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1.  iTRAQ-based proteomic profiling of Vibrio parahaemolyticus under various culture conditions 
Proteome Science  2015;13:19.
Background
Vibrio parahaemolyticus is a common pathogen infecting humans and marine animals; this pathogen has become a major concern of marine food products and trade. In this study, V. parahaemolyticus isolated from sewage was exposed to different culture conditions and analyzed by isobaric tag for relative and absolute quantitation (iTRAQ) based reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Our goal is to gain further insights into the proteomics of V. parahaemolyticus, particularly differentially expressed proteins closely correlated with growth conditions and pathogenicity associated proteins.
Results
In this study, a total of 2,717 proteins including numerous membrane proteins were significantly identified, and 616 proteins displayed significant differential expression under different conditions. Of them, 12 proteins mainly participating in metabolism showed the most elastic expression differentiation between different culture conditions. Some membrane proteins such as type I secretion outer membrane protein, TolC, lipoprotein, efflux system proteins iron-regulated protein A and putaive Fe-regulated protein B, ferric siderophore receptor homolog and several V. parahaemolyticus virulence-associated proteins were differentially regulated under different conditions. Some differentially regulated proteins were analyzed and confirmed at gene expression level by quantitative real time polymerase chain reaction (qRT-PCR).
Conclusions
Proteomics analysis results revealed the characteristics of V. parahaemolyticus proteome expression, provided some promising biomarkers related with growth conditions, the results likely advance insights into the mechanism involved in the response of V. parahaemolyticus to different conditions. Some virulence-associated proteins were discovered to be differentially expressed under different conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12953-015-0075-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12953-015-0075-4
PMCID: PMC4518887
Vibrio parahaemolyticus; Quantitative proteomics; iTRAQ; Biomarker; Pathogenicity
2.  New Insight into Phase Formation of MxMg2Al4+xSi5−xO18:Eu2+ Solid Solution Phosphors and Its Luminescence Properties 
Scientific Reports  2015;5:12149.
Here we reported the phase formation of MxMg2Al4+xSi5−xO18:Eu2+ (M = K, Rb) solid solution phosphors, where M+ ions were introduced into the void channels of Mg2Al4Si5O18 via Al3+/Si4+ substitution to keep the charge balance. XRD results revealed that the as-prepared phosphors with different M+ contents were iso-structural with Mg2Al4Si5O18 phase. The combined analysis of the Rietveld refinement and high resolution transmission electron microscopy (HRTEM) results proved that M+ ions were surely introduced into the intrinsic channels in Mg2Al4Si5O18. The emission peaks of MxMg2Al4+xSi5−xO18:Eu2+ (M = K, Rb) phosphors with various x values performed a systematic red-shift tendency, which was ascribed to the elongation of [MgO6] octahedra. The temperature stable photoluminescence and internal quantum efficiency (QE) of MxMg2Al4+xSi5−xO18:Eu2+ (M = K, Rb) phosphors were enhanced owing to the filling of M+ in the void channels suggesting a new insight to design the solid solution phosphors with improved photoluminescence properties.
doi:10.1038/srep12149
PMCID: PMC4507260  PMID: 26190348
3.  Review of advanced catheter technologies in radiation oncology brachytherapy procedures 
The development of new catheter and applicator technologies in recent years has significantly improved treatment accuracy, efficiency, and outcomes in brachytherapy. In this paper, we review these advances, focusing on the performance of catheter imaging and reconstruction techniques in brachytherapy procedures using magnetic resonance images and electromagnetic tracking. The accuracy of catheter reconstruction, imaging artifacts, and other notable properties of plastic and titanium applicators in gynecologic treatments are reviewed. The accuracy, noise performance, and limitations of electromagnetic tracking for catheter reconstruction are discussed. Several newly developed applicators for accelerated partial breast irradiation and gynecologic treatments are also reviewed. New hypofractionated high dose rate treatment schemes in prostate cancer and accelerated partial breast irradiation are presented.
doi:10.2147/CMAR.S46042
PMCID: PMC4507789  PMID: 26203277
catheter technologies; catheter reconstruction; electromagnetic tracking; hypofractionated high dose rate treatment; accelerated partial breast irradiation
4.  MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing 
PLoS ONE  2015;10(7):e0131336.
Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights into future investigations aimed to explore the in-depth mechanisms of miRNAs and other small RNAs including piRNAs in penile carcinogenesis regulation and effective target-specific theragnosis.
doi:10.1371/journal.pone.0131336
PMCID: PMC4497725  PMID: 26158897
5.  Light Trapping Enhancement in a Thin Film with 2D Conformal Periodic Hexagonal Arrays 
Applying a periodic light trapping array is an effective method to improve the optical properties in thin-film solar cells. In this work, we experimentally and theoretically investigate the light trapping properties of two-dimensional periodic hexagonal arrays in the framework of a conformal amorphous silicon film. Compared with the planar reference, the double-sided conformal periodic structures with all feature periodicities of sub-wavelength (300 nm), mid-wavelength (640 nm), and infrared wavelength (2300 nm) show significant broadband absorption enhancements under wide angles. The films with an optimum periodicity of 300 nm exhibit outstanding antireflection and excellent trade-off between light scattering performance and parasitic absorption loss. The average absorption of the optimum structure with a thickness of 160 nm is 64.8 %, which is much larger than the planar counterpart of 38.5 %. The methodology applied in this work can be generalized to rational design of other types of high-performance thin-film photovoltaic devices based on a broad range of materials.
Electronic supplementary material
The online version of this article (doi:10.1186/s11671-015-0988-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s11671-015-0988-y
PMCID: PMC4495099  PMID: 26153124
Light trapping; Photonic crystals; Nanospheres; Plasmonics; Rear reflectors
6.  Development of optical probes for in vivo imaging of polarized macrophages during foreign body reactions 
Acta biomaterialia  2014;10(7):2945-2955.
Plasticity of macrophages (MΦ) phenotypes exist in a spectrum from classically activated (M1) cells, to alternatively activated (M2) cells, contributing to both the normal healing of tissues and the pathogenesis of implant failure. Here, folate- and mannose-based optical probes were fabricated to simultaneously determine the degree of MΦ polarization. In vitro tests show the ability of these probes to specifically target M1 and M2 cells. In an in vivo murine model, they were able to distinguish between M1-dominated inflammatory response to infection and M2-dominated regenerative response to particle implants. Finally, the probes were used to assess the inflammatory/ regenerative property of biomaterial implants. Our results show that these probes can be used to monitor and quantify the dynamic processes of MΦ polarization and their role in cellular responses in real time.
doi:10.1016/j.actbio.2014.04.001
PMCID: PMC4041819  PMID: 24726956
Macrophage polarization; In vivo imaging; Optical probe; Inflammation; Biomaterials
7.  IMMUNE CHECKPOINT BLOCKADE FOR GLIOBLASTOMA: PRECLINICAL ACTIVITY OF SINGLE AGENT AND COMBINATORIAL THERAPY 
Neuro-Oncology  2014;16(Suppl 3):iii11-iii12.
BACKGROUND: Outcome for glioblastoma (GBM) remains dismal and innovative treatment strategies are desperately needed. Growing data support the contribution of local and systemic immune checkpoint molecules to regulating immunosuppression by GBM tumors. METHODS: Luciferized GL261 cells (GL261-Luc) were stereotactically implanted intracranially in albino C57BL/6 mice. Cohorts of mice with growing tumors (increasing bioluminescence) were treated with murine monoclonal antibodies (MAb) against PD-L1, PD-L2, CTLA-4 and combinations thereof every 3 days X 8 doses beginning 6 days following tumor implantation. Treated mice and appropriate controls were followed for overall survival and subsets of mice underwent magnetic resonance imaging (MRI) as well as analysis of tumor infiltrating immune cells, and evaluation of systemic immune cells and immunocytokines. Tumor re-challenge experiments were performed among long-term surviving treated mice. RESULTS: Improved survival was noted among mice treated with immune checkpoint blockade compared to appropriate controls. The most robust survival benefit was noted among mice treated with combinatorial therapy. MRI imaging among long-term surviving animals demonstrated clear evidence of initial tumor growth followed by regression and eradication of tumors. Long-term surviving mice appeared fully intact neurologically and exhibited no evidence of tumor growth following re-challenge of GL261-Luc cells injected subcutaneously. Characterization of tumor infiltrating immune cells as well as systemic immune cells and immunocytokines is ongoing. CONCLUSIONS: Immune checkpoint blockade provides significant survival benefit and appears safe in this immunocompetent, orthotopic GBM model. Re-challenge experiments demonstrate evidence of long-term immunologic memory and further elucidation of underlying mechanisms of immune-mediated anti-tumor activity is ongoing. These data strongly support the evaluation of immune checkpoint inhibitors among patients with GBM. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.
doi:10.1093/neuonc/nou206.40
PMCID: PMC4144507
8.  Activation of the MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response to oxidative stress induced by a novel copper complex in HeLa cells 
Autophagy  2014;10(7):1285-1300.
