Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (< 1 ng) starting genomic material for sequencing. Current solutions for amplifying sufficient DNA for metagenomics analyses include linear amplification for deep sequencing (LADS), which requires more DNA than is normally available, linker-amplified shotgun libraries (LASLs), which is prohibitively low throughput, and whole-genome amplification, which is significantly biased and thus non-quantitative. Here, we adapt the LASL approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a ‘reconditioning PCR’ step to increase yield and minimize late-cycle PCR artefacts, and empirically documenting the quantitative capability of the optimized method with both laboratory isolate and wild community viral DNA. Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with G + C content amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent, the first of which we tested and present data for here. Microbial processes drive much of the biogeochemistry that fuels the planet . FurtherYet, in most environments, there are few such model systems available, and ultimately a community-scale context is needed to understand the extent to which model system findings are a reliable proxy for wild populations. Researchers commonly turn to whole community sequencing, metagenomics  or wholeTwo recent developments set the stage for progress, particularly in environmental viral genomics. First, a new precipitation method improves aquatic viral concentration efficiencies from < 25%  to nearly 100% .Second, we identified an alternative high-fidelity polymerase (LA TaKaRa HS) to complement that previously used , as it can not be used to bound the upper size range (leaving DNA > 600 bp) due to a suboptimal ratio of PEG:DNA, the mechanism used to remove DNA fragments (Of the three sizing fractionation methods tested for target recovery efficiency (fraction recovered DNA in target 400–600 bp size range), throughput (ease of applicability to numerous samples simultaneously), and risk of cross-sample contamination, Pippin Prep, an automated optical electrophoretic system that does not require gel extraction, was the most efficient and reproducible  was 5.7% ± 2.3% efficient, with respect to total DNA recovery . We haveAfter optimizing the LA method, we sought to determine quantitatively the influence of starting DNA concentration and amplification parameters, such as cycle number and reconditioning . However, this was due to the unintentional reduced sequencing effort of the reconditioned samples . Aiming for average genome coverage of 15× should help to minimize these low coverage areas and avoid scaling issues that can interfere with cross-dataset comparisons when coverage is disparate. Based on this absence of discernible biases imposed by reconditioning, this step is highly recommended, as it has been shown to minimize heteroduplex formation during amplification  and using different cycling conditions are quantitatively comparable  and 4, wAs a common goal of ecology is cross-community comparisons of diversity, we also examined how LA impacted diversity profiles, represented as rarefaction curves of protein clusters with at least 20 members . Regardlnot occur in similarly prepared samples where the Pfu Turbo master mix was used, with the resultant amplified datasets thus appearing more diverse . Further, template mixtures can be used to sequence with Illumina technologies to circumvent issues with the non-random linker sequence on linker-amplified templates, which causes erroneous base-calling by the Illumina software. To this end, we recently pooled linker amplified DNA from 20 freshwater cyanophage isolates at a ratio of 1:1 with known phi29 DNA template (our template mixture) and successfully generated high quality, full-length Illumina sequence data  metagenomes appropriate for comparative analyses from samples with limiting amounts (< 1 ng) of DNA.Pseudoaltermonas phage H105/1  purified, as previously described (−1) for 2 h at room temperature, then DNase activity inactivated with 100 mM EDTA/EGTA. This virus preparation was purified on a CsCl gradient (recovering 7.1 × 1011 viruses ml-1 from the ρ = 1.4–1.52 CsCl fraction)   and DNA raction) , dilutedraction)  per the Covaris Adaptive Focused Acoustics (AFA) was used to shear DNA to 400–800 bp, with the following parameters: 130 µl of DNA in Tris EDTA (TE) buffer , duty cycle of 5%, intensity of 3200 cycles per burst, at 6–8°C for 62 or 120 s, for the Covaris E210 and S2 models respectively. DNA fragment sizes were determined before and after shearing on a DNA 7500 or High Sensitivity chip in the Agilent Bioanalyzer 2100 . Following shearing, low yield (< 500 ng) samples were concentrated two- to threefold  with Amicon Ultra-0.5100 kDa filter units , according to manufacturer's directions.not phosphorylated in order to promote unidirectional ligation) in a 50 mM NaCl buffer. Equal volumes of forward and reverse linkers were heated to 100°C, slowly cooled on the bench to room temperature, and placed on ice for 5 min. Resultant Linker-A was diluted to 10 µM in TE and stored at −20°C for subsequent use.Hemi-phosphorylated Linker-A  was prepBlunt-end repair of sheared DNA, ligation to Linker-A and required cleanup reactions  were per−1) was sheared to 400–600 bp (Covaris) to test three sizing methods in triplicate: gel electrophoresis, SPRI and a digital optical electrophoretic system, Pippin Prep . Each method was tested with 13.7 µl of sheared DNA at a concentration of 14.08 ng µl−1 . For gel extraction, sheared DNA was run on a 1.5% agarose gel stained with ethidium bromide for 90 min at 80 V. Gel fragments in the 400–600 bp size range were excised, purified to remove agarose (Qiagen MinElute Gell Extraction kit), and eluted in 20 µl EB buffer, as described (Results and discussion: LA method optimizations). To specifically retain the desired greater than 400 bp fragments a bead to DNA ratio of 55:100 was used, as determined per Roche  and TaKaRa LA Taq Hotstart , both with 3′ to 5′ exonuclease (‘proof-reading’) activity and an antibody quencher for hotstart capability, were assessed for efficiency and sensitivity. Both enzyme reactions used 1–2 µl Linker-A ligated and size-fractionated DNA with 0.5 µl (5 pmol) of the 10 µM PCR phos-A primer. For the Pfu Turbo Hotstart system, 12.5 µl Pfu Turbo Hotstart 2× Master Mix (0.1 U Pfu Turbo µl−1) was used, while the TaKaRa LA HS system required 2.5 µl 10× PCR buffer, 4.0 µl of 2.5 mM dNTP mix (10 nmol each), and 0.25 µl TaKaRa LS Taq HS (5 U LA TaKaRa µl−1). Both reactions were brought to 25 µl with nuclease-free water. For all reactions prepared for the sequence analysis , the TaKaRa LA Taq Hotstart system was used.Two high fidelity polymerases, A series of 5 bp barcodes was added to the ‘phos-A PCR primer’ (5′-p-CCACACAGATCACGAAGCATAC-3′) . The barTo determine the minimum number of PCR cycles needed for amplification of each sample, a small-scale PCR titration was performed with varying cycle numbers . Generally, three cycle numbers were tested according to the amount of DNA available after ligation and sizing, as a log-linear relationship exists between input DNA and cycle number needed for amplification . General−1 ethidium bromide, run in 1× Tris-acetate-EDTA (TAE) buffer at 90 V for 30 min, using 5 µl PCR product mixed with 1 µl 6× Blue/Orange Loading Dye (Promega). Quick Load 100 bp