Micrococcus luteus strain NDB3Y10, which utilizes 1,2-dichloroethane as a carbon source, was isolated from a bore well that produces coal seam gas. The draft genome size of the strain was 2.49 Mb with a G+C content of 72.97%. Genes involved in the metabolism of halogenated substrates, including halogenated hydrocarbons, were identified. Micrococcus, a member of the phylum Actinobacteria, currently consists of 17 taxonomically valid species (http://www.bacterio.net). Members of the genus Micrococcus are found in a wide range of diverse environments, including the type species Micrococcus luteus, a known commensal that is found in humans and can contribute to food spoilage (Micrococcus sp. strain NDB3Y10 was isolated from a water sample collected from a newly drilled (65 h) gas bore well located in the upper Surat Basin, Roma, Queensland, Australia. Strain NDB3Y10 grew optimally under aerobic conditions at 37°C and pH 8 in basal PL medium  showed that the strain metabolized several different carboxylic acids, saccharides, pyruvic acid methyl ester, and succinic acid mono-methyl-ester. We report here the first genome sequence of the 1,2-DCE-utilizing Micrococcus sp. strain NDB3Y10, isolated from a water sample collected from a newly drilled bore well producing coal seam gas.The genus L medium  supplemeDe novo assembly was performed using SPAdes version 3.5.0, which produced a high-quality draft genome consisting of 84 contigs (≥500 bp in length) with an N50 of 64,137 bp and an average genome coverage of 247×. The contigs had a G+C content of 72.97% and a combined length of 2,497,512 bp, which is estimated to cover 99.85% of the total genome length.High-molecular-weight DNA was extracted from a late log phase culture of strain NDB3Y10 using a modification of Marmur’s method . RAST (Micrococcus sp. strain NDB3Y10 in which potential hydrolytic dehalogenases have been identified.Annotation of the draft genome of strain NDB3Y10 using Prokka version 1.12b  revealedLQAC00000000. The version described in this paper is the first version, LQAC00000000.1.This whole-genome shotgun project has been deposited in GenBank under the accession number


Tnfα, Il1β, Ifnγ, Il6, Il17a, Il23a, Il4, and Il12a). The protective effect of anti-S100a9 antibody treatment was also observed in azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) mouse model. The inflammatory response, tumor cell proliferation, and immune cells infiltration in the colon tissues were suppressed by anti-S100a9 antibody. Gene expression profiling showed that key pathways known to be involved in CAC development, such as Wnt signaling pathway, PI3K–Akt signaling pathway, cytokine–cytokine receptor interaction, and ECM–receptor interaction pathway, were suppressed after treatment with anti-S100a9 antibody in CAC mice. In view of the protective effect of neutralizing anti-S100a9 antibody against DSS-induced colitis and AOM/DSS-induced CAC in mouse model, this study suggests that anti-S100a9 antibody may provide a novel therapeutic approach to treat ulcerative colitis and may decrease the risk for developing CAC.The association between chronic inflammation and cancer has long been recognized. The inflammatory bowel disease ulcerative colitis frequently progresses to colon cancer; however, the underlying mechanism is still unclear. S100a9 has been emerged as an important pro-inflammatory mediator in acute and chronic inflammation, and the aberrant expression of S100a9 also contributes to tumorigenic processes such as cell proliferation, angiogenesis, metastasis, and immune evasion. We previously revealed that S100a8 and S100a9 are highly activated and play an important role in the process of colitis-associated carcinogenesis, which suggests an attractive therapeutic target for ulcerative colitis and related colon cancer. Here, we report that administration of a neutralizing anti-S100a9 antibody significantly ameliorated dextran sulfate sodium (DSS)-induced colitis and accompanied by diminished cellular infiltrate of innate immunity cells  and production of pro-inflammatory cytokines ( Ulcerative colitis and Crohn’s disease are collectively referred to as inflammatory bowel disease (IBD). Ulcerative colitis is characterized by pathological mucosal damage and ulceration. IBD ranks as a high-risk condition for the development of colorectal cancer (CRC), with a standardized incidence ratio of 2.4 95% CI 0.6–6.0) in patients with extensive or pan ulcerative colitis , 2. More–6.0 in pThe combination of azoxymethane (AOM), a colonic genotoxic carcinogen, and dextran sulfate sodium (DSS), an inducer of colitis, has been proven to be a powerful tool for investigating the pathogenesis and chemoprevention of CAC , 11. In 2+ binding protein S100a9 belonging to the S100 family, released in abundance of non-resolving inflammation, such as IBD, rheumatoid arthritis, psoriasis, and solid tumors by neutrophils, activated monocytes, macrophages, dendritic cells (DCs) and myeloid-derived suppressor cells , AOM/DSS + IgG Ab , and AOM/DSS + anti-S100a9 Ab . Five to six mice per cage. Mice were intraperitoneal injected with a single dose of 10 mg/kg AOM  on day 1. One week after the AOM injection, mice were given three cycles of DSS  in their drinking water, and then distilled water until the end of the experiment. Antibodies were administered intravenously every 2 days during the three cycles of DSS treatment. Mice were sequentially killed randomly at the end of the 13th and 18th week, and at least five mice were killed for each group at each time point. The study procedure was shown in Figure Acute colitis was induced with 4% DSS  for 6 consecutive days. Sixty 5-week-old male ICR mice housed under specific pathogen free conditions were divided into four groups with 15 in each group: control , DSS + IgG Ab , DSS + anti-S100a9 Ab , and DSS + TNFα Ab . Five mice per cage, each cage only held one group. Experimental procedure was shown in Figure The disease activity index (DAI) was calculated as the sum of weight loss, diarrhea, and bleeding, according to the criteria described by Murthy et al . The weiLamina propria mononuclear cells were obtained from colonic specimens using Weigmann et al.’s protocol . Brieflyp < 0.05. Only genes with greater than twofold change and p-values of less than 0.05 were selected for pathway analysis. Gene ontology (GO) enrichment and KEGG pathway enrichment analysis of differently expressed genes were, respectively, performed using R based on the hypergeometric distribution. Hierarchical cluster analysis of differently expressed genes was performed to explore genes expression pattern.Total RNA from colon tissues was extracted using the mirVana™ miRNA Isolation Kit  following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer . The samples with RNA Integrity Number ≥7 were sequenced by the Solexa high-throughput sequencing service . Data were extracted and normalized according to the manufacturer’s standard protocol. The RNA-seq raw expression files and details have been deposited in NCBI GEO under accession No. GSE104614. Differentially expressed genes were identified using the DESeq (2012) functions estimateSizeFactors and nbinomTest . Log-folhttp://www.broadinstitute.org/gsea/index.jsp) is a computational pathway analysis tool that determines whether a set of genes show statistically significant, concordant differences between two biological states  and the last 25% samples  were subject to GSEA analysis. GSEA was used to associate the gene signatures with the S100A9 expression status (S100A9low vs S100A9high). A significance threshold was set at a nominal p-value < 0.05.Gene set enrichment analysis  were preincubated with S100a9 antibody (1 µg/ml) or IgG antibody as a negative control in a 6-well plate for 1 h at 37°C. Following the incubation, mouse macrophage RAW264.7 cells were added in 6-well plates at 5 × 105 cells per well and incubated with 5% CO2 at 37°C for 24 h. Then the cells were harvested and the mRNA levels of Tnfα and Il1β were measured by quantitative real-time PCR (qRT-PCR). Recombinant mouse S100a9 proteins were from Abnova .Rabbit anti-S100a9 polyclonal neutralizing antibody was purchased from Beijing Protein Innovation . The anti-S100a9 antibody is made against the mouse S100a9 protein and is produced by immunizing New Zealand rabbit with full-length mouse S100a9 protein (Swiss-Prot #P31725) expressed in The paraffin-embedded sections of colon were deparaffinized and rehydrated. Immunohistochemical staining was performed to detect the expression of S100a9 (NB110-89726) , myeloperoxidase  , CD11c (clone HL3) , p-Smad2  , β-catenin (clone D10A8), c-Myc (clone D3N8F), p-Akt  , CD68 (clone KP1), and CXCL5 (#ab9983)  as described previously . OmissioIn Vivo Imaging Kit . For assessing cell proliferation, mice were injected intraperitoneally with 5 mg/kg 5-EdU 5 h before sacrifice. At the time of sacrifice, the colon was removed and perfused with PBS to remove the contents. Frozen sections (5 µm) of colon tissues were fixed in 4% (w/v) paraformaldehyde at 4°C for 10 min, and carefully washed three times with PBS. Sections were treated with 0.5% Triton X-100 at room temperature for 10 min, and carefully washed three times with PBS before stained. 