstream gene expression in tissues or organs. The human involucrin (hINV) promoter (pINV) that contains 2474 bp of hINV upstream sequence is able to regulate tissue-specific gene expression. This tissue specificity may be important for the prevention and treatment of human papilloma virus infections. pINV was cloned by polymerase chain reaction and the human papillomavirus (HPV)16 E6/7 gene was obtained from the cancer tissue samples of patients with cervical carcinoma at the Yangzhou Maternal and China Health-Care Center of Jinagsu Province (Yangzhou, China). First, specific primers were designed according to the genomic DNA sequence of the HPV16-type standard strain that has been reported and the E6/7 gene was acquired by PCR. The carcinogenic fraction of the E6/7 gene was removed and the remaining section was cloned into T vectors, sequenced correctly and then cloned into the eukaryotic expression vector pCEP4, which was lacking the CMV promoter. The positive recombinants were identified using blue-white screening and endonuclease digestion, subsequent to sequencing and analysis, and the tissue-specific recombinant pINV-HPV16E6/7 plasmids was detected.
doi:10.3892/ol.2014.2736
PMCID: PMC4301524  PMID: 25621060
human papilloma viruses; E6/7; pINV-HPV16 E6/7; tissue-specificity
19.  Potential therapeutic mechanism of genistein in breast cancer involves inhibition of cell cycle regulation 
Molecular Medicine Reports  2014;11(3):1820-1826.
Genistein can prevent tumorigenesis and reduce the incidence of diseases that are dependent upon estrogen. Previous research, however, has shown that genistein can also increase the risk of breast cancer. Thus, the aim of the present study was to investigate the mechanism underlying the effect of genistein in breast cancer and to determine whether genistein produces a therapeutic effect or promotes the development of breast cancer. Gene microarray data obtained from three samples treated with alcohol (control group), three samples treated with 3 μmol/l genistein and three samples treated with 10 μmol/l genistein for 48 h, were downloaded from the Gene Expression Omnibus database. Analysis of the differentially expressed genes (DEGs) and functional enrichment in the two genistein groups was performed. The interaction networks of the DEGs were constructed and the overlapping network was extracted. Finally, the functions and pathways of the DEGs in the overlapping network were enriched. In total, 224 DEGs coexisted in the two genistein groups, and the most significant function of these was the cell cycle. The number and the fold change of expression values of the DEGs in the 10 μmol/l genistein group were significantly higher compared with that of the 3 μmol/l genistein group. The most significant function and pathway of the DEGs in the overlapping network was the cell cycle involving several genes, including GLIPR1, CDC20, BUB1, MCM2 and CCNB1. Thus, genistein stimulation resulted in gene expression changes in breast cancer cell lines and discrepancies increased with higher doses of genistein. The DEGs were most significantly associated with cell cycle regulation.
doi:10.3892/mmr.2014.2907
PMCID: PMC4270317  PMID: 25385471
genistein; breast cancer; differentially expressed genes; cell cycle
20.  Optimized rapeseed oils rich in endogenous micronutrients ameliorate risk factors of atherosclerosis in high fat diet fed rats 
Background
Micronutrients in rapeseed such as polyphenols, tocopherols, phytosterols and phospholipids in rapeseed exert potential benefit to atherosclerosis. Some part of these healthy components substantially lost during the conventional refining processing. Thus some new processing technologies have been developed to produce various endogenous micronutrient-enriched optimized rapeseed oils. The aim of this study is to assess whether optimized rapeseed oils have positive effects on the atherosclerosis risk factors in rats fed a high-fat diet.
Methods
Rats received experiment diets containing 20% fat and refined rapeseed oil or optimized rapeseed oils obtained with various processing technologies as lipid source. After 10 weeks of treatment, plasma was assayed for oxidative stress, lipid profiles and imflammation.
Results
Micronutrients enhancement in optimized rapeseed oils significantly reduced plasma oxidative stress, as evaluated by the significant elevation in the activities of CAT and GPx as well as the level of GSH, and the significant decline in lipid peroxidation. Optimized rapeseed oil with the highest micronutrient contents obtained by microwave pretreatment-cold pressing reduced the levels of TG, TC and LDL-C as well as IL-6 and CRP in plasma.