DNA Ladder , 250 bp DNA Ladder , or 1 kB Plus DNA Ladder (Invitrogen) was used for fragment size determination.PCR products were analysed on 1.5% agarose gels with 0.5 ng µlSample DNA was amplified (per PCR protocol above) using the number of cycles determined by the small-scale titration. Depending on quantity of starting DNA, six to ten reactions were performed in this step, resulting in up to 250 µl PCR product, to ensure sufficient DNA for sequencing (1–5 µg per standard 454 pyrosequencing or Illumina library).sensunot reconditioned  using gsMapper (v 2.5.3). Read depths were divided by the total number of nucleotides per treatment to normalize for sequencing effort. %G + C and average read depths were calculated over a 500 bp sliding window to generate ‘GC bias’ plots.k-mer length of 21. Genes were predicted on all raw reads and contigs greater than 100 bp using Prodigal   contained an ambiguous base, (ii) were at least two standard deviations from the mean sequence length per plate, (iii) were at least two standard deviations from the mean quality score per plate, or (iv) were identified by cd-hit454 (default parameters) as emulsion-PCR replicates. %G + C was calculated per read. For the rarefaction analysis, reads from all treatments were assembled using Velvet  with autdefault)  with wordefault)  at 90% wt-tests. To quantify the magnitude of read depth and %G + C bias, the area between the unamplified and amplified curves was calculated by trapezoid integration. Pairwise tests were performed to compare (i) all reconditioned versus all non-reconditioned samples (n = 14 pairs), (ii) the reconditioned and non-reconditioned per cycle number (n = 3 pairs), (iii) all treatments of a cycle number against each of the remaining cycle number treatments, reconditioned and non-reconditioned combined (n = 6 pairs), and (iv) ‘c’ repeated, testing reconditioned and non-reconditioned separately (n = 3 pairs).Tests comparing the relative read depths and %G + C bias between unamplified and amplified treatments were performed using a series of paired two-tailed Student's http://eebweb.arizona.edu/faculty/mbsulli/protocols.htm. All sequence data is available on the CAMERA web portal, tracking number CAM_P_0000912.The LA protocol is available at


Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A plethora of lipid mediators exist which makes the development of generic analytical methods challenging. Here we developed a robust and sensitive targeted analysis platform for oxylipins and applied it in a biological setting, using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) operated in dynamic multiple reaction monitoring (dMRM). Besides the well-described AA metabolites, oxylipins derived from linoleic acid, dihomo-γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were included. Our comprehensive platform allows the quantitative evaluation of approximately 100 oxylipins down to low nanomolar levels. Applicability of the analytical platform was demonstrated by analyzing plasma samples of patients undergoing cardiac surgery. Altered levels of some of the oxylipins, especially in certain monohydroxy fatty acids such as 12-HETE and 12-HEPE, were observed in samples collected before and 24 h after cardiac surgery. These findings indicate that this generic oxylipin profiling platform can be applied broadly to study these highly bioactive compounds in relation to human disease.The online version of this article (doi:10.1007/s00216-012-6226-x) contains supplementary material, which is available to authorized users. Oxylipins are oxygenated metabolites derived from poly-unsaturated fatty acids (PUFAs) such as arachidonic acid (AA), linoleic acid (LA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and dihomo-γ-linolenic acid (DGLA). The oxygenated 20-carbon products, collectively referred to as eicosanoids, are the best-described lipid mediators. Among their many functions, these lipids are potent inflammatory modulators and thus have been associated with responses to cardiovascular diseases, host defense, tissue injury, and surgical intervention –3.2 and TXB2. Class 1 and 3 PGs and TX are similarly produced through the oxidation of DGLA and EPA, respectively −. The water loss, especially for the hydroperoxides from AA, was also described previously [For each compound, optimal transitions were determined to assure optimal peak detection , reproducibility, matrix effect, and recovery were determined Table .Table 1VR2 ranged from 0.991 to 0.999, followed by 0.981–0.99 for 26 % and >0.9 for 17 %  were spiked with academic standards (eight concentrations). We determined the linearity, LOD, and LOQ of each compound (104 targets). For the majority of the analytes, the 1α, PGK2, Hepoxilin A3, 5,6-Lipoxin A4, LTE4, 5-HpETE, 12-HpETE, 15-HpETE, 9-HpODE, 13-HpODE, and PGF3α need to be noted  of the expected values were observed, prohibiting this approach to calculate matrix effects. Since water is not suitable as blank matrix, matrix variation tests are recommended before application to the various matrices. Although no matrix effect was determined, final concentrations were not effected as the calibration lines were made in matrix as well.4, the recovery was lower than 60 %. In contrast to the other selected oxylipins, LTE4 contains a cysteinyl group, and like LTDs and LTCs, it belongs to the cysteinyl leukotriens. While recoveries differed between chemical class, inter-batch reproducibility was acceptable with RSD values between 4.3 % and 9.6 %, except for (d3) LTE4 and (d6) 20-HETE with more than 10 % (10.3 % and 12.9 %).For the recovery calculations, a set of deuterated ISTDs was used to prevent endogenous influences. Therefore, the standards were spiked either before or after the extraction and ratios of the peak areas were calculated. Recoveries ranged from 84 % (±4.4) to 45 % (±5.8), depending on the compound .Our findings indicate that the analytical platform developed here allows the detection of consistent alterations in oxylipin profiles, even within a small patient population Below is the link to the electronic supplementary material.


Hyptis albida (CHA) on inflammatory responses in mouse lipopolysaccharide (LPS) induced peritoneal macrophages. Our findings indicate that CHA inhibits LPS-induced production of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). During the process, levels of cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), and nitric oxide (NO) increased in the mouse peritoneal macrophages; however, the extract suppressed them significantly. These results provide novel insights into the anti-inflammatory actions of CHA and support its potential use in the treatment of inflammatory diseases.We examined the effects of a chloroform extract of  Inflammatory disorders are treated using conventional anti-inflammatory drugs such as steroidal anti-inflammatory drugs and nonsteroidal anti-inflammatory drugs (NSAIDs) [Inflammation is an immediate response to many injuries produced by pathogens, noxious stimuli such as chemicals, or physical injury. Inflammation involves the activation and recruitment of phagocytes , NK cells, the complement system, and the secretion of cytokines such as IL-1(NSAIDs) . Thus, i(NSAIDs) . Hyptis consists of approximately 400 species distributed from Southern United States to Argentina [Hyptis has many species that are important in Mexican folk medicine. In particular, Hyptis albida is commonly used in remedies for the treatment of gastrointestinal disturbances, skin infections, rheumatism, cramps, and muscular pains [The genus rgentina . They hargentina –8. The gar pains , 10. Thrar pains  and the ar pains . The preH. albida were collected in Guadalcazar, San Luis Potosi state, México. The plant was identified by taxonomist José García Pérez. A voucher specimen (SPLM 20419) was deposited in the Isidro Palacios Herbarium of the Universidad Autónoma de San Luis Potosi.Aerial parts of The shade-dried aerial parts were reduced to a powder, and 100 g of the powder sample was refluxed for 4 h with 400 mL chloroform. The extract was filtered, and the solvent was removed under reduced pressure (yield 5.3%). The extract showed positive results on Liebermann-Burchard, Tortelli-Jaffe, and Tschugaeff tests for terpenes and positive results on boric acid and citric acid for flavonoids .