100 µl of Apollo reaction mixture were added to the samples at room temperature for 30 min, and the nuclei were stained with Hoechst (33342) for 30 min according to the manufacturer’s protocol. For quantification, the numbers of EdU positive cells were captured with a fluorescence microscope , and the ratio of proliferation cells was determined with Image Pro Plus software.Cell proliferation was measured using the Cell-Light™ EdU Apollo 567 A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was carried out to detect cell apoptosis. TUNEL staining was performed using the “One-Step TUNEL Apoptosis Assay Kit” . Paraffin sections (4 µm) were heated at 60°C for 1 h, washed in xylene and rehydrated through a series of ethanol and double distilled water. Sections were treated with proteinase K (40 µg/ml) for 25 min at 37°C before staining, and carefully washed four times with PBS. 50 µl of TUNEL reaction mixture  were added to the samples. For negative control, 50 µl label solution  were added to the slides. All slides were incubated in a humidified atmosphere for 60 min at 37°C, and the nuclei were stained with 4,6-diamidino-2-phenylindole  for 10 min. The numbers of TUNEL positive cells were captured with a fluorescence microscope, and the ratio of apoptosis cells was determined with Image Pro Plus software.Blood and stool of experimental mice were harvested at the indicated times. For cytokine measurement, homogenates from mice stool samples were sonicated and centrifuged to obtain supernatant. The protein concentration of S100a8/S100a9 in serum and stool was determined by ELISA assay according to the manufacturer’s instructions .2O (9.2 µl). The qRT-PCR analysis was performed using a Bio-Rad CFX96 Real-Time System . The data were analyzed using Bio-Rad CFX manager 2.0 software. The relative target gene mRNA levels were expressed as the ratio of target to Gapdh and calculated in relation to the standard curve. All reported results were the average ratios of three independent experiments. The primers for qRT-PCR were listed in Table S1 in Supplementary Material.Total RNA from colon tissues was extracted using Trizol . The cDNA was synthesized using 2 µg of total RNA and Revert Aid First Strand cDNA Synthesis Kit . Expression of the mRNAs was determined by qRT-PCR using SYBR Premix Ex TaqTM II  according to the manufacturer’s recommendations. The PCR mix includes SYBR Premix Ex TaqTMII , primer mix , cDNA (0.4 µl), and ddHt-test or one-way ANOVA with Bonferroni correction. Significance parameters were set at p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001 compared with indicated control group mice.Statistical analysis was performed using GraphPad Prism 5. Data were typically expressed as the mean ± SEM, and the differences between groups were analyzed using either unpaired two-tailed Student’s Tnfα and Il1β’s expressions in macrophages, while anti-S100a9 antibody significantly blocked this effect. To investigate the effect of neutralizing S100a9 antibody on DSS-induced colitis mice, neutralizing S100a9 antibody (1.5 mg/kg) or negative control IgG antibody (1.5 mg/kg) was administered by intraperitoneal injection for two times. Infliximab is a chimeric mouse–human monoclonal antibody that binds to soluble and membrane bound TNFα and prevents it from binding to its receptors  Figures B,C. InjeHistological analysis of the colon tissues from IgG-treated colitis mice showed severe inflammation with superficial ulceration, crypt destruction, mucosal damage, and leukocyte infiltration of epithelium and LP, whereas the administration of either anti-S100a9 Ab or anti-TNFα Ab alleviated histological colonic damage, and had preferential effect on histological subscores for inflammatory infiltrate and epithelial damage in colon tissues Figures A,B. The S100a9, Tnfα, Il1β, Ifnγ, Il6, Il17a, Il23a, Il4, and Il12a, but these effects were repressed by anti-S100a9 Ab treatment . Anti-S100a9 Ab-treated CAC mice presented a low level of DAI scores compared with IgG-treated mice . However, anti-S100a9 Ab treatment did not trigger colon cancer cell apoptosis in vivo . In addition, neutralize the S100a9 protein also reduced the intra-colonic infiltration of inflammatory cells, such as macrophages (CD68+) and neutrophils (MPO+) . These observations reveal that blockage of S100a9 represses the inflammation cells infiltration and tumor cell proliferation in the CAC model.Previous studies revealed that exogenous S100a8/a9 protein increases the proliferation of colon cancer cells in vitro , 30. We Cxcl1, Cxcl2, Tnfα, Il6, Saa3, Mmp7, Mmp9, and Lcn2 (vs Control), which was consistent with our previous findings in the gene expression profile of CAC mouse model (vs AOM/DSS + IgG Ab) . These data further indicate that the S100a9 signaling is blocked by the neutralizing antibody in the “inflammation–cancer link” experimental mice.To further investigated the role of anti-S100a9 Ab in CAC mice model, we analyzed the global transcriptome change of intestinal mucosa tissues cells from three groups of mice: normal control mice, AOM/DSS mice treated with IgG Ab, AOM/DSS mice treated with anti-S100a9 Ab. RNA-seq analyses showed that the expression of S100a9 and its downstream target molecules, such as and Lcn2 , 31–35, se model . The expvs Control). Whereas only 385 genes showed significant elevation, and 164 genes were downregulated in the anti-S100a9 Ab treatment colon mucosa compared with control group . The transcriptome analysis suggests that AOM/DSS-induced inflammation-carcinogenesis triggers a robust globe gene expression change in the colon tissues, whereas anti-S100a9 Ab treatment can significantly reverse this effect.Compared with normal colon mucosa, gene expression profile changes burst distinctly under the influence of AOM and DSS stimulation, as 1,017 genes showed significant elevation, and 815 genes were downregulated in the colon adenocarcinoma tissues , cell adhesion and migration , proliferation and metastasis , stem cell regulation   Figure . We prevignaling . In thislow and S100A9high human colon tumors  and human tumors  , 44. S10+-myeloid cells in vivo and in vitro , and through interaction with their receptor, TLR4 or RAGE, play essential roles in the process of colitis-associated carcinogenesis, including initiation, promotion, and progression , 30. Prein vitro . Quinoliin vitro , 52, andin vitro . HoweverTnfα, Il1β, Il6, Il17a, Ifnγ, and Il12a, all of them are master regulators of inflammation and tumorigenesis. Besides, anti-S100a9 antibody decreased DSS-induced cell death and maintained the regenerative proliferation of intestinal epithelial cell, suggesting that neutralizing S100a9 antibody had a protective effect on the intestinal mucosal barrier. These results suggest that beneficial potency of neutralizing S100a9 antibody may be attributed to the reduced number of infiltration immune cells and the inhibited expression of pro-inflammatory cytokines in the colon of DSS-treated mice. Anti-TNFα agents have been the first line treatment for moderate to severe IBD over the past 10 years  that were inactivated in the S100a9 blocked mice. The products of these pathways promote inflammation, leukocyte recruitment, angiogenesis, stem cell characteristics, tumor migration, and proliferation. Since the chemical induced colitis and CAC mouse models cannot completely mimic the natural of inflammation and tumor development, our results may also require to be replicated in other animal models and may need to be validated in clinical specimens in the future. We used data from GEO and revealed that expression levels of human s Figure .In conclusion, treatment of mice with anti-S100a9 antibody during colitis and CAC development exerted a protective effect on intestinal inflammation and tumorigenesis, suggesting that the neutralizing S100a9 antibody is a potential therapeutic agent for treating human IBD. Therefore, blockade of S100a9 may feasibly be a promising biological strategy to inhibit inflammatory symptoms associated with IBD and attenuate the progression of CAC.All animal experimental procedures were performed in accordance with the guidelines of the Animal Welfare and Research Ethics Committee of Central South University.Xuemei Zhang and JM conceived and designed the experiments. Xuemei Zhang, LW, Jing Wang, ZQ, Jia Wang, YL, Xiang Zheng, QP, QY, FA, PL, and SW performed experiments and analyzed the data. GL and SS were responsible for guiding and supporting the experiments. Xuemei Zhang and JM interpreted the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clostridium botulinum secretes a potent neurotoxin that causes devastating effects when ingested, including paralysis and death if not treated. In the United States, some clinically significant strains produce toxin type A while also harboring a silent B gene. These are the first two closed genome sequences published for this subset. Clostridium botulinum type A strains isolated in the United States also harbor a silent toxin type B gene (bont/B) (our unpublished data). These strains have been difficult to distinguish by traditional subtyping methods, such as pulsed-field gel electrophoresis or multilocus variable-number tandem-repeat analysis , with 5,926 bp, and pNPD12_2 (accession no. CP014147), with 59,360 bp. These plasmids contain 6 and 109 coding regions, respectively. The chromosome has 3,954,777 bp and includes 3,602 coding regions and a G+C content of 28.2%. Both toxin genes were found in the chromosome rather than on the plasmids.CDC69094 was isolated from a stool specimen from an infant botulism case. Interestingly, this genome did not include a plasmid, indicating that the unexpressed B gene is contained on the chromosome with the toxin type A gene. The chromosome (accession no. CP013246) included 4,089,027 bp, a G+C content of 28.1%, and 3,711 coding regions.CP013246 for CDC69094 and accession numbers CP014146, CP014147, and CP014148 for CDC67190.The finished sequences have been deposited in GenBank, under accession no.