Transition metal copper (Cu) can exist in oxidized or reduced states in cells, leading to cytotoxicity in cancer cells through oxidative stress. Recently, copper complexes are emerging as a new class of anticancer compounds. Here, we report that a novel anticancer copper complex (HYF127c/Cu) induces oxidative stress-dependent cell death in cancer cells. Further, transcriptional analysis revealed that oxidative stress elicits broad transcriptional changes of genes, in which autophagy-related genes are significantly changed in HYF127c/Cu-treated cells. Consistently, autophagy was induced in HYF127c/Cu-treated cells and inhibitors of autophagy promoted cell death induced by HYF127c/Cu. Further analysis identified that the MAPK11/12/13/14 (formerly known as p38 MAPK) pathway was also activated in HYF127c/Cu-treated cells. Meanwhile, the MAPK11/12/13/14 inhibitor SB203580 downregulated autophagy by inhibiting the transcription of the autophagy genes MAP1LC3B, BAG3, and HSPA1A, and promoted HYF127c/Cu-induced cell death. These data suggest that copper-induced oxidative stress will induce protective autophagy through transcriptional regulation of autophagy genes by activation of the MAPK11/12/13/14 pathway in HeLa cells.
doi:10.4161/auto.28789
PMCID: PMC4203553  PMID: 24905917
copper; HYF127c/Cu; autophagy; MAPK11; MAPK12; MAPK13; MAPK14; p38 mitogen-activated protein kinase; transcription; oxidative stress
9.  Bevacizumab plus Ipilimumab in Patients with Metastatic Melanoma 
Cancer immunology research  2014;2(7):632-642.
Ipilimumab improves survival in advanced melanoma and can induce immune-mediated tumor vasculopathy. Besides promoting angiogenesis, vascular endothelial growth factor (VEGF) suppresses dendritic cell maturation and modulates lymphocyte endothelial trafficking. This study investigated the combination of CTLA-4 blockade with ipilimumab and VEGF inhibition with bevacizumab. Patients with metastatic melanoma were treated in four dosing cohorts of ipilimumab (3 or 10 mg/kg) four doses at 3-week intervals and then every 12 weeks, and bevacizumab (7.5 or 15 mg/kg) every 3 weeks. Forty-six patients were treated. Inflammatory events included giant cell arteritis (1), hepatitis (2), and uveitis (2). On-treatment tumor biopsies revealed activated vessel endothelium with extensive CD8+ and macrophage cell infiltration. Peripheral blood analyses demonstrated increases in CCR7+/−/CD45RO+ cells and anti-galectin antibodies. Best overall response included 8 partial responses, 22 stable disease, and a disease-control rate (DCR) of 67.4%. Median survival was 25.1 months. Bevacizumab influences changes in tumor vasculature and immune responses with ipilimumab administration. The combination of bevacizumab and ipilimumab can be safely administered and reveals VEGF-A blockade influences on inflammation, lymphocyte trafficking, and immune regulation. This provides a basis for further investigating the dual roles of angiogenic factors in blood vessel formation and immune regulation as well as future combinations of anti-angiogenesis agents and immune checkpoint blockade.
doi:10.1158/2326-6066.CIR-14-0053
PMCID: PMC4306338  PMID: 24838938
10.  Meta-Analysis Comparing Zero-Profile Spacer and Anterior Plate in Anterior Cervical Fusion 
PLoS ONE  2015;10(6):e0130223.
Background
Anterior plate fusion is an effective procedure for the treatment of cervical spinal diseases but is accompanied by a high incidence of postoperative dysphagia. A zero profile (Zero-P) spacer is increasingly being used to reduce postoperative dysphagia and other potential complications associated with surgical intervention. Studies comparing the Zero-P spacer and anterior plate have reported conflicting results.
Methodology
A meta-analysis was conducted to compare the safety, efficacy, radiological outcomes and complications associated with the use of a Zero-P spacer versus an anterior plate in anterior cervical spine fusion for the treatment of cervical spinal disease. We comprehensively searched PubMed, Embase, the Cochrane Library and other databases and performed a meta-analysis of all randomized controlled trials (RCTs) and prospective or retrospective comparative studies assessing the two techniques.
Results
Ten studies enrolling 719 cervical spondylosis patients were included. The pooled data showed significant differences in the operation time [SMD = –0.58 (95% CI = −0.77 to 0.40, p < 0.01)] and blood loss [SMD = −0.40, 95% CI (−0.59 to –0.21), p < 0.01] between the two groups. Compared to the anterior plate group, the Zero-P group exhibited a significantly improved JOA score and reduced NDI and VAS. However, anterior plate fusion had greater postoperative segmental and cervical Cobb’s angles than the Zero-P group at the last follow-up. The fusion rate in the two groups was similar. More importantly, the Zero-P group had a lower incidence of earlier and later postoperative dysphagia.