μg/mL), in an atmosphere of 5% CO2 at 37°C.Macrophages were obtained from the peritoneal cavity of male BALB/c mice. Each mouse was injected with 1.5 mL of 4% thioglycollate in the peritoneal cavity. After 72 h a peritoneal lavage was performed with 10 mL cold 1x PBS buffer. The injected buffer was recovered and centrifuged to isolate cells. Cells were quantified using a Neubauer chamber and were cultured in plates for 24 h. Nonadherent cells were removed, and adherent cells were cultured in fresh medium. Peritoneal macrophages were maintained with RPMI supplemented with inactivated fetal bovine serum (FBS) at 10% and antibiotics, penicillin (100 units/mL) and streptomycin (100 6 cells/well) were cultured in a 12-well plate for 24 h at 37°C in 5% CO2. After the medium was removed and replaced with fresh medium, CHA was added and incubated for 24 h. Viable cells were assessed with 0.4% crystal violet staining solution. Briefly, 200 μL crystal violet was added to each well, and the cells were incubated for 30 min at room temperature until the crystal violet solution was changed to acid (33%). The solution was removed, and the absorbance was measured at 540 nm in a microplate reader [Cells  was added followed by incubation for a further 24 h. The cell supernatant was collected for nitrite analysis, and the pellet cell was used for the PCR assay. Nitrite production, an indicator of NO synthesis, was measured in the supernatant of cultured macrophages using the Griess reaction [μL) of treated culture supernatant and Griess reagent  were mixed and incubated at room temperature for 5 min, and the absorbance was measured at 540 nm in a microplate reader. The amount of nitrite in the sample was determined using a sodium nitrite standard curve. Peritoneal macrophages were cultured at a density of 1 × 10reaction . Brieflyμg) was carried out with M-MuLV reverse transcriptase  and oligo-(dT)18 primers. PCR was performed in a reaction mixture containing the resulting cDNA, dNTP mixture (Promega), 10 pmol of target gene-specific primers, and 0.25 units of Taq DNA polymerase (Promega). Primers were designed using the Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast)  . PCR pro6 cells/well and incubated overnight. Cell cultures were pretreated with different concentrations of CHA for 2 h thereafter; LPS (1 μg/mL) was then added followed by incubation for a further 24 h. The inhibitory effect of CHA on the production of proinflammatory cytokines (IL-6 and TNF-α) was determined in the supernatants. Quantities of cytokines were measured using a mouse ELISA kit (eBioscience).Peritoneal macrophages were cultured at a density of 2 × 10t-test). Statistical significance was accepted at P < 0.05.All values are expressed as the mean ± SEM. Differences between mean values of normally distributed data were assessed with one-way ANOVA . When cells were exposed to 1 μg/mL LPS for 24 h, the nitrite concentration increased markedly, to 22.42 ± 1.41 μM. CHA inhibited the production of NO after LPS stimulation in a dose-dependent manner by 36.62, 51.42, and 61.0% at 25, 50, and 100 μg/mL, respectively,  at 100 μg/mL  and induce the expression of proinflammatory mediators, which can promote inflammation by increasing cGMP levels and vascular permeability [γ), tumor growth factor beta (TGF-β), and proinflammatory cytokines [However, overproduction of NO during inflammation can activate nuclear factor kappa B  [α, was inhibited by flavonoids in LPS-treated human blood monocytes and RAW 264.7 cells [Many terpenoids and flavonoids possess anti-inflammatory activities in various animal models of inflammation. Many investigators have proposed cellular mechanisms of action explaining the activity . Severalactivity , and inhactivity  and NF-κtivation . Some sttivation . On the 2 (PLA2) , 48, COX2 (PLA2) , and lip2 (PLA2) –52 and i2 (PLA2) . Flavone2 (PLA2) , 55. The.7 cells , 57.H. albida and characterize their mechanism of action.In conclusion, we demonstrate that CHA is a potent inhibitor of inflammation. It inhibited the release of inflammatory mediators from macrophages and suppressed the overexpression of relevant genes. Further studies are underway to isolate the active components from


Seventy-two hours of preoperative fasting (F) or 2 weeks of 30% dietary restriction (DR) offers robust protection against renal ischemia-reperfusion injury (IRI) in mice. However, the mechanism remains to be elucidated. We hypothesize that immunomodulation plays a pivotal role. Innate immunity, especially the complement system, is crucial in the pathophysiology of IRI. Therefore, we investigated the impact of fasting and dietary restriction on complement activation pathways.ad libitum (AL) or underwent 72 hours fasting or 30% dietary restriction for 2 weeks (n=8/group). Consequently blood was drawn and serum was aliquoted and stored at -80°C. Functional activity of the complement activation pathways , lectin (LP) and alternative pathway (AP)) was assessed by ELISA using immobilized ligands. Deposition of C3 and C9 as a measure of complement activity along with concentration of upstream complement initiating proteins  was determined.Male C57BI/6 mice were fed A significant downregulation in CP and LP activity by dietary restriction and in CP, LP and AP activity by fasting was observed, compared to the ad libitum group. The activation of both C3 and C9 in the dietary restriction and fasting group was significantly downregulated (p≤0.002) in CP, LP and AP (except for C3 activation in the AP of the dietary restriction group). The MBL-A concentrations were significantly lower (p≤0.001) after dietary restriction and fasting; 15.4 µg/ml (DR) and 12.4 µg/ml (F) compared to 19.9 µg/ml in ad libitum mice. MBL-C concentrations were also significantly lower (p≤0.0001) in the dietary restriction and fasting groups; 89.4 and 49.5 µg/ml respectively compared to 109.6 µg/ml in the ad libitum group. C1q concentration was only significantly lower in the fasted group (p≤0.0001).Dietary interventions downregulate complement activation pathways. Compared to dietary restriction, fasting has a more pronounced effect. CP seems to be more affected by dietary restriction while AP is most affected by fasting. To our knowledge, our data for the first time show that DR and fasting cause downregulation of complement activation pathways. Therefore, we conclude that complement downregulation may be one of the mechanisms by which dietary interventions protect against renal IRI.