Different treatment conditions (presence or absence of serum) and differentiation stimuli  were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the  The skin is a continuously self-renewing organ that dynamically manages the outside-inside-outside relationships of the human body and actively participates in the host defenses . Keratin2+) gradient in the epidermis, increasing from the basal to the granular layer, represents one of the most important triggers of KC differentiation [Under homeostatic conditions, KCs differentiate and mature from proliferating nucleated basal cells to the highly differentiated, nucleus-free corneocytes. Each stage of differentiation is characterized by the expression of structural proteins, such as keratins (K) and lipids , 4. For Resting KCs produce epidermal growth factor receptor (EGFR) ligands and vascular endothelial growth factor (VEGF), but when activated by bacterial products or by direct damage by UV light or chemicals, the expression of cytokines and chemokines changes , 6. In sin vitro at low Ca2+ concentration retain a basal phenotype, and they differentiate upon addition of Ca2+ > 0.1 mM [in vitro; secondly, once induced to differentiate, they rapidly die and do not allow long-term investigation of the differentiation signals [in vitro responses, different plating efficiencies, the short lifetime in culture, and the changes in proliferation and differentiation characteristics with increasing number of passages, complicates the interpretation of experimental data.Cultured human KCs are frequently employed for studies of KC functions in chronic inflammatory skin diseases , 10. Pri> 0.1 mM , 12. How signals . Moreove2+ concentration in the medium [in vivo [To minimize these problems, the spontaneously immortalized human KC cell line HaCaT from adult skin has been proposed as a model for the study of KC functions. HaCaT is a nontumorigenic monoclonal cell line, adapted to long-term growth without feed-layer or supplemented growth factors , 14; it e medium ; they re[in vivo .in vitro model to evaluate, at different stages of differentiation, the production of proinflammatory mediators, chosen among those mostly involved in skin inflammation and angiogenesis.The aim of the present study was to investigate and optimize the best conditions to use HaCaT cells as a reliable 2 at 37°C in regular Dulbecco's Modified Eagle's Medium (DMEM)  containing 1.8 mM Ca2+, or with DMEM  at low concentration of Ca2+ (0.07 mM). Both media were supplemented with 10% heat-inactivated fetal bovine serum, glutamine (2 mM), penicillin (100 U⁄ml) (Euroclone), and streptomycin (100 mg⁄ml) (Euroclone). For all experiments, cells were seeded at a density of 5.7 × 103 cells⁄cm2 and cultured with DMEM at high or low Ca2+concentration for 6 or 14 days. The samples were labeled as follows: A6, cells cultured for 6 days with low Ca2+ concentration (0.07 mM) and tested when 80% confluent; A14, cells cultured for 14 days with low Ca2+ concentration (0.07 mM) and tested when overconfluent; C6, cells cultured for 6 days with high Ca2+ concentration (1.8 mM) and tested when 80% confluent; and C14, cells cultured for 14 days with high Ca2+ concentration (1.8 mM) and tested when overconfluent. The medium was changed every 2 days. A flow chart with details of the experimental protocol is reported in HaCaT cells, spontaneously immortalized human keratinocyte line , were ki2+ concentration (<0.07 mM), and supplemented with human keratinocyte growth factors (Gibco BRL). When the monolayer reached 60%–70% confluence, cells were split by trypsinization. For all the experiments, keratinocyte cultures between the third and fourth passages were used. Informed consent was obtained from all donors providing tissue samples, and ethical approval was obtained from the Ethics Committee of “La Sapienza” University, Rome, Italy.Primary KCs were isolated from nonlesional skin biopsies obtained from adult psoriatic patients not receiving either topical or systemic therapies for at least 6 months, or at the time of sample collection. To separate the epidermal layer from the basement membrane, the 0.4 mm punch biopsy was treated with dispase . After 18 h at 4°C, the epidermal sheet was separated mechanically and dissociated with TrypLE  for 20 min at 37°C. The obtained primary cells were then plated on 6-well tissue culture plates (Costar), precoated with coating matrix , cultured using a specific keratinocyte-serum-free media at low Caμl of medium/well. Cells were seeded at an initial density of 1.0 × 104 cells/cm2, and, after 1, 6, 9, and 14 days, incubation medium was removed and replaced by 100 μl of fresh medium. Then, 10 μl of stock MTT solution (5 mg/ml in PBS) was added and plates were incubated at 37°C for 4 h. Finally, 100 μl of 10% sodium dodecyl sulfate (SDS) , in 0.01 M HCl, was added to each well and the amount of formazan formed was measured at 540 nm using a Benchmark microplate reader . Sixteen wells per time-point were analyzed in three independent experiments.The proliferation of HaCaT cells was determined at the indicated intervals using the MTT colorimetric assay as described . This te4 cells/well in 500 μl were seeded in 24-well plate and cultured in low or high Ca2+ medium, with medium changes every two days. After 6 or 14 days, the medium was removed and replaced with 250 μL of medium or serum-free medium, supplemented with TNFα (10 ng/ml) or IL-1β (10 ng/ml) .For the stimulation of HaCaT cells, an amount of 1.0 × 102+, and magnesium-free medium, dried, and stored at −20°C for the analysis of DNA content.After different lengths of time  at 37°C, the supernatants from two wells for each different treatment and time were pooled in a single tube and frozen at −20°C to be subsequently analyzed for the presence of cytokines, chemokines, and growth factors. The cell monolayers were then washed twice with PBS, Ca4 cells/cm2 in 3 ml were seeded in 6-well plate and cultured in low Ca2+ medium, with medium changes every two days. When the cells reached 60%–70% confluence, the medium was removed and replaced with 3 ml of serum-free medium, supplemented with TNFα (10 ng/ml). After 48 hours of incubation at 37°C, the supernatant was collected, frozen at −80°C, and subsequently analyzed for the presence of cytokines, chemokines, and growth factor as described in the multiplex system section.For the stimulation of keratinocytes from skin biopsies, 8–10 × 10μl of samples was transferred in duplicate into wells at room temperature for 2 hours. The results were detected by spectroscopy  using biotinylated and streptavidin-HRP conjugate antibodies, evaluating 3,5,3,59-tetramethylbenzidine (TMB) substrate reaction. The quantification of analytes was done using an optimized standard curve supplied with the ELISA sets. The data are expressed as pg/106 cells. Results are mean ± SD of at least three independent cell culture experiments in duplicate.The release of CXCL8/IL8 and VEGF from HaCaT cells was quantified by using two different high sensitivity human ELISA set  following the method described below. Briefly, Corning 96-well EIA/RIA plates from Sigma-Aldrich  were coated with the antibodies provided, overnight at 4°C. Matrix metalloproteinase-9 (MMP-9) secretion from HaCaT cells was evaluated by a different ELISA set  using a precoated 96-well plate, supplied with the kit. In all the three cases, 300 α (10 ng/ml) for 48 hours, were analyzed for the presence of chemokines, cytokines, and growth factors. Regarding chemokines, we assayed CXC chemokine ligands  and CC chemokine ligands . Among cytokines, we assayed TGFα, TNFβ, IFN-α2, IFN-γ, G-CSF, GM-CSF, IL-10, IL-12p70, IL-15, and IL-33. All factors were quantified simultaneously by Bio-Plex Pro human cytokine assays according to the manufacturer's instructions . The analyte levels were determined using Bio-Plex array reader  and the Bio-Plex manager software. The relative concentration of each analyte was obtained through the establishment of standard curves, and results are expressed as pg/106 cells.Supernatants obtained from HaCaT cells and from primary keratinocytes, stimulated or not with TNFμL of distilled H2O was added to each well. After three cycles of freezing-thawing, 100 μl was then transferred into a 96-well plate and used for the assay. In parallel, the standard calibration curve was made using increasing concentrations (0–1000 ng/100 μl) of DNA from calf thymus diluted in TE buffer . The amount of 100 μL of Hoechst dye was then added to each well containing the sample or standard, previously diluted 1 : 400 in TNE buffer . The emitted fluorescence was measured with microplate reader TECAN F500  using excitation and emission wavelengths at 346 nm and 460 nm, respectively. The DNA content was calculated by interpolation of the absorbance of the samples on the calibration curve. The DNA content was expressed as μg DNA/106 cells counted, in each well, using a Burker chamber and trypan blue.Analysis of DNA content was performed using the commercial kit fluorimetric “FluoReporter Blue Fluorometric DNA Quantitation Kit”  in 96-well plates following the manufacturer's instructions. Plates with treated HaCaT cells were thawed, and 200 2VO4, and 8.5 μg⁄ml of aprotinin, in PBS), shaking for 20 min at 4°C. Samples were collected by scraper, incubated for 60 min at 4°C, and centrifuged at 12.000 rpm for 15 min at 4°C, and the supernatant was collected and frozen at −20°C until use. The soluble proteins in the extract were quantified according to the method described by Lowry et al. [For total protein extraction, HaCaT cells were cultured for 6 or 14 days with proper medium. For each time-point of the differentiation, cells were washed with cold PBS and lysed in RIPA lysis buffer , 1 mM Nay et al. .