Conclusions
Compared to anterior plate fusion, Zero-P is a safer and effective procedure, with a similar fusion rate and lower incidence of earlier and later postoperative dysphagia. However, the results of this meta-analysis should be accepted with caution due to the limitations of the study. Further evaluation and large-sample RCTs are required to confirm and update the results of this study.
doi:10.1371/journal.pone.0130223
PMCID: PMC4466022  PMID: 26067917
11.  Effect of Cytochrome b5 Content on the Activity of Polymorphic CYP1A2, 2B6, and 2E1 in Human Liver Microsomes 
PLoS ONE  2015;10(6):e0128547.
Human cytochrome b5 (Cyt b5) plays important roles in cytochrome P450 (CYP)-mediated drug metabolism. However, the expression level of Cyt b5 in normal human liver remains largely unknown. The effect of Cyt b5 on overall CYP activity in human liver microsomes (HLM) has rarely been reported and the relationship between Cyt b5 and the activity of polymorphic CYP has not been systematically investigated. In this study, we found that the median value of Cyt b5 protein was 270.01 pmol/mg from 123 HLM samples, and 12- and 19-fold individual variation was observed in Cyt b5 mRNA and protein levels, respectively. Gender and smoking clearly influenced Cyt b5 content. In addition, we found that Cyt b5 protein levels significantly correlated with the overall activity of CYP1A2, 2B6, and 2E1 in HLM. However, when the CYP activities were sorted by single nucleotide polymorphisms (SNP), the effect of Cyt b5 protein on the kinetic parameters varied greatly. There were significant correlations between Cyt b5 content and Vmax and CLint of CYP1A2 wild-types (3860GG, 2159GG, and 5347CC) as well as homozygous mutants (163AA and 3113GG). In contrast to Vmax and CLint, the Km of CYP2B6 516GG and 785AA genotypes was inversely associated with Cyt b5 content. Correlations between Cyt b5 content and Vmax and CLint of CYP2E1 -1293GG, -1293GC, 7632TT, 7632TA, -333TT, and -352AA genotypes were also observed. In conclusion, Cyt b5 expression levels varied considerably in the Chinese cohort from this study. Cyt b5 had significant impact on the overall activity of CYP1A2, 2B6, and 2E1 in HLM and the effects of Cyt b5 protein on polymorphic CYP1A2, 2B6, and 2E1 activity were SNP-dependent. These findings suggest that Cyt b5 plays an important role in CYP-mediated activities in HLM and may possibly be a contributing factor for the individual variation observed in CYP enzyme activities.
doi:10.1371/journal.pone.0128547
PMCID: PMC4457846  PMID: 26046844
12.  Effects of transforming growth factor β-1 infected human bone marrow mesenchymal stem cells on high- and low-metastatic potential hepatocellular carcinoma 
Background
This study investigates the effects of human bone marrow-derived mesenchymal stem cell (hMSC) on migration and proliferation ability of hepatocellular carcinoma (HCC) with high- and low-metastatic potential.
Methods
The hMSC and transforming growth factor-β1 (TGFβ-1) gene infected hMSC were co-cultured with hepatoma cells. The ability of cells migration was assessed by Transwell assay. The ability of cells proliferation was detected using CCK-8 assay. The mice were engrafted with hMSC and TGFβ-1 gene infected hMSC, respectively, after hepatoma cells inoculation 15 days, twice a week for 6 weeks successively. The tumor inhibition rate was calculated. TGFβ-1, osteopontin (OPN), and programmed cell death protein 4 (PDCD4) genes expression of hepatoma cells were detected by quantitative real-time polymerase chain reaction (qPCR) before and after co-cultured experiments.
Results
TGFβ-1 infected hMSC or hMSC co-culture with hepatoma cells groups can significantly promote hepatoma cells proliferation (P < 0.05). The migration numbers of hepatoma cells with TGFβ-1 infected hMSC co-culture groups were significantly reduced compared with the other two groups (P < 0.05). The tumors weight inhibition rates of MHCC97-H and MHCC97-L animal models were the highest in the third week by hMSC engraftment. But the highest tumor inhibition rate of MHCC97-H animal models was observed in the fourth week and MHCC97-L animal models in the fifth week after TGFβ-1 infected hMSC engraftment. OPN gene relative quantitative expression of hepatoma cells was significantly down-regulated after co-cultured with hMSC and TGFβ-1 gene infected hMSC groups (P < 0.05). TGFβ-1 gene relative quantitative expression of MHCC97-H and MHCC97-L cells was significantly up-regulated after co-cultured with TGFβ-1 gene infected hMSC groups (P < 0.05). PDCD4 expression had no statistical differences among groups.