Adipose tissue (AT) contributes to metabolic dysfunction through imbalanced production of adipokines, including cytokines. Visceral AT in particular is associated with metabolic disorders, indicating a specific secretory status. The relative significance of different human AT depots in adipokine release is not fully known. Further, previous in vitro systems usually included medium containing bovine serum albumin (BSA), which may induce cytokine release. Our aim was to compare release of a number of adipokines/cytokines – all implicated in insulin resistance – from human subcutaneous and visceral AT in a short-term incubation system minimizing cytokine induction and including repeated measurements during 24 h. A prerequisite was to evaluate a potential alternative to BSA in the incubation medium.2) undergoing elective surgery was incubated for 2, 4, 6, 8, and 24 h in medium with or without 1% BSA or human serum albumin (HSA). Medium concentrations of adiponectin, chemerin, nine cytokines, dipeptidyl peptidase 4 (DPP4), and omentin were analyzed by multiplex immunoassay or ELISA. Adipocyte size, AT macrophage density, and medium concentrations of endotoxin were determined.Subcutaneous and/or visceral AT from 17 patients , and DPP4 from the visceral depot, while adiponectin release was higher from subcutaneous than visceral AT. Release was linear for a maximum of 2–6 h. Macrophage density was higher in visceral than subcutaneous AT. Levels of endotoxin in the medium were negligible.Adiponectin, chemerin, many cytokines, and DPP4 are released from human AT in a depot-dependent manner. These results highlight functional differences between visceral and subcutaneous AT, and a mechanistic link between regional fat accumulation and metabolic disorders. Supplementation of human AT incubation medium with HSA rather than BSA is recommended to minimize induction of cytokine release. Adipose tissue is a major signaling organ that produces and releases many proteins with autocrine, paracrine, and endocrine activity, the so-called adipokines ,2. DysreAccumulation of visceral fat in particular is strongly associated with metabolic disorders ,7, indicAddition of bovine serum albumin (BSA) to the medium is often recommended in protocols for adipose tissue incubation, since albumin binds fatty acids that are mobilized and exert toxic effects when unbound . In 2006In this study, our aim was to compare the release of adipokines, including cytokines and other inflammatory/metabolic biomarkers, from human subcutaneous and visceral adipose tissue in a short-term incubation system minimizing induction of cytokine release, and including repeated measurements during 24 h. Further, a prerequisite was to first evaluate the influence of different albumin preparations regarding induction of cytokine release from human AT. Using our final incubation protocol, we investigated nine pro-inflammatory cytokines, the anti-inflammatory and intensively studied adiponectin, and chemerin and dipeptidyl peptidase 4 (DPP4), all with incompletely investigated, controversial or unexplored depot-dependent release.2 (range 22.6–56.7 kg/m2). Subcutaneous abdominal adipose tissue biopsies were obtained from all subjects. Paired biopsies of subcutaneous and visceral (omentum) adipose tissue were collected from the 11 subjects (5 men and 6 women) undergoing gall bladder surgery or RYGB; these subjects had a mean age of 52.2 ± 15.1 yr (range 20–68 yr) and a mean BMI of 41.6 ± 8.9 kg/m2 (range 27.0–56.7 kg/m2). No subjects with malignant disease were included. All participants gave oral and written consent. The study was conducted in accordance with the Declaration of Helsinki and approved by the Regional Ethical Review Board, University of Gothenburg.Adipose tissue biopsies were obtained from 17 patients (6 men and 11 women) undergoing elective abdominal surgery  in 9). The mean age was 48.7 ± 13.1 yr (range 20–68 yr), and the mean BMI was 35.8 ± 10.8 kg/m3, 0.15 μM adenosine, 1% HSA , 0.1 mg/mL cephalotin , pH adjusted to 7.4. Medium was then removed and centrifuged for 5 min at 450 g. Aliquots of medium were stored at –80°C until analysis.Biopsies were initially handled as described in our protocol for long-term incubation ,22. In bTo evaluate the influence of albumin preparation, exploratory incubations for 2, 4, 6, 8, and 24 h were carried out in medium lacking albumin and in medium supplemented with 1% HSA, 1% BSA Fraction V, or 1% BSA Essentially Fatty Acid Free .The influence of 0.1% BSA was also studied. For practical reasons, only one tube of tissue was incubated with each medium, which was replaced after 4 h. After additional 20 h incubation, the second portion of incubation medium was removed.2 atmosphere in an incubator ; the medium was stirred gently with a rotator.All incubations were performed at 37°C in a 5% COA multiplex immunoassay was used to measure the levels of nine cytokines in the incubation medium: granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, and TNF-α . ELISA was used to measure adiponectin , chemerin , omentin , IL-6 , and DPP4 . Adipokine concentrations were expressed per g adipose tissue.About 500 mg of each fresh biopsy was digested with collagenase , and thePaired subcutaneous and visceral adipose tissue specimens were fixed in phosphate-buffered formalin, dehydrated, embedded in paraffin and sectioned at 4 μm. After hydration and heat-induced antigen retrieval, a monoclonal antibody targeted at human CD68 was added to detect macrophages . MACH 3 Mouse AP-Polymer and Warp Red was used as secondary and detection reagents . The sections were counter stained with hematoxylin and mounted. Slides were digitalized  and macrophages were counted and normalized for analyzed section area . Origin of the tissue was unknown to the observer.The endotoxin concentrations in unconditioned medium with or without 1% of the different albumin preparations were analyzed at the Laboratory of Clinical Bacteriology, Sahlgrenska University Hospital, using a LAL assay with quantitative chromogenic end point . The lower limit of detection was 0.5 pg/mL. All determinations were performed at least in duplicate.t test and Bonferroni correction. The influence of 0.1% BSA was evaluated by two-way analysis of variance  followed by Dunnett’s test. Differences in adipokine release from subcutaneous and visceral adipose tissue over time were assessed by three-way analysis of variance . To assess differences in adiponectin release between obese and nonobese subjects, we designed a split-plot experiment with subjects as main plots and times as subplots. Differences between groups were estimated from main plots, and differences between times were estimated from subplots [t test. The relationship between adiponectin release and adipocyte size was investigated with Pearson correlation analysis. Simple regression was used to investigate the relationship between IL-6 concentrations in the medium determined by multiplex immunoassay and ELISA (non-transformed data). P < 0.05 was considered significant. All statistical analyses were conducted in SPSS .Values are expressed as mean ± SEM. To stabilize variances, values were logarithm-transformed. The influence of different albumin preparations (1%) on adipokine release over time was determined by three-way analysis of variance  followed by paired subplots . AdipocyIn three experiments, subcutaneous adipose tissue was incubated for 2, 4, 6, 8, and 24 h in albumin-free medium or medium supplemented with 1% HSA, 1% BSA Fraction V, or 1% BSA Essentially Fatty Acid Free. Both preparations of BSA markedly induced the release of all nine cytokines as analyzed by multiplex immunoassay , as analyzed by multiplex immunoassay. After additional 20 h of incubation, concentrations of all nine cytokines were higher in both media with 0.1% BSA than in albumin-free medium  (data not shown).The endotoxin concentration in a batch of unconditioned medium without albumin was <0.5 and 1.6 pg/mL (duplicate). In three batches of unconditioned medium supplemented with 1% HSA, the endotoxin concentration was 6.2, 9.1, and 3.8 pg/mL, respectively. The corresponding concentrations of endotoxin in medium containing 1% BSA Fraction V and 1% BSA Essentially Fatty Acid Free were 9200 and 11800 pg/mL.Adipocyte size in 11 paired biopsies of subcutaneous and visceral adipose tissue was analyzed with a computerized technique. The mean adipocyte volume was similar in subcutaneous and visceral adipose tissue . The mean