μl of total protein was separated on 7.5% SDS-PAGE gel and transferred to a polyvinylidenedifluoride transfer membrane (PVDF)  for 16 h at 150 mA, using transfer buffer . The membranes were blocked by incubation in blocking buffer (PBS containing 0.1% Tween and 5% dried nonfat milk) for 2 h and 30 min at room temperature. Then, membranes were blotted overnight at 4°C with various dilutions of primary antibodies, specifically, rabbit polyclonal IgG anti-involucrin , mouse polyclonal IgG anti-cytokeratin 14 , and rabbit monoclonal IgG anti-cytokeratin 10 . Mouse monoclonal anti-β-actin antibody  was used to normalize gel loading. Blots were washed six times with PBS-0.1% Tween and incubated for 1 h at room temperature with horseradish peroxidase-linked secondary antibodies. Involucrin and K10 were detected with a donkey anti-rabbit IgG . For K14 and actin, a goat anti-mouse IgG  was used. All blots were developed by ECL Western blotting detection LiteAblot® plus Kit Reagent , following the manufacturer's protocol. Immunoreactive proteins were visualized by autoradiography on Hyperfilm ECL . The relative intensities of band signals were quantified by digital scanning densitometry, and β-actin was used to normalize the results to protein content.For Western blot analysis, 40 HaCaT and primary keratinocytes, cultured on 8-well slide chambers, were washed with PBS, fixed, and permeabilized in ice-cold methanol for 5 min. First, cells were incubated in PBS containing 0.1% bovine serum albumin (BSA) for 10 min, then were incubated with primary antibodies against K10  for 60 min in PBS containing 1% BSA. The cells were subsequently incubated with secondary antibody Alexa Fluor® 488-labeled goat anti-rabbit IgG and Alexa Fluor 488-labeled goat anti-mouse IgG  for 30 min, respectively. The nuclei of the cells were stained with 4′,6-diamidino-2-phenylindole , which specifically recognizes DNA. The slides were observed by using Nikon Eclipse TE200 inverted microscope with immersion objective at 60x magnification and photographed with Nikon digital camera .p < 0.05 was considered statistically significant.Data are expressed as mean ± SD of at least three experiments performed in duplicate. Data were analysed by unpaired one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Statistical analysis was done using GraphPad Prism 5.0 software . in vitro model to study the inflammatory response of human keratinocytes, two known stimuli of KC differentiation, cell density and extracellular Ca2+ concentration, were used. HaCaT cells were plated at the same density in low (0.07 mM) and high (1.8 mM) Ca2+ medium, and cell proliferation was assessed by both MTT assay and cell counts at day 6 and day 14.To set up and validate HaCaT cells as an 2+ medium, a steady increase in metabolic activity and cell number was observed over time, while in high Ca2+ medium, cell growth was slower. Comparing low to high Ca2+ medium, about 27.5% and 70.3% decrease of MTT activity and 40% and 60% decrease in cell count were seen at days 6 and 14, respectively (1S). However, no significant changes in cell morphology were observed in the different conditions. Phase contrast images showed that HaCaT cells were flat and spread out after 6 days of growth both in low (A6) or high (C6) Ca2+ medium when they were at 80% confluence; at day 14 both in low (A14) and high (C14) Ca2+ concentration, they became more cubical in shape with higher cell-to-cell packing and stratification . As reported in Table 1S, no significant changes were observed in the DNA and protein content in HaCaT cells grown in medium with different Ca2+ concentrations at the 6th or 14th day.In low Ca2+ levels was assessed by Western blot analysis. Physiologically, the expression of K14 is restricted to the epidermal basal layer, while the presence of K10 and involucrin indicates a more differentiated phenotype.The expression of three classical KC differentiation markers  in response to changes in cell density and extracellular Ca2+ medium was more evident for involucrin than for K10 , whereas in high Ca2+ medium, the increase was 4.7-fold versus 3.2-fold, respectively.As shown in 2+, at day 6, HaCaT cells showed similar levels of K14, K10, or involucrin in A6 and C6 culture conditions, suggesting that Ca2+ concentration did not significantly influence their expression at 80% cell confluence (2+ medium (1S).Regarding the effect of Canfluence . Unexpec+ medium . This re2+ medium both in 80% confluent or overconfluent cells. Control samples without primary antibodies were negative, confirming specificity (data not shown).The acquisition of HaCat cell-differentiated phenotype was confirmed by immunofluorescence experiments . The exp2+ and serum-free medium. Interestingly, as shown in 2+ medium (A6).The pattern of K10 expression was also evaluated on primary human keratinocytes grown in low CaHaCaT cells grown in different culture conditions described above were then utilized to evaluate the production of a series of bioactive molecules, known to be released in the skin during inflammation or repair, both in basal conditions and upon proinflammatory stimulation.α and IL-1β were used as stimuli. Influence of serum in the medium was also verified.In a first set of experiments, we focused on three main mediators: CXCL8/IL8, VEGF, and MMP-9, which are crucial for inflammatory cell recruitment, angiogenesis, and matrix remodeling, respectively. TNF2+ medium and treated, after 6 or 14 days of culture, with 10 ng/ml TNFα or IL-1β for 6 or 24 h, as indicated, in absence  . Higher levels of CXCL8/IL8, VEGF, and MMP-9 were seen at day 6, compared to day 14, independently of serum or Ca2+ concentration. In the presence of serum, unstimulated cells produced higher levels of mediators than without serum, and, except for IL8, the increase of VEGF or MMP-9 triggered by IL-1β was not significant.Similar profiles were observed when cells were stimulated with IL-1α (10 ng/ml) for 48 h resulted in the upregulation of secretion of almost all the 18 mediators tested in the different culture conditions, although the level of stimulation varied among them. In particular, 6 days of culture in high Ca2+ concentration seem to represent the best combination for optimal production of the majority of mediators, except for CCL4/MIP1b, INFα2, INFγ, and G-CSF. Of note, CCL7/MCP3 seemed to be the only cytokine strongly upregulated by TNFα independently of the day of culture and Ca2+ concentration, whereas CCL22/MDC was the only cytokine for which overconfluency, and not Ca2+ levels, contributed to its production. On the contrary, Ca2+ concentration, but not the days in culture, seemed to be particularly relevant for TNFα-induced release of GM-CSF. No measurable values for CXCL12/SDF-1, CX3CL1/fractalkine, or IL-33 were obtained from any samples (data not shown).To extend these observations, different chemokines and cytokines were studied by magnetic bead suspension array using the Bio-Plex Pro technology. The experiments were conducted in serum-free medium to reduce the interferences described in the previous paragraph. As shown in Figures 2+ medium and, when tested at 60–70% confluence, showed K10 expression comparable to A6 HaCaT cells  confirm and extend previous observations that the increase in cell density and extracellular Ca2+ concentration favors HaCaT differentiation [2+ conditions and less than 80% confluent (A6), while they begin to differentiate expressing high levels of K10 and involucrin after a long-term culture (A14), that is when, although maintained in low Ca2+ medium, they are overconfluent. Different from previous observations, we noticed that HaCaT cells maintain their basal phenotype also when the cells are cultured in high Ca2+ conditions, but at less than 80% confluence. This observation suggests that, although extracellular Ca2+ > 0.1 mM appears to be a major regulator of HaCaT differentiation, the role of cell density is also relevant and it might have been underestimated in previous in vitro studies.With this in mind, we first tested the differentiation potential of HaCaT cells in response to the increase in cell density and in extracellular Cantiation . HaCaT c2+ condition, the overconfluence promotes a slight decrease of both K10 and involucrin levels and a weak increase of K14. Although this observation may be a consequence of the lower density of C14 with respect to A14, these data are in agreement with the changes of Ca2+ and confluence-dependent expression of K1, a biomarker of more differentiated cells [Moreover, in our study, the Western blot analysis of K10 and involucrin expression showed that when HaCaT cells are maintained in high Caed cells , but noted cells . Altoget2+ switch on the proliferation of HaCaT cells is poorly investigated. Our viability assays and cell counts highlight that the extracellular Ca2+ concentration strongly influences the proliferative ability of HaCaT cells. Up to day 2 of culture, no differences in the proliferation rate of HaCaT cells grown in low or high Ca2+ medium were seen, whereas a difference in the growth rate was evident in low Ca2+ medium starting from day 6, which became significantly higher at day 14. These findings are at variance of those showing a progressive, time-dependent increase of HaCaT proliferation in high Ca2+ medium [2+ concentration used in the present study (1.8 mM) compared to 1.2 mM in [2+ concentration above 1.5 mM results in a reduced growth rate [The effect of the Cawth rate .2+ medium for different lengths of time, were used to investigate the release of CXCL8/IL8, VEGF, and MMP-9 in response to two proinflammatory stimuli, TNFα, and IL-1β. These cytokines were chosen for their predominant role in the pathogenesis of skin inflammation, since they both regulate genes previously shown to be specifically overexpressed in psoriasis [2+ concentrations slightly enhanced the secretion of these mediators, especially when IL-1β was used as proinflammatory stimulus; this effect could be partially explained by the activation of the Ca2+ responsive promoter of activator protein (AP)-1 [In the second part of this study, HaCaT cells, cultured in low or high Casoriasis , 22–24. n (AP)-1 .