Conclusions
hMSC and TGFβ-1 gene infected hMSC can promote hepatoma cells proliferation and inhibit hepatoma cells migration. hMSC and TGFβ-1 gene infected hMSC exhibit anti-tumor activity in a time-dependent manner. TGFβ-1 cytokine may be the main factor in HCC proliferation. OPN makes a significant contribution to the changes of hepatoma cells metastasis.
doi:10.1186/s40001-015-0144-2
PMCID: PMC4464870  PMID: 26003220
hMSC; Hepatoma cells; TGFβ-1; Genetically modified; OPN; PDCD4
13.  miR-19b downregulates intestinal SOCS3 to reduce intestinal inflammation in Crohn’s disease 
Scientific Reports  2015;5:10397.
Although aberrant microRNA (miRNA) expression has frequently been observed in inflammatory bowel disease (IBD), its biological functions and targets remain largely unknown. Present study found that miR-19b was significantly downregulated in active Crohn’s disease (CD). Using bioinformatics analysis, suppressor of cytokine signalling 3 (SOCS3), a physiological regulator of innate and adaptive immunity that controls several immuno-inflammatory diseases, was predicted to be a potential target of miR-19b. An inverse correlation between miR-19b and SOCS3 protein levels, but not mRNA, was identified in active-CD intestinal tissue samples. By overexpressing or knocking down miR-19b in Caco2 cells and HT29 cells, it was experimentally validated that miR-19b is a direct regulator of SOCS3. Using a luciferase reporter assay, it was confirmed that miR-19b directly recognizes the 3’-untranslated region (3’-UTR) of SOCS3. Furthermore, overexpression of miR-19b decreased SOCS3 expression, leading to increased production of macrophage-inflammatory protein-3α (MIP-3α) in Caco2 cells. In contrast, knockdown of miR-19b increased SOCS3 and decreased MIP-3α. Finally, intracolonically delivered miR-19b decreased the severity of colitis induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS). Taken together, our findings suggest that miR-19b suppresses the inflammatory response by inhibiting SOCS3 to modulate chemokine production in intestinal epithelial cells (IECs) and thereby prevents the pathogenesis of CD.
doi:10.1038/srep10397
PMCID: PMC4441154  PMID: 25997679
14.  Manipulation of the Xanthophyll Cycle Increases Plant Susceptibility to Sclerotinia sclerotiorum 
PLoS Pathogens  2015;11(5):e1004878.
The xanthophyll cycle is involved in dissipating excess light energy to protect the photosynthetic apparatus in a process commonly assessed from non-photochemical quenching (NPQ) of chlorophyll fluorescence. Here, it is shown that the xanthophyll cycle is modulated by the necrotrophic pathogen Sclerotinia sclerotiorum at the early stage of infection. Incubation of Sclerotinia led to a localized increase in NPQ even at low light intensity. Further studies showed that this abnormal change in NPQ was closely correlated with a decreased pH caused by Sclerotinia-secreted oxalate, which might decrease the ATP synthase activity and lead to a deepening of thylakoid lumen acidification under continuous illumination. Furthermore, suppression (with dithiothreitol) or a defect (in the npq1-2 mutant) of violaxanthin de-epoxidase (VDE) abolished the Sclerotinia-induced NPQ increase. HPLC analysis showed that the Sclerotinia-inoculated tissue accumulated substantial quantities of zeaxanthin at the expense of violaxanthin, with a corresponding decrease in neoxanthin content. Immunoassays revealed that the decrease in these xanthophyll precursors reduced de novo abscisic acid (ABA) biosynthesis and apparently weakened tissue defense responses, including ROS induction and callose deposition, resulting in enhanced plant susceptibility to Sclerotinia. We thus propose that Sclerotinia antagonizes ABA biosynthesis to suppress host defense by manipulating the xanthophyll cycle in early pathogenesis. These findings provide a model of how photoprotective metabolites integrate into the defense responses, and expand the current knowledge of early plant-Sclerotinia interactions at infection sites.
Author Summary
In recent years, the role of the chloroplast in the defense against microbes has been intensively investigated and is of high interest to both plant-microbe interaction and photosynthesis research. The xanthophyll cycle is well known to be involved in dissipating excess light energy to protect the photosynthetic apparatus in a process commonly assessed via non-photochemical quenching (NPQ) of chlorophyll fluorescence. Recent studies show that NPQ can be positively or negatively affected by pathogen attack. However, knowledge about the regulatory processes by which pathogens affect NPQ, as well as their impact on plant defense responses, is incomplete. This work characterized the impact of infection of Arabidopsis leaves by the necrotrophic pathogen Sclerotinia sclerotiorum on the xanthophyll cycle. Our research revealed for the first time that Sclerotinia uses a novel strategy involving manipulation of the xanthophyll cycle to weaken host defense responses and increase its successful colonization of host cells. These findings contribute to understanding the plant-Sclerotinia interactions in early pathogenesis, which will provide new sights into the development of strategies to increase Sclerotinia resistance in plants for practical applications.
doi:10.1371/journal.ppat.1004878
PMCID: PMC4439079  PMID: 25993128
15.  Chemerin C9 peptide induces receptor internalization through a clathrin-independent pathway 
Acta pharmacologica Sinica  2014;35(5):653-663.