diameter of reference microspheres was 97.63 ± 0.25 μm . On average, 601 subcutaneous adipocytes (range 322–1245) and 515 visceral adipocytes (range 232–1164) were analyzed in each biopsy.2, p < 0.05)  was incubated for 2, 4, 6, 8, and 24 h in medium containing 1% HSA according to our final incubation protocol. Eleven paired biopsies of subcutaneous and visceral adipose tissue was also incubated accordingly. Adiponectin and omentin concentrations in the media were analyzed by ELISA. As expected, adiponectin was more abundantly released from adipose tissue of nonobese than of obese subjects (p < 0.05) Figure A, and adEleven paired biopsies of subcutaneous and visceral adipose tissue was incubated according to our final protocol. As shown by multiplex immunoassay, release of all nine cytokines but IL-6 were more abundant from visceral than subcutaneous adipose tissue over 24 h of incubation  (data not shown).Paired biopsies of subcutaneous and visceral adipose tissue were incubated according to our final protocol, and concentrations of adiponectin, chemerin, IL-6, and DPP4 were determined by ELISA. Adiponectin was more abundantly released from subcutaneous than visceral adipose tissue  , indicating almost complete agreement between the methods while the multiplex technique systematically underestimated the IL-6 concentration in the samples by about 30%.This study shows that adiponectin, chemerin, eight pro-inflammatory cytokines, and DPP4, all inflammatory and/or metabolic biomarkers, are released from human adipose tissue in a depot-dependent manner. Chemerin, cytokines, and DPP4 were more abundantly released from visceral than subcutaneous adipose tissue, while adiponectin release was higher from the subcutaneous depot. Our findings also showed that cytokine release from human adipose tissue was markedly induced by BSA but not by HSA which was therefore used in our final protocol.In particular, when comparing adipose tissue biopsies—from different groups of subjects, from different depots, or from an individual at different time points—with respect to adipokine/cytokine release, in vitro conditions must be standardized and selected with care . In testIt may be noted that no differences in cytokine release was observed between medium containing HSA and medium without albumin. In a short-term incubation system like ours, with a high medium-to-adipose tissue ratio, supplementation of the medium with albumin may not be necessary.As in a previous study of isolated adipocytes , cytokinAs mentioned above, measures of obesity are associated with reduced plasma levels of adiponectin, an adipokine predominantly produced by adipocytes ,28,31,38To our knowledge, the possibility that release of chemerin is depot-dependent has not been investigated by incubation of human adipose tissue. Chemerin is a rather novel adipokine that regulates adipogenesis and may induce insulin resistance ,47. In oDPP4, another recently discovered adipokine, may also impair insulin sensitivity . Serum DAccumulating evidence suggests that obesity—in particular central/visceral accumulation of fat—and its co-morbidities correlate with increases in circulating levels of inflammatory proteins such as C-reactive protein and IL-6 ,5,52,53.Another possible limitation of this study is the rather heterogeneous group of patients included. However, analysis of paired biopsies from the obese subjects separately did not affect the results obtained. Further, the multiplex immunoassay is mainly intended for screening purposes. However, we observed an almost complete agreement between the multiplex immunoassay and the specific ELISA regarding analysis of IL-6 although the multiplex technique systematically underestimated the IL-6 concentration by about one third.Taken together, our findings show that human adipose tissue is characterized by depot-dependent release of many adipokines. Chemerin, GM-CSF, IFN-γ, IL-1β, IL-2, IL-8, IL-10, IL-12p70, TNF-α, and DPP4 were more abundantly released from visceral than subcutaneous adipose tissue, while adiponectin release was higher from the subcutaneous depot. These results highlight functional differences between visceral and subcutaneous adipose tissue, and a mechanistic link between regional fat accumulation and metabolic disorders. Our results also demonstrate that the system applied, including HSA instead of BSA, is suitable for further investigations of the human adipose tissue secretome and relative differences between depots. Further, for any in vitro system for human adipose tissue or adipocytes, potential albumin effects on tissue/cell function should be considered.The authors declare that they have no competing interests.SE and ML designed the study. HS and BO collected patient material and performed the experiments. HS, SE and ML analyzed and interpreted the data. HS, BO, SE drafted the manuscript. ML revised the final version of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/14/7/prepub


Lasiopodomys brandtii), a rodent species from Inner Mongolia grassland in China.Early postnatal environments may have long-term and potentially irreversible consequences on hypothalamic neurons involved in energy homeostasis. Litter size is an important life history trait and negatively correlated with milk intake in small mammals, and thus has been regarded as a naturally varying feature of the early developmental environment. Here we investigated the long-term effects of litter size on metabolic phenotype and hypothalamic neuropeptide mRNA expression involved in the regulation of energy homeostasis, using the offspring reared from large (10–12) and small (3–4) litter sizes, of Brandt's voles  mRNA in the hypothalamus at weaning or in adulthood, however, hypothalamic suppressor of cytokine signaling 3 (SOCS3) mRNA in adulthood increased in small litters compared to that in large litters. As a result, the agouti-related peptide (AgRP) mRNA increased in the offspring from small litters.These findings support our hypothesis that natural litter size has a permanent effect on offspring metabolic phenotype and hypothalamic neuropeptide expression, and suggest central leptin resistance and the resultant increase in AgRP expression may be a fundamental mechanism underlying hyperphagia and the increased risk of overweight in pups of small litters. Thus, we conclude that litter size may be an important and central determinant of metabolic fitness in adulthood. Early-life environmental influences on the adult metabolic phenotype are of interest both scientifically and clinically, as it relates to the risk factors contributing to the obesity epidemic Lasiopodomys brandtii), which have a mean litter size of seven (litter size varies from 2 to 14) Litter size is an important life history trait The mediobasal hypothalamus is the site of energy homeostasis, and can exquisitely sense and integrate peripheral metabolic cues to coordinate peripheral metabolism In the present study, we investigated the long-term effects of natural litter sizes on offspring metabolic phenotype and biomarkers, such as food intake, resting metabolic rate (RMR), nonshivering thermogenesis (NST), uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), body compositions, serum leptin and tri-iodothyronine (T3) and thyroxine (T4) levels in adulthood. In addition, we analyzed gene expression of OB-Rb, SOCS3, and orexigenic and anorexigenic neuropeptides in hypothalamus from young and adult voles raised in large and small litters. We hypothesized that postnatal litter size would permanently influence offspring metabolic phenotype and hypothalamic neuropeptide expression. We predicted that offspring reared from small litters would exhibit hyperphagia, excessive weight gain and hyperleptinemia in adulthood as compared to the counterparts from large litters, and that these phenotypes in small litters would be related to greater expression in hyperthalamic orexigenic neuropeptides.All experimental protocols were reviewed and approved by the Animal Care and Use Committee of Institute of Zoology, the Chinese Academy of Sciences. The institute does not issue a number to any animal study, but each study requires the permit to use animals from the ethical committee. The animal facility must be licensed by the experimental animal committee of Beijing, and all staff, fellows and students must receive appropriate training before performing animal studies.ad libitum.Brandt's voles were the offspring of our laboratory breeding colony founded