α-induced release of MMP-9 and enhanced the VEGF secretion, both at basal and stimulated levels. As previously reported, growth factors derived from fetal calf serum may influence a variety of parameters involved in KC proliferation, differentiation, and wound healing processes [The presence of serum reduced the TNFrocesses –28. Baseα in KC regulates different genes involved in inflammation and angiogenesis, such as IL-1, ICAM-1, VEGF [β, chemokines , and members of the CXCL8 family, including CXCL1, CXCL2, and CXCL3 [2+ concentration and cellular density on the secretion of different cytokines, chemokines, and growth factors in HaCaT cell line. All the analyzed parameters were upregulated to a great extent by TNFα at day 6 in comparison to day 14; only CCL22/MDC seemed to be preferentially released in more differentiated HaCaT cells. CCL4, INFα2, INFγ, and G-CSF had a higher release in low Ca2+ condition, whereas, on the opposite, in high Ca2+ concentration, a comparable or higher secretion of CCL2, CCL3, CCL5, CCL7, CCL11, CCL22, CXCL1, CXCL10, TGFα, TNF-β, IL-10, and IL-15 was seen. To summarize, both long-term culture and Ca2+ concentration in the medium affect the ability of HaCaT cells to release chemokines and growth factors.Several papers demonstrated that TNF-1, VEGF , 30, TGFnd CXCL3 , 23. The2+medium and in the absence of serum.Finally, to validate HaCaT cells as a reliable model to dissect the inflammatory/repair response of human KCs, we measured the release of inflammatory mediators in primary nonlesional epidermal KCs obtained from psoriatic patients. These cells were assayed in low Caα on human KCs  correlated relatively well to the values released by HaCaT cells in low Ca2+, considering the fold induction. Similar results were obtained on growth factors released . Not so close was the correlation with the CC family of chemokines some of which  were highly released in A6 and C6 HaCaT cells, but quite low in normal human KCs; CCL2 was higher in C6 HaCaT as in normal KCs, whereas CCL3 was similar. Our results also show that HaCaT cells especially at day 14 of culture produce more CCL22/MDC than normal human KCs, thus confirming previously published data [The levels of the CXC family of chemokines, induced by TNFhed data , 32. Thihed data .2+ concentration, the cell density, the differentiation state, and the presence of serum, were considered as factors that may influence release of proinflammatory mediators by KCs. Our results support the use of HaCaT cell line, under carefully optimized in vitro condition, as a reliable model, with respect to normal KCs, to screen for new anti-inflammatory compounds for skin diseases. Indeed, HaCaT cell line has been successfully used for studying those pathologies in which skin keratinocytes are involved, such as infectious diseases or tumors, or as in vitro carcinogenesis model of human skin [2+ concentration in culture medium and maintaining 80% confluence, HaCaT cells are in the conditions of producing cytokines at medium/low levels (A6 condition) or at medium/high levels (C6 condition) as expected in highly activated KCs from skin lesions. This in vitro system has the advantage of being reproducible and reliable and definitively less invasive and with less variability than KC from skin biopsies. However, one of the drawbacks is the inability of reproducing the skin complexity and cellular heterogeneity in basal or inflammatory conditions. 2D-3D culture systems have been proposed which mimic the differentiation process [In conclusion, this study is the first report where several variables, all together, including the influence of Caman skin . By modu process . It cann


Pyrolysis of these polymer functionalized inorganic nanoparticles leads to TiO2 nanoparticles coated with a thin carbonaceous shell. Such materials may, e.g., be interesting as battery materials.This paper describes the controlled radical polymerization of an ionic-liquid monomer by RAFT polymerization. This allows the control over the molecular weight of ionic liquid blocks in the range of 8000 and 22000 and of the block-copolymer synthesis. In this work we focus on block copolymers with an anchor block. They can be used to control the formation of TiO Most of them have a melting point below 100 °C . These o−, BF4−) . Because−, BF4−) . Propert−, BF4−) , they ca−, BF4−) .2 absorbing materials, microwave absorbing materials and porous materials [Polymeric ionic liquids (PILs) are made of ionic liquids with a polymerizable group, like a vinyl or acrylate group. They build a new class of macromolecules with unique properties. Alternatively, it is possible to coordinate low molar mass ionic liquids to polymers by complexation of their anions to cyclodextrin side chains. This can have an influence on their lower critical solution temperature (LCST) –11. Besiaterials . Most ofaterials . This laaterials –15, a caaterials . There aaterials , nanotubaterials . Due to aterials . Furtheraterials ,20–21.2, ZnO, Si or LiFePO4 [2 or ZnO with respect to the reversible storage of lithium or sodium ions [Independently from the work on polymeric ionic liquids, thin shells of carbonaceous materials around inorganic nanoparticles have been intensively investigated recently –25. This LiFePO4 . However LiFePO4 . While n LiFePO4 . In this LiFePO4 –28 can bium ions –25. UsinAn approach to coat nanoparticles with either (i) a thin film of PILs or (ii) a homogeneous carbonaceous layer derived from ionic liquids requires – at first – a synthetic route to block copolymers, which possess besides an anchor block , a block2 nanoparticles using block copolymers is displayed in The schematic synthesis route to carbon-coated TiO1) as an IL monomer, which was prepared following a literature procedure [1 was polymerized with 2-dodecylsulfanylthiocarbonylsulfanyl-2-methylpropionic acid  [1H NMR). The corresponding SEC elugrams (< 1.20). All the data regarding molecular weight and polydispersity are listed in 1H NMR spectroscopy).In a first step the PIL block is synthesized using 1-vinyl-3-cyanomethylimidazolium bromide   as a chaelugrams  reveal t1H NMR spectroscopy, which is shown in 4) as the anchoring unit. Dopamine has been proven to coordinate well on transition metal oxide surfaces [N-Acryloxysuccinimide  was chosen as a reactive ester because of its tolerance towards trace amounts of water present in DMSO, which is required for the block copolymerization as a polar solvent to solubilize the PIL macro-CTA. Optimized reaction conditions using 2,2-azobis (AMDVN) as an initiator, resulted in the successful block copolymerization. This was confirmed by 1H NMR spectroscopy after stirring for 20 hours at 45 °C. The broad signal which is typical for the NAS block can be observed at 2.8 ppm as shown in −1 and is assigned to the carbonyl group of the reactive ester  and the precipitated product was centrifuged. The process was repeated three times to remove solvent and unbound ligand. The product was dried under vacuum at room temperature. To examine the content of ligands on the surface, thermogravimetric analysis was performed (TGA) after several centrifugation steps, as shown in 2, phase transitions of the anatase TiO2 might occur, which leads to a mixture of anatase and rutile TiO2. XRD measurements , and corresponding images are shown in 2 particles with the block copolymer as a ligand on the surface. Raman spectroscopy and TEM images show that PILs are suitable carbon precursors and the herein introduced materials can be further applied as anode material in lithium or sodium ion batteries.In conclusion, we were able to synthesize well-defined block copolymers containing a PIL block and a reactive ester block. Besides, we showed the post-polymerization modification of these polymers, while remaining the block copolymer structure and simultaneously introducing an anchor group. Afterwards, we showed the successful in situ synthesis of TiO−1 K+TFA− as eluent. Modified silica was used as stationary phase and a refractive index detector, JASCO G1362A RID, was used. Poly(methyl methacrylate) (PMMA) was used as calibration standard. TGA was performed with a Perkin Elmer Pyris 6 instrument with an oxygen flow. Raman spectroscopy was conducted with Horiba Jobin Y LabRAM HR spectrometer with a frequency doubled neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. X-ray diffraction was performed on a Siemens D 5000 diffractometer equipped with Cu Kα radiation source (wavelength of 1.54056 Å) for both as synthesized as well as carbon coated TiO2 nanoparticles. TEM samples were prepared by dispersing the sample in ethanol and drop casting on 300 mesh carbon coated copper grids. The images were captured with a transmission electron microscope, a Tecnai G2 Spirit with an acceleration voltage of 120 kV.All chemicals were acquired from commercial sources (Acros or Sigma-Aldrich) and used without further purification. Synthesis and structural characterization: NMR spectroscopy was applied with a Bruker ARX 400 spectrometer. Fourier-transform infrared (FTIR) spectroscopy was conducted on a Jasco FT/IR 4100 spectrometer with an attenuated total reflectance (ATR) unit. The SEC measurements were carried out at 40 °C with a solution of HFIP with 3 g LSynthesis of PIL: The IL and also DMP which was used as chain transfer agent, were synthesized as already described in the literature [P1A, 0.02 equiv for P1B, 0.013 equiv for P1C) and the initiator AIBN  were mixed together and dissolved in DMSO, followed by three freeze-pump-thaw cycles. The reaction mixture was stirred for 20 h at 70 °C. Afterwards the mixture was purified by precipitation in acetone. 1H NMR  δ (ppm) 9.85 , 8.01 , 5.63 , 4.62 , 2.91 , 1.23 , 0.85 ; FTIR ν: 2973 (w), 2255 (w), 1626 (m), 1553 (s), 1425 (m), 1159 (s), 1019 (m), 748 cm−1 (w).terature ,30. For Synthesis of P (IL-b-NAS): PIL was used as macro-CTA. Together with NAS (20 equiv) and 2,2-azobis (0.2 equiv) PIL was dissolved in DMSO. After three freeze-pump-thaw cycles the mixture was stirred at 40 °C for 20 h. Afterwards the polymer was worked up by precipitation in acetone. 1H NMR  δ (ppm) 9.85 , 7.85 , 5.64 , 4.51 , 2.91 , 2.80 , 1.23 , 0.85 ; FTIR ν: 2969 (w), 2255 (w), 1808 (m), 1732 , 1553 (s), 1204 (m), 1161 cm−1 (m); SEC (eluent: HFIP): 23 098 g mol−1, PDI = 1.17.Synthesis of P (IL-b-DAAM): P (IL-b-NAS) (1 equiv) and lithium bromide (50 equiv) were dissolved in DMSO in a Schlenk flask. Dopamine hydrochloride (50 equiv) and triethylamine (50 equiv) were also dissolved in DMSO. The two solutions were combined and stirred overnight at 50 °C. For work-up, the polymer was precipitated in acetone. 1H NMR  δ (ppm) 9.97 , 8.85 , 7.94 , 6.58–6.36 , 5.66 , 4.58 , 3.15 , 2.91 , 1.23 , 0.85 ; FTIR ν: 2969 (w), 2255 (w), 1691 (m), 1645 , 1553 (s), 1434 (m), 1160 (m), 1020 cm−1 (m); SEC (eluent: HFIP): 23 180 g mol−1, PDI = 1.22.Synthesis of in situ functionalized TiO2 nanoparticles: 400 mg of catechol containing polymeric ligand was dissolved in 10 mL of DMSO (Sigma-Aldrich) and added to 70 mL of benzyl alcohol (Acros). The content of the flask was heated to 80 °C. The solution was degassed and filled with argon using a Schlenk line. The process was repeated three times. To this argon filled solution 3.2 mL of TiCl4 was slowly injected under vigorous stirring. The dark red solution was kept at 80 °C for 72 hours while constantly stirring at 750 rpm. The resulting brown suspension was precipitated using CHCl3 and hexane (1:3) and the precipitated product was centrifuged. The process was repeated three times to remove the solvent and unbound ligand. The product was dried under vacuum at room temperature.Pyrolization of as-functionalized TiO2 nanoparticles: All samples were pyrolyzed using the same conditions. 40 mg of the as-functionalized TiO2 nanoparticles were filled in a corundum boat, which was placed in a tube furnace. The heating rate was 5 °C/min up to a temperature of 650 °C, which was held for 1 h under a constant flow of argon. After that, the samples were cooled down naturally.File 1Additional spectra.