Conclusions
These results suggest that optimized rapeseed oils may contribute to prevent atherogenesis and make them very promising functional food in cardiovascular health promotion.
doi:10.1186/1476-511X-13-166
PMCID: PMC4232689  PMID: 25358951
Optimized rapeseed oils; Micronutrients; Atherosclerosis; Oxidant stress; Plasma lipids; Inflammation
21.  Extracts and compounds with anti-diabetic complications and anti-cancer activity from Castanea mollissina Blume (Chinese chestnut) 
Background
Castanea mollissima Blume (Chinese chestnut), as a food product is known for its various nutrients and functional values to the human health. The present study was carried out to analyze the anti-diabetic complications and anti-cancer activities of the bioactive compounds present in C. mollissima.
Methods
The kernels (CK), shells (CS) and involucres (CI) parts of C. Blume were extracted with 90% alcohol. The water suspension of these dried alcohol extracts were extracted using EtOAc and n-BuOH successively. The n-BuOH fraction of CI (CI-B) was isolated by silica gel column, Sephadex LH 20 column and preparative HPLC. The isolated compounds were identified by 1H-NMR, 13C-NMR, HMBC, HMQC and ESI-Q-TOF MS, All the fractions and compounds isolated were evaluated on human recombinant aldose reductase (HR-AR) assay, advanced glycation end products (AGEs) formation assay and human COLO 320 DM colon cancer cells inhibitory assay.
Results
CI-B was found to show a significant inhibitory effect in above biological screenings. Six flavonoids and three polyphenolic acids were obtained from CI-B. They were identified as kaempferol (1), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (2), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-galactopyranoside (3), kaempferol-3-O-[2''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (4), kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-glucopyranoside (5) and kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-galactopyranoside (6), casuariin (7), casuarinin (8) and castalagin (9). Compounds 2–9 were found to show higher activity than quercetin (positive control) in the AR assay. Compounds 3–6, 8, and 9 showed stronger inhibitory effects than amino guanidine (positive control) on AGEs production. Compounds 4–6, 7, and 8 showed much higher cytotoxic activity than 5-fluorouracil (positive control) against the human COLO 320 DM colon cancer cells.
Conclusions
Our results suggest that flavonoids and polyphenolic acids possesses anti-diabetes complications and anti-cancer properties, and they were presumed to be the bioactive components of Castanea mollissima Blume.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6882-14-422) contains supplementary material, which is available to authorized users.
doi:10.1186/1472-6882-14-422
PMCID: PMC4226895  PMID: 25351676
Anti-diabetic complications; Anti-cancer; Castanea mollissina Blume; Phenolic acids; Flavonoids
22.  Circulating tumor cells as early predictors of metastatic spread in breast cancer patients with limited metastatic dissemination 
Introduction
Traditional factors currently used for prognostic stratification do not always adequately predict treatment response and disease evolution in advanced breast cancer patients. Therefore, the use of blood-based markers, such as circulating tumor cells (CTCs), represents a promising complementary strategy for disease monitoring. In this retrospective study, we explored the role of CTC counts as predictors of disease evolution in breast cancer patients with limited metastatic dissemination.
Methods
A total of 492 advanced breast cancer patients who had a CTC count assessed by CellSearch prior to starting a new line of systemic therapy were eligible for this analysis. Using the threshold of 5 CTCs/7.5 ml of blood, pretreatment CTC counts were correlated in the overall population with metastatic site distribution, evaluated at baseline and at the time of treatment failure, using Fisher’s exact test. Time to visceral progression and time to the development of new metastatic lesions and sites were estimated in patients with nonvisceral metastases and with single-site metastatic disease, respectively, by the Kaplan-Meier method. Survival times were compared between groups according to pretreatment CTC count by logrank test.
Results
In the overall population, a pretreatment level ≥5 CTCs/7.5 ml was associated with an increased baseline number of metastatic sites compared with <5 CTCs/7.5 ml (P = 0.0077). At the time of treatment failure, patients with ≥5 CTCs/7.5 ml more frequently developed new metastatic lesions and sites compared with those with <5 CTCs/7.5 ml (development of new lesions: P = 0.0002; development of new sites: P = 0.0031). Among patients with disease originally confined to nonvisceral sites, ≥5 CTCs/7.5 ml was associated with remarkably shorter time to visceral metastases (P = 0.0021) and overall survival (P = 0.0006) compared with <5 CTCs/7.5 ml. In patients with single-site metastatic disease, ≥5 CTCs/7.5 ml was associated with a significant reduction of the time to development of new metastatic sites (P = 0.0051) and new lesions (P = 0.0002) and with worse overall survival (P = 0.0101).