Aim
The chemerin receptor CMKLR1 is one type of G protein-coupled receptors abundant in monocyte-derived dendritic cells and macrophages, which plays a key role in the entry of a subset of immunodeficiency viruses including HIV/SIV into lymphocytes and macrophages. The aim of this work was to investigate how CMKLR1 was internalized and whether its internalization affected cell signaling in vitro.
Methods
Rat basophilic leukemia RBL-2H3 cells, HEK 293 cells and HeLa cells were used. CMKLR1 internalization was visualized by confocal microscopy imaging or using a FACScan flow cytometer. Six potential phosphorylation sites (Ser337, Ser343, Thr352, Ser344, Ser347 and Ser350) in CMKLR1were substituted with alanine using site-directed mutagenesis. Heterologous expression of wild type and mutant CMKLR1 allowed for functional characterization of endocytosis, Ca2+ flux and extracellular signal-regulated kinase (ERK) phosphorylation.
Results
Chemerin and the chemerin-derived nonapeptide (C9) induced dose-dependent loss of cell surface CMKLR1-GFP fusion protein and increased its intracellular accumulation in HEK 293 cells and RBL-2H3 cells stably expressing CMKLR1. Up to 90% of CMKLR1 was internalized after treatment with C9 (1 µmol/L). By using different agents, it was demonstrated that clathrin-independent mechanism was involved in CMKLR1 internalization. Mutations in Ser343 for G protein-coupled receptor kinase (GRK) phosphorylation and Ser347 for PKC phosphorylation abrogated CMKLR1 internalization. Loss of CMKLR1 internalization partially enhanced the receptor signaling, as shown by increased Ca2+ flux and a shorter latency to peak level of ERK phosphorylation.
Conclusion
CMKLR1 internalization occurs in a clathrin-independent manner, which negatively regulated the receptor-mediated Ca2+ flux and ERK phosphorylation.
doi:10.1038/aps.2013.198
PMCID: PMC4075970  PMID: 24658352
chemerin; CMKLR1; receptor trafficking; endocytosis; calcium flux; ERK phosphorylation
16.  Epithelioid inflammatory myofibroblastic sarcoma with recurrence after extensive resection: significant clinicopathologic characteristics of a rare aggressive soft tissue neoplasm 
A case of epithelioid inflammatory myofibroblastic scarcoma (EIMS) developing in an 8-year-old boy who presented with a bulky intra-abdominal occupying lesion with recurrence undergoing a radical resection was reported. Histologically, the tumor cells arranged in cords, strands or sheets of round-to-epithelioid cells with a vesicular nuclear chromatin pattern, prominent nucleoli and weakly eosinophic or basophilic cytoplasm embedded in the abundant myxoid stroma with lymphocytes infiltration. They were positive for ALK, Desmin, SMA, CD30, but negative for AE1/AE3, LCA, CD2, CD3, CD5, CD7, S-100, CD34, CD31, EMA, MyoD1, and myogenin. An elevated proliferation index was demonstrated by Ki-67 comparing the first and the second lesion. Fluorescence in situ (FISH) showed the presence of chromosomal translocation involving ALK. This case show EIMS is a rare variant of inflammatory myofibroblatic tumor with aggressive biological behavior and unfavourable prognosis. To be familiar with its significant clinicopathologic characteristics could prompt us to take it into consideration when facing the relevant dieases.
PMCID: PMC4503172  PMID: 26191301
Epithelioid inflammatory myofibroblastic scarcoma; soft tissue neoplasm; ALK; FISH
17.  Primary adrenal microcystic/reticular schwannoma: clinicopathological and immunohistochemical studies of an extremely rare case 
A case of primary adrenal microcystic/reticular schwannoma affecting the right adrenal gland in a 31-year-old female was reported. Histologically, the tumor significantly consisted of strikingly anastomosing strands of spindle cells imparting a microcystic and reticular pattern with a focal transition to epithelioid nests. Immunohistochemically, S-100 and CD56 protein showed a uniform and strong positivity, GFAP and EMA weakly and focally expressed, but AE1/AE3, CK5/6, CD31, CD34, calrentin, D2-40, WT1, CgA, Melan-A and Neu-N were negative. The patient live through a calm period for 4 months after the whole right paranephros was removed with no evidence of relapse. This interesting case showed primary adrenal microcystic/reticular schwannoma is characteristic of a distinctive and infrequent morphology compared to another subtype of schwannoma involved in the paranephros. To be familiar with its significant histological features would prompt us to take it into consideration when facing the mimickers.