Kocuria polaris strain CD08_4, an isolate from the duodenal mucosa of a celiac disease patient. The genome consists of specific virulence determinant genes, antibiotic resistance genes, genes for coping with oxidative stress, and genes responsible for iron acquisition and metabolism, suggestive of its pathogenic attributes.We report here the 3.8-Mb genome sequence of  Kocuria polaris from a clinical source, which may help understand the molecular basis of its pathogenic behavior for the future. Previously, we reported Kocuria palustris CD07_3 from a CD patient, and K. polaris CD08_4 is another species of the genus . The assembled genome of strain CD08_4 had a total length of 3,833,718 bp, mean coverage of 100×, an N50 value of 221,530 bp, and a mean G+C content of 72.6%.Genomic DNA was extracted from a 48-h-old culture using the ZR fungal/bacterial DNA miniprep kit. Strain CD08_4 was identified as K. polaris CD08_4 had 3 genes coding for enzyme peroxidase, catalase, and superoxide dismutase, which may provide protection against reactive oxygen species generated during host-microbe interactions, thereby leading to self-protection.The Rapid Annotations using Subsystems Technology (RAST) server was used for functional annotation of the genome –6, and t–LQBK00000000. The version described in this paper is the first version, LQBK01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. Multivesicular bodies are compartments inside cells different for endosomes and lysosomes. In these compartments there are intralumenal vesicles, which once secreted to the extracellular medium, are termed exosomes. These exosomes have a particular lipid and protein composition, and a size between 40 to 100 nm. These structures were described for the first time during maturation of erythrocytes . ThereafOur group characterized previously that FasL and Apo2L/TRAIL are stored inside human T cell blasts in multivesicular bodies , and thahttp://exocarta.org/index.html; http://www.microvesicles.org). In some cases, the secretion of exosome-associated FasL by tumoral cells has been correlated with tumor counterattack mechanisms to escape immunosurveillance by T cells -acetate metabolic labeling of the cells during 72h. After that, cells were stimulated as indicated above for exosome secretion, cells separated from supernatants by centrifugation, and exosomes isolated as indicated above. After that, cell or exosome lipids were extracted with chloroform/methanol  and lipids separated by thin-layer chromatography (TLC). Phospholipid separation was made using chloroform/methanol/32% ammonia  as elue:5, v/v; ). Radiol6 cells were washed with PBS and solubilized in 100 μl lysis buffer ) by incubating on ice during 30 minutes and centrifugation 15 minutes at 13,500 rpm. Supernatant was collected and protein concentration was quantified with Coomasie® Plus Protein Assay Reagent following the manufacturer's protocol. Exosomes from 250 × 106 Jurkat or 500 × 106 T-cells blasts were lysed in 500 μl of the same lysis buffer and protein concentration was quantified with Coomasie® Plus Protein Assay Reagent following the manufacturer's protocol.5×10Analytical loadings of 30 μg of protein were suspended in rehydration buffer  to a final volume of 350 μl. Samples were applied to IPG strips 18 cm pH 3-10 by in-gel rehydration, over-night at room temperature. Four-step-focusing  was performed with an IPGphor system  for 65 kVh. Strips were equilibrated for 60 min in 50 mM Tris-HCl pH 8,8 buffer containing 6 M urea, 30% glycerol, 2% SDS, 1% DTT and 0,01% bromophenol blue, then treated for 60 min in the same buffer containing 2,5% iodoacetamide instead of DTT. The strips were then transferred onto 10% SDS-PAGE gels (200 × 250 × 1.00 mm) for the 2-D separation .Silver staining was performed as described before . Silver Extracts with urea were dialyzed in two steps. In the first step, extracts were dialyzed against 4M urea, 137 mM sodium chloride, 20 mM tris, 1 mM EDTA and 1% Triton X-100 during 4 hours at 4°C. In the second step, extract were dialyzed against 137 mM sodium chloride, 20 mM tris, 1 mM EDTA and 1% Triton X-100 over night at 4°C. 5 μg of protein were analyzed by NuPAGE™ 12% Bis-Tris Gels (Invitrogen) and separated proteins transferred to PVDF membranes (Millipore).® Plus Protein Assay Reagent following the manufacturer's protocol. 10 μg of protein were analyzed by NuPAGE™ 10% Bis-Tris Gels (Invitrogen) and separated proteins transferred to PVDF membranes (Millipore).To analyze supernatants before and after ultracentrifugation, supernatants were treated with 10% trichloroacetic acid  during 15 min on ice. Supernatant were centrifuged during 15 min to 13000g at 4°C, washed with ice-cold acetone and centrifuged during 5 min to 13000g at 4°C. Precipitates were solved in PBS with 1% SDS, 1% triton X-100 and Complete Mini (EDTA free) during 30 min on ice. Protein concentration was quantified with CoomasieThe antibodies used were mouse mAb against human Grp94, Hsp90, Hsc70/Hsp70, Hsp60, Hsp40 and TCP-1α , Rab5 and Lck (BD Biosciences), β-actin, α-tubulin , and α-actin . CD63 was detected using the mAb TEA3/18, a kind gift from Dr. Noa Martín-Cófreces, from Fundación CNIC, Madrid. Apo2L/TRAIL was revealed using the rabbit polyclonal antibody C35, kind gift from Avi Ashkenazi, Genentech, South San Francisco, USA. FasL was detected in cell and exosome extracts using a rabbit polyclonal antibody produced previously in our laboratory using cDNA immunization . VCP was30 μg protein were runned in the same conditions as for silver staining. Gels were fixed with 50% methanol and 2% o-phosphoric acid over night. After that, gels were washed three times with distilled water during 20 min and incubated with 34% methanol, 2% o-phosphoric acid and 17% ammonium sulfate during 1 hour. Gels were stained with 34% methanol, 2% o-phosphoric acid, 17% ammonium sulfate and 0,066% Coomasie G-250 during at least 5 days. Finally, gels were distained with 25% methanol during 1 min and kept in distilled water until excising. Identified spots were excised from the gel and spotted on a MALDI AnchorChip target plate according to the manufacturer's instructions. Analysis was performed on an OrbitrapXL MALDI TOF instrument (Thermofisher) using Bruker Flex-control, Flex-analysis, and Biotools software. Calibration was performed externally using SIGMA Proteo- Mass™ Peptide MALDI–MS Calibration Kit. MALDI mass spectra were recorded within an m/z range of 700–3500 in the positive ion reflector mode. Mass spectra were obtained by averaging up to 300 individual laser shots. Each analysis was performed in triplicate.P< 0.05 was used as the detection criteria taken for the protein identification, meaning that all proteins identified has a probability of less than 5% to be false positives.Protein samples were digested overnight at 37°C with 60 ng/μl trypsin in 50 mM ammonium bicarbonate, pH 8.8, containing 10% acetonitrile and 0.01% 5-cyclohexyl-1-pentyl-β-D maltoside. Tryptic peptide mixtures were separated using a nanoAcquity UPLC system . The nano UPLC system was coupled online to an LTQ Orbitrap XL mass spectrometer . Data were acquired by scan cycles of one FTMS scan with a resolution of 60000 at 400 m/z and a range from 370 to 2000 m/z in parallel with six MS/MS scans in the ion trap of the most abundant precursor ions. The mgf-files were used for database searches with the MASCOT search engine . Using this software, http://david.abcc.ncifcrf.gov/list.jsp). Interaction profiles between the different proteins identified were represented using the STRING software (http://string-db.org/), and the significance for clustering in a given group was also calculated, being P≤ 0.012 for all the interaction nodes identified.Using the uniprot ID numbers obtained, the protein name, species and function, together with their inclusion in different metabolic pathways was analyzed using the David software (2, N4-Ndibenzylquinazoline-2,4-diamine, also termed DBeQ [To estimate the functional value of VCP expression in the exosomes of tumoral Jurkat cells but not in those from normal T cell blasts, we performed several functional assays using the VCP inhibitor med DBeQ .Jurkat cells or human 6-day T-cell blasts were incubated overnight with increasing doses of DBeQ, from 3 to 6 μM, and cellular growth in comparison with non-treated control cells estimated by the MTT method, and cell death determined by Trypan blue staining and counting in an hemocytometer. Previously to establish this range of concentrations, which are not extremely toxic, DBeQ concentrations from 1 to 30 μM were tested.To estimate the effect of DBeQ on this functional response, we used a bioassay previously optimized by our group , 56. As


It is well known that mutations in the gene coding for amyloid precursor protein are responsible for autosomal dominant forms of Alzheimer’s disease. Proteolytic processing of the protein leads to a number of metabolites including the amyloid beta peptide. Although brain amyloid precursor protein expression and amyloid beta production are associated with the pathophysiology of Alzheimer’s disease, it is clear that amyloid precursor protein is expressed in numerous cell types and tissues. Here we demonstrate that amyloid precursor protein is involved in regulating the phenotype of both adipocytes and peripheral macrophages and is required for high fat diet-dependent weight gain in mice. These data suggest that functions of this protein include modulation of the peripheral immune system and lipid metabolism. This biology may have relevance not only to the pathophysiology of Alzheimer’s disease but also diet-associated obesity. Obesity is characterized, in part, by excessive fat accumulation throughout the body and increased levels of adipokines and inflammatory cytokines1123456789101112131415161718192023242527−/− mice to define the requirement for APP in regulating adipose tissue hypertrophy and macrophage activation. To further assess the tissue changes, individual cell types were isolated to better define the function of APP in regulating fatty acid uptake, inflammatory secretion, and phenotypic response.Although APP is a ubiquitously expressed transmembrane protein, its function remains unclear beyond being the parent protein of Aβ peptides. It has been suggested to function as a receptor or growth factor303132333436373841232425−/− mice consistent with their smaller stature , FAS, SREBP1, and adiponectin in perirenal adipose tissue compared to wild type mice , fatty acid synthase (FAS), peroxisome proliferator activated receptor γ (PPARγ), APOE, adiponectin, SCD1, and DLK. Consistent with macrophage activation and infiltration during adipose tissue hypertrophy during high fat feeding, multiple macrophage marker proteins also increased in wild type mice including TLR2, CD68 and arginase-1 . Under cype mice . Under hype mice . These de tissue  and subce tissue  were sig−/− compared to wild type tissue to be a reflection of diminished macrophage infiltration and activation−/− mice demonstrated robust macrophage immunoreactivity even without high fat diet-feeding in both perirenal , anti-rabbit (goat), anti-goat (bovine), anti-rat (goat), and anti-mouse (bovine) horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology . FABP4, CD36, PPARγ, SREBP1, UCP2, FXR, LXRα, LXRβ, TLR4, caveolin, arginase-1, VLDLR, GAPDH, α-tubulin and actin antibodies were purchased from Santa Cruz Biotechnology . Elite Vectastain ABC Avidin and Biotin, Vector VIP, Vector DAB, biotinylated anti-rabbit, anti-mouse, and anti-rat antibodies were purchased from Vector Laboratories Inc . CD68 antibody was purchased from AbD Serotec . pAKT, and AKT antibodies were purchased from Cell Signaling Technology Inc . APP (Y188), TNFα, FAS, LPL, DLK, FABP4, Adiponectin, Leptin, and APOE were purchased from Abcam Inc . TLR2 antibody was purchased from Imgenex . Aβ clone 4G8 was purchased from Covance . BAM-10 was purchased from Sigma-Aldrich Corp. . GLUT-4 antibody was purchased from EMD Millipore . SCD1 antibody was purchased from Thermo Fisher Scientific Inc. . Alexa Fluor 594 donkey anti-mouse IgG, Alexa Fluor 594 donkey anti-rat IgG, Alexa Fluor 350 goat anti-rat IgG, and Alexa Fluor 350 donkey anti-rabbit IgG antibodies were purchased from Life Technologies . LRP (H4 clone) antibody was generously provided by Dr. Isa Hussaini .tm1Dbo/J homozygous (APP−/−) mice and wild type (C57BL6) mice were purchased from Jackson Laboratory . Mice were provided food and water ad libitum and housed in a 12 hour light/dark cycle. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Animal use was approved by the University of North Dakota IACUC.APP−/− mice were placed on either a 21.2% by weight high fat diet (Harlan Teklad TD.88137) or a 5.5% by weight regular fat diet (Harland Teklad 8640), ad libitum. Animals were weighed each week for 22 weeks. Food was weighed each week during weeks 13–17. Food consumed was multiplied by the energy density  3.0 Kcal/g for control diet and 4.5 Kcal/g for high fat diet. After 22 weeks of diet feeding the animals were perfused with PBS and liver, visceral  fat, and subcutaneous fat was collected.At six weeks of age, 11 male C57BL6 wild type mice and 10 male APPAt 22 weeks mice were fasted for 6 hrs (water only) and then glucose, triglycerides, HDL and total cholesterol were measured using a CardioChek meter  and corresponding strips via tail lancet or True Result glucose meter and corresponding test strips. After baseline measurements were recorded mice were orally gavaged with 58 mg/mouse of glucose followed by 15, 30, 60, 120 min glucose measurements. One week later, mice were again fasted for 5–6 hrs (water only) and then glucose was measured. After baseline measurements were recorded mice were injected intraperitoneally with 0.02157 U/mouse insulin followed by 15, 30, 60, 120 min glucose measurements.Prior to collection, total stool weight was measured by placing the animals in a clean empty cage and collecting all stool produced over a 1 hr time period at the same time of day per animal.Fecal samples were air-dried and mixed with a mortar and pestle to uniform particle size. Total energy was determined in 0.5 g samples by adiabatic oxygen bomb calorimetry using a Parr 6200 Isoparibol Calorimeter Human adipose tissue was obtained from Dr. Michael D. Jensen at the Mayo Clinic Rochester, MN. Ten male samples were obtained, five lean (BMI 23.2–26.91) and five obese (BMI 40.25–51.90) from both a subcutaneous and omental fat depot were examined. Written, informed consent was obtained from all participants. The study was approved by the Institutional Review Board of the Mayo Clinic. These studies were done in compliance with the principles of the Declaration of Helsinki.6 cells/well onto tissue culture plastic for 3 hours in DMEM/F12. Plates were rinsed with ice cold DMEM/F12, any floating debris was removed.Macrophages were isolated by washing the peritoneal cavity with sterile PBS. The PBS was centrifuged, cells were counted and plated 0.5 × 10Subcutaneous abdominal adipose tissue was removed from the mice, rinsed with sterile Hanks basal saline solution (HBSS), cut into small pieces, placed in 35 mm plate, incubated for 1 hr @ 37 C on a rocker in sterile enzyme solution . Following incubation adipocytes were sucked up, placed in Eppendorf tubes, spun at 186 × g for 2 min, the upper layer was removed for adipocyte and fibroblast isolation and the lower layer was removed for macrophage isolation, both layers were resuspended in sterile HBSS to rinse, spun at 186 × g for 2 min, again separating the lower and upper layers. The upper layer containing adipocytes and fibroblasts was resuspended in DMEM/F12 + 10% FBS + 5% horse serum + antibiotics (PSN) and added to a T-25 flask with media or plated in 96 well plates, covered with sterile parafilm. The cells were grown in inverse position (bottom tissue culture treated side up) cell culture for 2 weeks. In T-75 flasks and 96 well plates, adipocytes in suspension floated to the tissue culture treated side up allowing the cells to adhere while fibroblasts sunk to the bottom non tissue culture treated side. For adipocyte use, the cultures were returned to right side up. The lower layer from the spin that contained adipose tissue macrophages was resuspended in DMEM/F12 without serum for 3 hours. Plates were then rinsed with ice cold DMEM/F12 and any floating debris was removed.3VO4, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton, 0.1% SDS, and 0.5% deoxycholate) with protease inhibitors . To remove insoluble material, cell lysates were sonicated and centrifuged . The Bradford method  was used to quantitify protein concentrations. Proteins were resolved by 7–10% SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blotting using anti- CD36, PPARγ, SREBP1, SREBP2, UCP2, FXR, LXRα, LXRβ, TLR4, CRP, caveolin, arginase-1, VLDLR, CD68, GAPDH, pAKT, AKT, APP (Y188), TNFα, GLUT-4, FAS, LPL, DLK, FABP4, adiponectin, leptin, APOE, TLR2, SCD1, LRP, α-tubulin and actin antibodies. Antibody binding was detected with enhanced chemiluminescence . In some instances, blots were stripped in 0.2 NaOH, 10 min, 25 °C, for reprobing. Western blots were quantified using Adobe Photoshop software. Optical density of bands were normalized against their respective loading controls and averaged (+/−SD).Tissues or cells were lysed using ice cold RIPA buffer  for immunostaining with anti-APP, CD68, 4G8, or BAM-10 antibodies or respective secondary only antibodies. Antibody binding in fat and liver was visualized using Vector VIP as chromogens . Images were taken using an upright Leica DM1000 microscope and Leica DF320 digital camera system. Figures were made using Adobe Photoshop software.Sectioned tissue was immunostained using anti-APP, CD68, 4G8 or BAM-10 antibodies or respective secondary only antibodies. Antibody binding was visualized using Alexa Fluor 594 or 350 secondary antibodies. Images were taken using a Zeiss LSM 510 Meta Confocal Microscope.Tissue was weighed then lysed in ice cold RIPA buffer with protease inhibitors. To remove insoluble material tissue lysates were sonicated and centrifuged. Cells were stimulated overnight in DMEM/F12 with or without 10 ng/mL LPS, 100 pM or 100 nM Aβ 1–40. Media from stimulated cells or tissue lysates were then plated for quantifying TNF-α, IL-6, IL-10, IL-4, IL-1β, MDC, IL-8, MCP-1, and adiponectin according to the manufacturer protocol . Tissue lysates were also used for commercial Aβ 1–40 and Aβ 1–42 ELISA .14C]16:0 were dissolved in ethanol and had a final specific activity of 55 nCi/nmol. Cells were incubated in serum free media containing fatty acid tracer (50 nCi/ml) for 5, 15 and 30 min at 37 °C. Ethanol concentration in the medium was 0.2%. At the end of incubation, cells were washed twice with ice-cold phosphate-buffered saline and extracted with hexane:2-propanol (3:2) and an aliquot of lipid extract was assayed for radioactivity using liquid scintillation counter.[1–Plated cells were used following the manufacturer protocol for Cholesterol Uptake Cell-Based Assay Kit, Glucose Uptake Cell-Based Assay Kit, Adipolysis Assay Kit and Adipogenesis Assay Kit Cayman Chemical .−/−, two-way repeated measures ANOVA with Holm-Sidak post hoc test for weight gain and glucose measurements, or two-way ANOVA with Holm-Sidak post hoc test when comparing diets or stimulations between wild type and APP−/− animals/cells.Data were analyzed using an unpaired t-test with Welch correction when comparing only wild type to APPHow to cite this article: Puig, K. L. et al. Amyloid precursor protein modulates macrophage phenotype and diet-dependent weight gain. Sci. Rep.7, 43725; doi: 10.1038/srep43725 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This study aimed to identify 18F-FDG PET/CT findings that might differentiate AIP from PC.FDG-PET/CT findings of 26 AIP and 40 PC patients were reviewed. Pancreatic and extra-pancreatic lesions related findings, including maximum standardized uptake values (SUVmax) and patterns of FDG uptake, were identified and compared.p < 0.05). AUCs were 0.700 (early SUVmax), 0.687 (delayed SUVmax), 0.683 (early lesions/liver SUVmax), and 0.715 (delayed lesion/liver SUVmax). Bile duct related abnormalities were found in 12/26 (46.15%) AIP and 10/40 (25.00%) PC patients, respectively. Incidentally, salivary and prostate gland SUVmax in AIP patients were higher compared with those of PC patients (p < 0.05). In males,an inverted “V” shaped high FDG uptake in the prostate was more frequent in AIP than PC patients . Increased FDG activity in extra-pancreatic bile duct was present in 4/26 of AIP patients, while was observed in none of the PC patients. Only in AIP patients, both diffuse pancreatic FDG accumulation and increased inverted “V” shaped FDG uptake in the prostate could be found simultaneously.All 26 patients with AIP had increased pancreatic FDG uptake. Focal abnormal pancreatic FDG activities were found in 38/40 (95.00%) PC patients, while longitudinal were found in 18/26 (69.23%) AIP patients. SUVmax was significantly different between AIP and PC, both in early and delayed PET/CT scans (18F-FDG PET/CT findings might help differentiate AIP from PC. Autoimmune pancreatitis (AIP) is a chronic pancreatitis characterized by pancreatic enlargement, irregular pancreatic duct stenosis, and increased serum IgG4 levels, mediated by autoimmune mechanisms . Althougselected from the population who were suspected of having pancreatic mass and underwent FDG PET/CT. Consecutive patients diagnosed with AIP from August 2010 to March 2014 at Changhai hospital were analyzed. The PC group comprised randomly selected age and sex matched patients to match the AIP group.In this retrospective study, the patients in our study were 18F-FDG PET/CT imaging results before treatment for AIP; (2) confirmed diagnosis of pancreatic cancer based on histological findings or liquid based cytology.Inclusion criteria were: (1) confirmed diagnosis of AIP based on the 14th International Association of Pancreatology diagnostic criteria  and Exclusion criteria were: (1) invasive examinations such as aspiration biopsy, ERCP, and stent placement before the PET/CT examination; (2) treatment for inflammation or cancer.Of the 36 enrolled AIP patients, 10 were excluded . Median age of the 26 AIP patients included was 60 years, ranging from 40 to 83 years; there was one female. Meanwhile, a total of 40 patients with pancreatic cancer  were enrolled. The study was approved by the ethics committee of Chnaghai hospital.Each of the 66 patients (26 AIP and 40 PC) underwent a whole body PET/CT. Meanwhile, 22/26 AIP patients and 36/40 PC patients had additional delayed PET-CT scans of the abdomen at 120 min after tracer injection.18F-FDG  was provided by Shanghai Atomic Sinovac Pharmaceutical Co., Ltd. Subjects were instructed to fast for more than 6 h, and 3.70-5.55 MBq/kg of 18F-FDG was intravenously