Conclusion
Our results suggest that baseline CTC counts can be used as an early predictor of metastatic potential in breast cancer patients with limited metastatic dissemination.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0440-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0440-8
PMCID: PMC4303121  PMID: 25223629
23.  T-type Ca2+ channels regulate the exit of cardiac myocytes from the cell cycle after birth 
T-type Ca2+ channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth.
Methods
Cardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca2+ currents were measured.
Results
All myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger.
Conclusions
The studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth.
doi:10.1016/j.yjmcc.2013.05.016
PMCID: PMC3888788  PMID: 23743021
T-type Ca2+ channels; Cardiac myocytes; Cell cycle
24.  Effectiveness and Complications of Ultrasound Guided Fine Needle Aspiration for Primary Liver Cancer in a Chinese Population with Serum α-Fetoprotein Levels ≤200 ng/ml - A Study Based on 4,312 Patients 
PLoS ONE  2014;9(8):e101536.
Background
Hepatocellular carcinoma (HCC) can be diagnosed by noninvasive approaches with serum α-fetoprotein (AFP) levels >200 ng/ml and/or a radiological imaging study of tumor mass >2 cm in patients with chronic liver disease. Percutaneous fine needle aspiration (FNA) under ultrasound (US) guidance has a diagnostic specificity of 95% and is superior to radiological imaging studies.
Aim
The aim of this study is to elucidate the effectiveness and complications of fine needle aspiration in a Chinese population with primary liver cancer and AFP levels ≤200 ng/ml.
Materials and Methods
A retrospective study was conducted over a period of 28 years. This selection period included patients with a suspected diagnosis of primary liver cancer whose AFP levels were ≤200 ng/ml and who underwent US-FNA. This data was then analyzed with cytomorphological features correlating with medical history, radiological imaging, AFP, and follow-up information.
Results
Of the 1,929 cases with AFP ≤200 mg/ml, 1,756 underwent FNA. Of these, 1,590 cases were determined malignant and the remaining 166 were determined benign. Further, 1,478 malignant cases were diagnosed by FNA alone, and of these, 1,138 were diagnosed as PLC. The sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy of the diagnoses were 92.96%, 100%, 100%, 59.71%, and 93.62% respectively. There was no significant difference in the sensitivity, specificity, PPV and NPV between the subgroups with tumor size<2 cm and ≥2 cm. Major complications included implantation metastasis and hemorrhage.
Conclusion
Patients with PLC, especially those who present with an AFP ≤200 ng/ml, should undergo FNA. If negative results are obtained by FNA, it still could be HCC and repeated FNA procedure may be needed if highly suspicious of HCC on imaging study. The superiority of FNA in overall accuracy may outweigh its potential complications, such like hemorrhage and implantation metastasis.
doi:10.1371/journal.pone.0101536
PMCID: PMC4149350  PMID: 25170868
25.  Heterochronic bilateral ectopic pregnancy after ovulation induction*  
Ectopic pregnancy is identified with the widely-applied assisted reproductive technology (ART). Bilateral ectopic pregnancy is a rare form of ectopic pregnancy which is difficult to be diagnosed at the pre-operation stage. In this paper, we presented an unusual case of heterochronic bilateral ectopic pregnancy after stimulated intrauterine insemination (IUI), where there has been a delay of 22 d between the diagnoses of the two ectopic pregnancies. Literature was reviewed on the occurrence of bilateral ectopic pregnancy during the past four years in the MEDLINE database. We found 16 cases of bilateral ectopic pregnancy reported since 2008, and analyzed the characteristics of those cases of bilateral ectopic pregnancy. We emphasize that ovulation induction and other ARTs may increase the risk of bilateral ectopic pregnancy. Because of the difficulty in identification of bilateral ectopic pregnancy by ultrasonography, the clinician should be aware that the treatment of one ectopic pregnancy does not preclude the occurrence of a second ectopic pregnancy in the same patient and should pay attention to the intra-operation inspection of both side fallopian tubes in any ectopic pregnancy case.
doi:10.1631/jzus.B1400081
PMCID: PMC4129096  PMID: 25091994
Heterochronic bilateral ectopic pregnancy; Assisted reproductive technology; Intrauterine insemination; Ovulation induction

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