PMCID: PMC4503173  PMID: 26191302
Microcystic/reticular schwannoma; adrenal gland; pathology; immunohistochemistry; differential diagnosis
18.  Therapy-related acute myeloid leukemia with eosinophilia, basophilia, t(4;14)(q12;q24) and PDGFRA rearrangement: a case report and review of the literature 
The myeloid and lymphoid neoplasms with eosinophilia and PDGFRA gene rearrangements usually show a good response to Imatinib and are typically associated with a normal karyotype, occasionally exhibiting a secondary chromosomal abnormality associated with clonal evolution. Five variant translocations involving PDGFRA have been reported. Here, we report a rare case of therapy-related acute myeloid leukemia with PDGFRA rearrangement after chemotherapy for prior B lymphoblastic leukemia (B-ALL). The patient had a history of BCR-ABL negative, hypodiploid B-ALL in complete remission after chemotherapy. However, 15 months later the patient developed acute myeloid leukemia with rapidly increasing eosinophilia, basophilia and a complex karyotype that included a novel t(4;14)(q12;q24). FIP1L1 was not associated with the PDGFRA rearrangement. The patient had a very aggressive clinical course, and died from the disease shortly after diagnosis. This is the first case of a primary therapy-related myeloid neoplasm with secondary PDGFRA rearrangement. The t(4:14)(q12;q24) is joining the growing list of the variant translocations involving PDGFRA.
PMCID: PMC4503174  PMID: 26191303
Acute myeloid leukemia (AML); PDGFRA; eosinophilia; basophilia; therapy-related myeloid neoplasm
19.  Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber 
BMC Biotechnology  2015;15:28.
Background
Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers.
Results
A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6–12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern.
Conclusion
This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-015-0143-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12896-015-0143-y
PMCID: PMC4414359  PMID: 25926043
Amylopectin branching; GBSS silencing; SBEII overexpression; Starch gelatinisation; Starch properties; Solanum tuberosum
20.  The Role of Toll-Like Receptor 9 in Chronic Stress-Induced Apoptosis in Macrophage 
PLoS ONE  2015;10(4):e0123447.
Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.
doi:10.1371/journal.pone.0123447
PMCID: PMC4401452  PMID: 25885582
21.  Preparation and Representation of Recombinant Mn-Ferritin Flower-Like Spherical Aggregates from Marine Invertebrates 
PLoS ONE  2015;10(4):e0119427.
Ferritin has important functions in the transition and storage of toxic metal ions, but its regulation and function in many invertebrate species are still largely unknown. In our previous work, the cDNA sequence of Sinonovacula constricta, Apostichopus japonicas and Acaudina leucoprocta were constructed and efficiently expressed in E. Coli BL21 under IPTG induction. In this follow-up study, the recombinant ferritins were exposed to heavy metal manganese. The manganese concentration levels in three recombinant ferritins were greater than horse spleen ferritin (HSF). Compared with HSF, the amount of manganese enrichment in the three recombinant ferritins was 1.75-fold, 3.25-fold and 2.42-fold increases in ScFER, AjFER, and AlFER, respectively. After phosphate stimulation, the concentration of manganese increased and was higher than the ordinary dialysis control groups. The ScFER was four times its baseline value. The AjFER and AlFER were 1.4- and 8-fold higher, respectively. The AlFER sample stimulated by phosphate was 22-fold that of HSF. The morphologies of the resulting Mn-Ferritin from different marine invertebrates were characterized with scanning electron microscopy. Surface morphologies were lamella flower-like and are consistent with changes in surface morphologies of the standard Mn-HSF. Invertebrate recombinant ferritin and HSF both can uptake manganese. We found that the structure of A. leucoproctarecombinant Mn-Ferritin aggregate changed over time. The surface formed lamella flower-like aggregate, but gradually merged to create a relatively uniform plate-like phase of aggregate spherically and fused without clear boundaries.
doi:10.1371/journal.pone.0119427
PMCID: PMC4399908  PMID: 25879665
22.  Characteristics of Human Amniotic Fluid Mesenchymal Stem Cells and Their Tropism to Human Ovarian Cancer 
PLoS ONE  2015;10(4):e0123350.