by field-captured animals. After weaning (21 days of age), voles were housed as same gender sibling pairs in plastic cages (30×5×20 cm) and maintained in temperature (23±1°C) and humidity-controlled rooms under a 16∶8 h light/dark photoperiod with lights on at 04:00 h. All animals were provided standard rabbit pellet chow  and water n = 12; SM, male from small litter, n = 9; LF, female from large litter, n = 13; SF, female from small litter, n = 10) were used in this study.At 3–4 months of age, virgin female voles were housed individually and acclimated for 2 weeks and then were paired with males for 4 days to allow mating. On the day of parturition, the dams with 10–12 pups (regarded as large) and 3–4 pups  were selected to compare the effect of litter size, since we found previously that there was a difference in pup body mass only between these two groups 2 overdose between 09:00 h and 11:00 h at 13 weeks of age. Trunk blood was collected and centrifuged at 4000 rpm for 30 min at 4°C and serum stored at −80°C until assayed. The whole brains were rapidly removed and placed on dry ice for slow freezing. A slice of brain tissue was cut between the optic chiasm and the mammillary bodies, and the hypothalamus was dissected by one horizontal cut immediately below the anterior commissure and sagittal cuts through the edge of the septum and perihypothalamic sulcus as previously described All the animals were sacrificed by CO2 consumption and CO2 production at 30°C  . A vole was placed in a chamber  for 2 h. The flow rate of incurrent and excurrent air  was approximately 300–400·ml·min–1 and 100 ·ml·min–1, respectively. The baseline of oxygen and carbon dioxide concentration were measured before and after each test. Oxygen consumption was recorded at intervals of 10 s. RMR was estimated from the stable lowest consecutive rate of oxygen consumption over 5 min.RMR and NST were measured by using an open-circuit respirometer . To avoid possible effects of circadian rhythm interfering with the group effects, two groups of animals were measured in an alternating manner between 08:00 h and 17:00 h. RMR was assessed from the rate of OThe voles stayed in the chamber for another 1 h for NST measurement. Maximum NST was induced by a subcutaneous injection of norepinephrine (NE) at 25±1°C and mass-dependent dosage of NE  was calculated according to Heldmaier O2 = 2, CO2), Fi = input fractional concentration, Fe = excurrent fractional concentration).VAfter dissection of the hypothalamus and iBAT, the following organs and tissues, including the heart, lungs, liver, kidneys, spleen, gonad, stomach, small intestine, caecum, colon, together with subcutaneous fat, epididymal fat, mesenteric fat, epigonadal fat were extracted and weighed (±1 mg). The organs and carcass with fat pads were then dried in an oven at 60°C to constant weight. Body fat extraction from dry grinded carcass was performed with a Soxhlet Fat Extraction System  with petroleum ether.125I Multi-species Kit , which had been validated in Brandt's voles Serum leptin levels were determined by radioimmunoassay (RIA) with the Serum T3 and T4 were quantified using RIA kits  according to the instructions and we have validated this kit for use in Brandt's voles previously Total protein content in iBAT was determined by Folin phenol method with bovine serum albumin as standard Primary antibodies used were as follows: rabbit anti-UCP1 , diluted 1∶10,000; mouse anti-COX4 , diluted 1∶1,000; rabbit anti-SIRT1 (H-300) , diluted 1∶1,000; mouse anti-β-tubulin , diluted 1∶5000. The secondary antibodies of goat anti-rabbit IgG  and goat anti-mouse IgG  were used.Total RNA was isolated using Trizol  following the manufacturer's instructions. After treating with DNase I , RNA was reverse transcribed  using a first-strand cDNA synthesis kit .Real time PCR reactions were performed in a 12.5 µL total volume comprised of 6.25 µL 2×SYBR Premix EX Taq™master mix, 1 µL cDNA templates and 0.2 µmoL/L primers using the SYBR Green I qPCR kit  in the Mx3005P quantitative PCR system . Thermal cycling conditions were: 95°C for 10 s, 40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 20 s. Samples were run in duplicate and all runs were accompanied by the housekeeping gene β-actin. Species-specific primers were designed  and veriData were analyzed using SPSS 13.0 . Prior to all statistical analyses, data were examined for assumptions of normality of variance using the Kolmogorov–Smirnov tests. Non-normally distributed data underwent logarithm or arcsine square root transformation. The temporal changes in body mass and food intake were assessed by repeated-measures ANOVA, followed by LSD post-hoc test. Group differences in body mass and food intake were assessed by two-way ANOVA and ANCOVA respectively. Differences among groups in serum leptin, T3, T4 levels, the mRNA levels of BAT UCP1, hypothalamic Ob-Rb, SOCS3, NPY, AgRP, POMC and CART in adult were assessed by two-way ANOVA, followed by Tukey post-hoc test. Differences in body compositions were analyzed by two-way ANCOVA with body mass as a covariate. At weaning, the differences in UCP1 and hypothalamic gene expression were assessed by independent samples t-test. Pearson correlation analyses were used to detect possible associations of serum leptin levels with body fat mass and food intake. Data are expressed as mean±SE. Values of P<0.05 were considered statistically significant.1, 42 = 79.420, P<0.001) , and the males were 20% heavier than females . After adjusting for the effect of body mass at weaning, body mass was no longer affected by litter size (P>0.05), but remained significantly influenced by gender (P<0.01) until the end of the experiment. During the course of experiment, all voles showed a continuous growth until weeks 10 or 11, after which, body mass stabilized   . At weekP<0.001) .1, 41 = 2.682, P>0.05), gender , or their interaction   . During 1, 41 = 12.503, P<0.001; 1, 41 = 3.602, P = 0.065; 1, 41 = 4.605, P>0.05) or NST  between litter sizes. Males from small litters had lower RMR compared to any other group , but there was no significant difference in NST among groups .The females had markedly higher RMR  or gender    . Adult f P>0.05) . In adul P>0.05) , but not P>0.05) .1, 35 = 4.367, P<0.05; 1, 35 = 7.010, P<0.05; 1, 35 = 1.745, P>0.05; 1, 35 = 2.466, P>0.05), T4  or the ratio of T3/T4 .We did not measure serum T3 and T4 in the offspring at weaning because of small volume of serum. The adult offspring from small litters had lower T3 , and 26% higher COX4 content  than males, which was in accordance with higher RMR and NST in females. Further, SIRT1 content in iBAT was influenced by the interaction of litter size and gender .At weaning , there wThere was no difference in hypothalamic OB-Rb, SOCS3, NPY, AgRP, POMC and CART mRNA expression between large and small litters at weaning .1, 22 = 3.600, P>0.05) or gender    . In cont P<0.05) .1, 22 = 2.572, P>0.05 , but there was no gender difference    nor was  P>0.05) .1, 39 = 14.592, P<0.001). In addition, BAT mass  and dry stomach mass  were higher in females as compared to males. However, there were no differences observed in other organs between either litter sizes or gender.The data for organ mass are presented in 1, 39 = 8.762, P<0.01), mesenteric , perinephric  and total fat mass , but less epigonadal fat , and other fat pads and total fat mass did not vary with litter size , while females has less dry carcass mass than males , but no changes in anorexigenic POMC and CART in the vole offspring from small litters in adulthood. Hypothalamic orexigenic NPY and AgRP mRNA expression also increased in high-fat fed rats from reduced litter size Taken together, voles from small litters showed greater food intake and body mass than voles from large litters. Litter size did not affect adult serum leptin levels, but did have long-term effects on hypothalamic leptin sensitivity. In addition, increased AgRP expression was associated with hyperphagia in the offspring from small litters. These findings provide further evidence that litter size can permanently influence leptin sensitivity and hypothalamic neuropeptides, resulting in bonafide changes in the adult metabolic phenotype. From a physiological point of view, this study also highlights the importance of litter size in evolution, and suggests that animals raised in natural litter sizes would not be susceptible to obesity unless subjected to artificial manipulation during the early postnatal period.