injected when blood glucose (BG) < 11.1 mmol/L. Then, after resting in the waiting room for 60 min, a body topogram scan was performed using an electric current of 35 mA at a voltage of 120 kV, a scan time of 10.5-15.6 s and a scan thickness of 0.6 mm. Then, whole-body CT scans were performed using an electric current of 170 mA at a voltage of 120 kV, with a scan time of 18.67-21.93 s and scan thickness of 3 mm. Then, whole-body PET scans were performed covering 5-6 bed positions, with an acquisition time of 2.0-2.5 min per bed position. The 3D scanning of the head was performed additionally. Delayed PET scans of the pancreas were carried out with 1-2 bed positions,120 min after injection with 18F-FDG, using the same parameters described above. Images were reconstructed by the post-processing workstation TureD System, including the direction of cross-sectional, coronal, and sagittal tomographic images and three-dimensional projection images.The Siemens Biograph64 PET/CT  was used for the PET/CT. 18F-FDG PET/CT images were interpreted by two experienced nuclear medicine physicians blinded to clinical and histopathological data. Images were evaluated by visual, subjective and semi-quantitative (SUVmax) methods. Any disagreement was resolved by discussions. Mean values from both physicians were considered as final results. The mean retention index (RI) was calculated as RI =  was calculated. Pancreatic lesions were grouped into three categories: a) diffuse, b) focal, and c) multifocal. The multifocal type was defined as more than 1 foci of non-continuous pancreatic lesions were present. The presence or absence of each of the following parameters was determined as “yes” (present) or “no” (absent) by the reviewers: (1) increased FDG activity in the pancreatic lesion; (2) dilated main pancreatic duct; (3) biliary duct abnormalities, including increased FDG activity in the extra-pancreatic bile duct and gallbladder, dilation and wall thickening of intra and extra-hepatic bile ducts; (4) abnormal mediastinal, pulmonary hilar, peri-pancreatic or retroperitoneal lymph nodes; (5) retroperitoneal fibrosis; (6) inverted “V” shaped high FDG uptake in the prostate.t-test. Data with abnormal distribution were presented in median and interquartile range (IQR), and compared by rank-sum test. Using statistically significant data of the above parameters, ROC curves were generated and areas under the curves (AUCs) were calculated. Cut-off values were determined by the Youden index. The corresponding sensitivity, specificity, and positive predictive value were calculated. Differences in SUVmax between PET60min and PET120min scans were analyzed by t-test, and the corresponding SUVmax retention index calculated. Differences in detection rates between the two groups were determined using chi-squared analysis or Fisher’s exact test.Statistical analyses were performed with SPSS version 17.0. Measurement data with normal distribution were shown as The 26 AIP patients included 25 men and 1 woman with a median age of 60.0 ± 10.7; two patients were diagnosed based on IgG4-positive plasma cells found by submandibular lymph node and pancreatic biopsies, respectively. In 17 patients, endoscopic ultrasonography assisted fine-needle aspiration revealed no malignant cells. Meanwhile, 22 patients had abnormally elevated serum IgG4 levels. All 26 patients had clinical follow-up with imaging studies at least 6 months.n = 23) and endoscopic ultrasonography assisted fine-needle aspiration (FNA) and/or laparoscopy (n = 17). In PC group, diameter of the tumour was 30.9 ± 12.2 mm, and 57.5% (23/40) located in the pancreatic head, 22.5% (9/40) located in the pancreatic body, 10% (4/40) located in the pancreatic tail, 10% (4/40) located in the junction of pancreatic body and tail; 20% (8/40) was classified as Stage T1, 10% (4/40) was classified as Stage T2, 30% (12/40) was classified as Stage T3, 40% (4/40) was classified as Stage T4; 50% (20/40) was classified as Stage N0, 50% (20/40) was classified as Stage N1; 72.5% (29/40) was classified as Stage M0, 27.5% (11/40) was classified as Stage M1.The forty pancreatic cancer patients included 35 men and 5 women with a median age of 60.7 ± 10.6; final diagnosis was confirmed by surgical pathology . Eighteen patients with a diffuse pattern of increased pancreatic FDG uptake, six patients with a focal FDG uptake lesion and two patients with multifocal pattern were observed in 26 AIP patients. Average SUVmax was 5.24 ± 1.81.All patients had increased FDG activity of pancreatic lesions on early PET/CT scan (PETn = 40), the patterns of increased FDG metabolic activity included: focal (n = 38), diffuse (n = 1), multifocal (n = 1) types, with an average SUVmax of 7.30 ± 3.21. FDG activity of the lesions in PC patients was higher than that of the AIP group, with a statistically significant difference .In the PC group (120min), with a statistically significant difference  between average SUVmax between the two groups.Twenty-two patients with AIP and 36 patients with PC had delayed PET/CT scans  to diagnose PC.P < 0.05), but not of pancreatic lesions (P > 0.05)  in AIP and PC patients, respectively. There was statistically significant difference between the two groups in the RI values of the liver .Pancreatic duct dilatation was observed in only 6 of 26 (23.1%) AIP patients, and in 22 of the 40 (55.0%) patients with PC. Fisher’s exact test confirmed the statistically significant difference between the two groups (P < 0.05) and inverted “V” shaped high FDG uptake in the prostate (P < 0.001); however, there was no statistically significant difference in the positive rate of retroperitoneal fibrosis, FDG uptake of mediastinal and hilar lymph node, or peri-pancreatic peritoneum lymph nodes. Increased FDG activity of the extra pancreatic portion of the bile duct was only observed in the AIP group (4/26 of patients), but not in PC patients (0/40); with pancreatic lesions showing diffuse FDG accumulation, inverted “V” shaped high FDG uptake in the prostate was observed in 11 of the 20 patients with AIP, while none of the 2 patients with PC showed this feature . The AUC of the SUVmax of salivary glands in diagnosing AIP was 0.716; with a cut-off value of 1.92, sensitivity and specificity were 84.6 and 57.5%, respectively.Median SUVmax of salivary glands in AIP patients was 2.36 (range: 1.95- 3.41), and was 2.02 ± 0.76 in PC patients, indicating a statistically significant difference between the two groups (P > 0.05). No significant difference in the average SUVmax of peri-pancreatic and retroperitoneum lymph nodes was observed between the two groups (2.28 (1.31-3.78) for patients with PC, 2.03 ± 1.23 for patients with AIP, P > 0.05).Median SUVmax of mediastinal and hilar lymph nodes in AIP patients was 3.52 (range: 2.46-4.67) and was 2.77 (range: 2.48-3.99) in PC patients. No significant difference between the two groups was found . The AUC of prostate SUVmax in diagnosing AIP was 0.776; with a cut-off value of 2.94; sensitivity, specificity, and accuracy value were 56.0, 97.1, and 97.1%, respectively.Mean SUVmax of the prostate was higher in the AIP group (3.11 ± 1.27) than that in the PC group . AUC of this ratio in diagnosing AIP was 0.729; with a cut-off value of 1.02, sensitivity, specificity, and accuracy value were 56.0, 97.1, and 97.1%, respectively.The ratios of prostate SUVmax to liver SUVmax (prostate/liver) were also evaluated. Compared with the PC group (0.73 ± 0.15), AIP patients showed significantly higher values  is a specific type of chronic pancreatitis, including two subtypes, which might be identified using tissue pathology, clinical features and/or diagnostic criteria –16. TypeIn the early years, due to the lack of awareness of AIP, it was often misdiagnosed as PC. In a study of 37 AIP patients utilizing conventional imaging modalities, six patients were misdiagnosed as PC and two cases as cholangiocarcinoma . AnotherIt is well documented that increased FDG accumulation might be used as a marker of inflammatory lesions –21. ThusA focal, nodular pattern of increased FDG activity was significantly more frequent in patients with pancreatic cancer, whereas an longitudinal abnormal pancreatic FDG activities was more suggestive of AIP , 9. In tn = 26) and PC (n = 40) patients, suggested that both SUVmax and the SUVmax ratio of pancreas to liver in the early and delayed phases were significantly different. On early PET/CT scans with a cut-off value of SUVmax of 5.94, diagnostic sensitivity and specificity were 70.0 and 76.9%, respectively, for PC diagnosis. The larger sample size of AIP in the present study might be the reason for disagreement with prior reports. In addition, the following reasons could be considered as well. (1) Technical differences between previously used PET scanners and modern PET-CT scanners, which could account for discrepant SUV values. (2) In the current study, FDG SUV were compared in age and sex matched two groups, unlike previous studies [Previous studies suggested that FDG SUV of a lesion was usually greater than 4.0 in patients with PC, 3.0 - 4.0 in chronic pancreatitis patients and below 3 in healthy volunteers . Althoug studies , 9. (3)  studies . (4) The studies  study waPatients with AIP may have autoimmune inflammation involving the liver and biliary tract , 24; infWhen pancreatic cancer invades the bile duct, the main manifestation is direct compression of the bile duct, stenosis, or even an obstruction. In pancreatic cancer, the bile duct can also be compressed by a metastatic lymph node. In AIP patients, the inflammatory swelling of the pancreatic head could also cause narrowing and occlusion of the distal common bile duct, eventually leading to secondary expansion/dilatation of the upstream bile duct. In the current study, 46.2% patients in the AIP group showed biliary duct changes, which was only present in 25.0% of PC patients. The higher positive rate for the AIP group is possibly related to frequent involvement of the pancreatic head (88.5 AIP vs 52.5% PC). Besides concomitant bile duct inflammation might result in higher proportion of biliary system change in AIP patients. Moreover, this study revealed that FDG accumulation in the extra pancreatic portion of the bile duct was only observed in AIP patients.P < 0.05). At a threshold value for salivary gland’s FDG SUVmax of greater than 1.92, diagnostic sensitivity was 84.6% in the AIP group, for a specificity of 57.5%. These results suggested the involvement of the salivary gland in patients with AIP.A number of studies have shown that AIP also involves salivary glands, and is characterized by increased FDG hyper metabolism on PET/CT. Ozaki’s comparative study  found thOur previous studies demonstrated that over half of AIP male patients show inverted “V” shaped high FDG uptake in the region of the prostate gland , 13. ThiIn contrast to prior reports , 9, no sDiffuse uptake of FDG in the pancreas might be used to differentiate AIP from PC. Furthermore, some parameters including the ratio of pancreatic lesion/liver SUV, the SUV of salivary glands, the SUV of prostate might help to differentiate, and some morphological and metabolic features of EPLs including inverted “V” shaped high FDG uptake in the prostate and increased FDG activity in extra-pancreatic bile duct have high specificity for diagnosing AIP. FDG PET/CT could provide various findings as supplements to CECT and MR, which might improve the diagnostic accuracy.