The mesenchymal stem cells (MSCs) derived from amniotic fluid (AF) have become an attractive stem cells source for cell-based therapy because they can be harvested at low cost and avoid ethical disputes. In human research, stem cells derived from AF gradually became a hot research direction for disease treatment, specifically for their plasticity, their reduced immunogenicity and their tumor tropism regardless of the tumor size, location and source. Our work aimed to obtain and characterize human amniotic fluid mesenchymal stem cells (AFMSCs) and detect their ovarian cancer tropsim in nude mice model. Ten milliliters of twenty independent amniotic fluid samples were collected from 16-20 week pregnant women who underwent amniocentesis for fetal genetic determination in routine prenatal diagnosis in the first affiliated hospital of Harbin medical university. We successfully isolated the AFMSCs from thirteen of twenty amniotic fluid samples. AFMSCs presented a fibroblastic-like morphology during the culture. Flow cytometry analyses showed that the cells were positive for specific stem cell markers CD73,CD90, CD105, CD166 and HLA-ABC (MHC class I), but negative for CD 45,CD40, CD34, CD14 and HLA-DR (MHC class II). RT-PCR results showed that the AFMSCs expressed stem cell marker OCT4. AFMSCs could differentiate into bone cells, fat cells and chondrocytes under certain conditions. AFMSCs had the high motility to migrate to ovarian cancer site but didn’t have the tumorigenicity. This study enhances the possibility of AFMSCs as drug carrier in human cell-based therapy. Meanwhile, the research emphasis in the future can also put in targeting therapy of ovarian cancer.
doi:10.1371/journal.pone.0123350
PMCID: PMC4400015  PMID: 25880317
23.  Gastric-jejunum pouch side-to-end anastomosis: a novel and safe operation of gastrojejunostomy for preventing reflux gastritis 
Purpose: This study aims to introduce a simple operation method of gastrojejunostomy for preventing reflux esophagitis --gastric-jejunum pouch side-to-end anastomosis. Methods: Based on Billroth II anastomosis (Billroth II) method, we designed a new technique of anastomosis between stomach wall and jejunal pouch. The technique was named gastric-jejunum pouch side-end anastomosis. We retrospectively analyzed the clinical data which was collected from March 2012 to February 2014. Among all the recruited 66 patients, 51 gastric cancer patients and 7 pyloric obstruction patients were implemented with hand-assisted laparoscopic distal gastrectomy plus D2 lymph node dissection. The remaining 8 patients were malignant pyloric obstruction; they were treated with small exploratory incision on the upper abdomen and distal gastric partial transection. All the patients were treated with gastric-jejunum pouch side-to-end anastomosis. Results: The surgical incision was small, the operative time was short, their bleeding volume was little, the recovery time of the bowel function and hospitalization time was relatively short. Postoperatively, there was neither mortality and gastroparesis, nor anastomotic leakage and jejunal pouch leakage. Minor postoperative complications were detected followed up for 12 months, no acid reflux and reflux esophagitis symptoms was reported. Conclusions: Gastric -jejunum pouch side-to-end anastomosis is a simple operation technique with highly surgical safety.
PMCID: PMC4484030  PMID: 26131135
Gastrojejunostomy; jejunal pouch; reflux gastritis
24.  Synthesis and Anticancer Activity of 4β-Triazole-podophyllotoxin Glycosides 
A series of novel 4β-triazole-podophyllotoxin glycosides were synthesized by utilizing the Click reaction. Evaluation of cytotoxicity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using MTT assay shows that most of these compounds show weak cytotoxicity. It was observed that compound 16 shows the highest activity with IC50 values ranging from 2.85 to 7.28 μM, which is more potent than the control drugs etoposide and cisplatin against four of five cancer cell lines tested. Compound 16 is characterized with an α-d-galactosyl residue directly linked to the triazole ring and a 4′-OH group on the E ring of the podophyllotoxin scaffold. HPLC investigation of representative compound indicates that incorporation of a sugar moiety seems to improve the chemical stability of the podophyllotoxin scaffold.
doi:10.1007/s13659-015-0057-3
PMCID: PMC4402586  PMID: 25869591
Podophyllotoxin; 4β-Triazole-podophyllotoxin; Glycosides; Click reaction; Anticancer; Synthesis
25.  Synthesis and Anticancer Activity of 4β-Triazole-podophyllotoxin Glycosides 
A series of novel 4β-triazole-podophyllotoxin glycosides were synthesized by utilizing the Click reaction. Evaluation of cytotoxicity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using MTT assay shows that most of these compounds show weak cytotoxicity. It was observed that compound 16 shows the highest activity with IC50 values ranging from 2.85 to 7.28 μM, which is more potent than the control drugs etoposide and cisplatin against four of five cancer cell lines tested. Compound 16 is characterized with an α-d-galactosyl residue directly linked to the triazole ring and a 4′-OH group on the E ring of the podophyllotoxin scaffold. HPLC investigation of representative compound indicates that incorporation of a sugar moiety seems to improve the chemical stability of the podophyllotoxin scaffold.
doi:10.1007/s13659-015-0057-3
PMCID: PMC4402586  PMID: 25869591
Podophyllotoxin; 4β-Triazole-podophyllotoxin; Glycosides; Click reaction; Anticancer; Synthesis

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