Edwardsiella tarda, a Gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from Gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1β, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p<0.0001) than were control fish. Investigation of OMVs may be useful not only for understanding the pathogenesis of E. tarda but also in development of an effective vaccine against edwardsiellosis.Infection with  Escherichia coli, Helicobacter pylori, Neisseria meningitidis, Pseudoaltermonas antarctica, Pseudomonas aeruginosa, Shigella flexneri, and Vibrio choleraeOuter membrane vesicles (OMVs) are spherical blebs of average diameter 10–300 nm that are naturally released from Gram-negative bacteria into the environment Edwardsiella tarda is the causative agent of edwardsiellosis in a variety of cultured freshwater and marine fish, including channel catfish Ictalurus punctatus, olive flounder Paralichthys olivaceus, Japanese eel Anguilla japonica, red sea bream Pagrus major, mullet Mugil cephalus, and turbot Scophthalmus maximusE. tarda infection in olive flounder are exophthalmia, enlargement of the spleen, malodorous ascites, and rectal hernia E. tarda infection E. tarda include several important virulence factors that play key roles in pathogenicity E. tarda that have been investigated include dermatotoxin, hemolysins, catalase, outer membrane proteins, EseDs, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) A number of studies have shown that vaccination using outer membrane proteins results in development of protective effects against Neisseria meningitidis serogroup B vaccine was successfully developed using derived OMVs; 55 million doses have been administered to date Previous proteomic studies indicated that both outer membrane proteins and LPS in OMVs might play roles as pathogen-associated molecular patterns (PAMPs) delivered to the host innate immune system, and could thus elicit immune responses E. tarda. Thus, we investigated the possibility of using OMVs released from E. tarda as a vaccine against edwardsiellosis, based on both a proteomic study and cytokine induction assays.However, none of OMV protein composition, antigenicity, or vaccine efficacy has been studied in bacteria pathogenic for fish, especially the bacterium in vivo immunogenicity testing. After centrifugation, two clear white bands were evident in the centrifugation tube, and the materials therein were examined by TEM  and TLR2 expression levels, which are pattern recognition receptors (PRRs) in olive flounder, were measured to determine whether the innate immune response could be elicited by OMVs E. tarda infection; control fish were injected with PBS  (p<0.0001).OMVs were administered to olive flounder as a candidate vaccine against with PBS . Fish inE. tarda was also shown to produce round OMVs, 10–40 nm in diameter, when grown in a liquid medium. Preparation of pure OMVs from bacterial supernatants is essential when studying the roles played by OMVs in both bacteria and their hosts E. tarda OMVs were separated from contaminants using such methods. Flagella, pili, and aggregated proteins were present in OMV samples before sucrose density gradient purification, but were removed during this step.Several Gram-negative bacteria produce OMVs E. tarda outer membrane identified only 21 proteins by 2-DE analysis, and only 1 protein was confined to this membrane Two representative proteomic analysis methods have been suggested for the study of OMVs. Of these, the first is two-dimensional electrophoresis (2-DE) followed by matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) spectrometry, and the other is 1D SDS-PAGE followed by LC-ESI-MS/MS Some reports have suggested that OMV proteomes consist mainly of outer membrane and periplasmic proteins E. tarda mutants could not invade human epithelial cell lines E. coli strain invaded brain microvascular endothelial cells only with difficulty E. tarda pathogenesis; these proteins are located on the cell surface and are responsible for pore formation E. tarda contain numerous virulence factors that may be important both for bacterial survival and to enhance immunogenicity in the natural host.LC-ESI-MS/MS analysis of OMVs showed that several proteins involved in pathogenesis, including hemolysin, outer membrane protein A (OmpA), GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein, were present. Hemolysin is known to be required for cellular invasion and cytotoxicity E. tarda, compared with fish injected with FKC, especially at early post-injection time points. Similarly, mice injected with OMVs of Bordetella pertussis showed upregulation of mRNAs encoding IL-6 and TNFα, compared with the levels seen in animals injected with formalin-killed B. pertussisE. tarda OMVs indicates that the OMVs may initiate a proinflammatory cytokine cascade, including both recruitment and activation of macrophages and stimulation of an adaptive immune response. IFNγ, a Th1 cytokine, is also upregulated in fish injected with OMVs, and is known to enhance cell-mediated immunity and antigen presentation to macrophages QRT-PCR demonstrated that the genes encoding all of IL-1β, IL-6, TNFα, and IFNγ were induced in fish injected with OMVs of E. tarda, may have played roles as PAMPs, thus interacting with the innate immune system. In addition, OMVs are known to possess LPS and bacterial DNA that function as ligands for host PRRs via myeloid differentiation factor 88 (MyD88)-dependent pathways Innate immunity is an important first line of defense against invading pathogens, and recognition of PAMPs is achieved principally by PRRs E. tarda, showed that OMVs exhibited high protective efficacy against E. tarda infection of olive flounder, with an RPS of 70% (p<0.0001). Other reports have described successful inhibition of E. tarda infection in laboratory experiments using various antigen preparations including formalin-killed and ghost cells, recombinant proteins, and outer membrane fractions The proteomic data, and the demonstrated immunostimulatory effects of OMVs from E. tarda contained various important virulence factors originating from diverse subcellular locations. Such vesicles were able to induce synthesis of several proinflammatory cytokines, and may stimulate the host innate immune system, thus serving as PAMPs. To demonstrate vaccine efficacy, we injected olive flounder with OMVs, and found that such fish were protected to a significantly higher extent than were control fish. The present study on OMVs may lead to development of more effective vaccines, and may enhance our understanding of the roles of OMVs in hosts.In conclusion, OMVs naturally released from E. tarda strain isolated from the spleen of infected olive flounder, was used in the present study; the LD50 value was 1.2×103 CFU/ml (data not shown). ED45 was cultured on Tryptone Soya Agar  or in Tryptone Soya Broth , supplemented with 2% (w/v) NaCl . ED45 was grown on TSA-2 at 25°C for 36 h; a single colony was inoculated into 25 ml TSB-2, and the culture grown with shaking to a cell density of 1×109 CFU/ml. Next, 20 ml of this culture was added to 4 l TSB-2 and growth proceeded in a shaking incubator at 25°C for 15 h. After centrifugation at 5,000×g for 20 min, the supernatant was used for isolation of OMVs.ED45, a virulent g for 3 h at 4°C in an XL-90 ultracentrifuge running a 70Ti rotor .OMVs were collected and purified from the supernatant as described previously, with several modifications g for 20 h at 4°C in a XL-90 ultracentrifuge running an SW41 Ti rotor, to obtain purified OMVs. Two clear bands were visualized, and the sucrose density in the vicinity of each band was measured by refractometry . The final preparations were stored at −80°C prior to use.Pelleted OMVs were resuspended in phosphate-buffered saline , layered onto a 30–60% (w/v) discontinuous sucrose gradient, and centrifuged at 200,000×ctometry . After w600 value of 1.0 was attained, centrifuged at 5,000×g for 30 min, and washed three times with PBS prior to preparation of ultrathin sections. After centrifugation at 5,000×g for 20 min, pellets were pre-fixed in 2% (v/v) paraformaldehyde for 3 h, washed, and fixed in 4% (w/v) osmium tetroxide for 2 h. After washing with PBS, the fixed pellets were dehydrated in a series of 70–100% (v/v) ethanol baths and embedded in epoxy resin. Sections were cut using a diamond blade  fitted to an ULTRACUT UCT , mounted on carbon-coated copper grids, and stained with 3% (w/v) uranyl acetate and lead citrate. To visualize OMVs after negative staining, samples were placed on 400- mesh carbon-coated grids for 2 min, washed with deionized sterile water (dd H20), and negatively stained with 3% (w/v) uranyl acetate for 30 sec. All TEM images were acquired using a Technai 12  operating at an acceleration voltage of 120 kV.ED45 cells were grown in TSB-2 at 25°C until an ODg for 30 min, and washed with PBS. Pelleted cell density was adjusted to 4 g/ml in 20% (w/v) sucrose dissolved in 20 mM Tris-HCl (pH 8.0), also with 0.1 M EDTA and lysozyme (600 µg/g cells). After incubation on ice for 40 min, 0.5 M MgCl2 (0.16 ml/g cells) was added and spheroplasts were removed by centrifugation . The supernatant containing PPs was stored at −80°C until use. Spheroplasts were resuspended in ice-cold 10 mM Tris-HCl (pH 8.0) and sonicated for purification of OMPs. Following centrifugation at 8,000×g for 5 min to remove cells, the supernatant was centrifuged at 40,000×g for 1 h to pellet cell membrane material. Pellets were washed in 10 mM Tris-HCl (pH 8.0), resuspended in dd H20, and freeze-thawed. The membranes were incubated in 0.5% (w/v) Sarkosyl  at 25°C for 20 min and OMP pellets were purified by centrifugation at 40,000×g for 1 h. Purified OMPs were resuspended in 10 mM Tris-HCl (pH 8.0) and stored at −80°C until use. Protein concentration was determined using a non-interfering protein assay .Periplasmic proteins (PPs) and outer membrane proteins (OMPs) were purified as described previously g for 20 min. Supernatants were subjected to SDS-PAGE analysis. Five microgram amounts of WCL, PP, OMP, and OMV proteins were loaded and proteins were detected by silver staining SDS-PAGE was performed using a 15% (w/v) acrylamide separating gel, according to the method of Laemmli To achieve in-gel digestion, 20 µg amounts of OMVs were electrophoresed on a 12.5% (w/v) separating gel and stained with Bio-safe Coomassie G-250 . After cutting the gel into 12 slices, each slice was destained with a 500 µl amount of 40% (v/v) ethanol in 75 mM ammonium bicarbonate (ABC) and treated with DTT (0.0039 g dithiothreitol in 5 ml 25 mM ABC) and IAA (0.0509 g iodoacetamide in 5 ml 25mM ABC) solutions. Each gel slice was dehydrated using 300 µl acetonitrile (ACN) for 30 min at 37°C, dried, and used for in-gel digestion employing 20 ng/ml of sequencing-grade modified trypsin  at 37°C overnight. Tryptic peptides were extracted into 30 µl 0.1% (v/v) formic acid and the solutions were sonicated for 10 min.2O and ACN, both containing 0.1% (v/v) formic acid, and delivered at a constant flow rate of 0.2 µl/min. The gradient commenced with 2.0% (v/v) ACN, rising linearly to 60% (v/v) ACN over 50 min, next increasing to 80% (v/v) ACN over the next 5 min, with a change to 100% H2O for the final 15 min. A data-dependent acquisition mode  was enabled, and each survey MS scan was followed by five MS/MS scans with the 30 sec dynamic exclusion option set. The spray voltage was 1.9 kV and the ion transfer tube temperature 195°C. The normalized collision energy was set to 35%.LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) analysis was carried out using a Thermo Finnigan Proteome X workstation with an LTQ linear ion trap and a MS apparatus equipped with NSI sources . Twelve microliter amounts of peptide mixtures were injected and loaded onto peptide trap cartridges . Trapped peptides were eluted onto a 10 cm-long reverse-phase PicoFrit column packed in-house with 5 µm C18 resin of pore size 300 Å, and the peptides were next separated on an RP column using gradient elution. The mobile phase solutions were HE. tarda database of NCBI downloaded on 9 July 2010, using the Mascot Deamon interface  supported by the Korea Basic Science Institute . The peptide tolerance of the parent ion was adjusted to be 1 Da and the MS/MS tolerance was 0.8 Da. During analysis, carbamidomethyl (C) modification was fixed whereas oxidation modification (M) was not. One missed cleavage was permitted, and peptide charges were set at 2+ and 3+. Individual ion scores of more than 15 (p<0.05) were considered reliable and were subsequently used.The mzData file of tandem mass spectra was used to search the http://www.ncbi.nih.gov/COG) http://www.psort.org) Functional annotations of OMV proteins were performed using the “clusters of orthologous groups” (COGs) functional classification (E. tarda cells (FKC) were prepared by addition of 1% (v/v) formalin to an ED45 culture, followed by adjustment to an OD600 value of 1.0 using PBS. OMVs were prepared as described above and used after confirmation that no colonies formed when OMVs were plated onto TSA-2. Aliquots (0.1 ml) of FKC , or 10 µg amounts of OMVs, were intraperitoneally injected into olive flounder. The administred dosage was determined based on previous studies on immunization with OMVs originating from several gram negative bacteria Naïve olive flounders (with an approximate weight of 55 g) were purchased from a commercial fish farm in Korea and acclimatized for 2 weeks at 21°C in aerated seawater. Formalin-killed All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Gyeongsang National University, Jinju, Republic of Korea .t-test was used to determine differences between tests using FKC and OMV; p<0.05 was viewed as significant.Total RNA was isolated from kidney samples using the TRIzol reagent  according to the manufacturer's protocol. After purification of 1 µg aliquots of mRNA using deoxyribonuclease I (Invitrogen), cDNAs were synthesized with the assistance of a high-capacity cDNA reverse transcriptase kit  according to the manufacturer's protocol. cDNA samples were diluted 1∶4 (v/v) into nuclease-free water prior to amplification using quantitative real-time PCR (QRT-PCR). Primers were designed employing the Primer Express software of Real-Time PCR version 3.0 (Applied Biosystems). Primer names, sequences, and GenBank accession numbers are listed in 4–109 CFU/ml) and mortality was recorded over the next 28 days. A group of fish injected with 1.2×104 CFU/ml of ED45 showed 70% mortality, and this dose was thus chosen as the challenge (data not shown). Twenty fish were vaccinated with 0.1 ml amounts of FKC  or 10 µg amounts of OMV, whereas fish injected with PBS served as controls. After acclimatization for 28 days, fish were challenged with 100 µl amounts of ED45 (1.1×104 CFU/ml) by intraperitoneal injection, and mortality was recorded over the following 31 days. Vaccine efficacy was calculated as relative percentage survival  Before vaccination, 10 naïve olive flounders in each group were intraperitoneally injected with ED45 (1.2×10


K-134 is a more potent antiplatelet drug with a selective inhibitory effect on phosphodiesterase 3 (PDE3) compared with its analogue, cilostazol.This study was performed to compare the ameliorating effects of K-134 and cilostazol on brain damage in an experimental photothrombotic cerebral infarction model.n = 12, 87.5±5.6 vs. 126.8±7.5 mm3, P<0.01), indicating its potent antithrombotic effect. On the other hand, the effects of cilostazol on MCA occlusion time and cerebral infarct size are relatively weak even at the high dosage of 300 mg/kg. Furthermore, K-134 blocked rat platelet aggregation more potently than cilostazol in vitro. Also in an arteriovenous shunt thrombosis model, K-134 showed an antithrombotic effect greater than cilostazol.We investigated the effects of oral preadministration of PDE3 inhibitors in a rat stroke model established by photothrombotic middle cerebral artery (MCA) occlusion. K-134 significantly prolonged MCA occlusion time at doses >10 mg/kg, and reduced cerebral infarct size at 30 mg/kg in the stroke model (These findings suggest that K-134, which has strong antithrombotic activity, is a promising drug for prevention of cerebral infarction associated with platelet hyperaggregability. The underlying pathophysiology in most cases of ischemic stroke involves thrombotic or thromboembolic arterial occlusion in vivo thrombotic models and beneficial effects on the photothrombotic stroke model compared to cilostazol. Hence, the aim of the present study was to further evaluate and compare the effects of oral preadministration of K-134 and cilostazol on MCA occlusion and infarct volume in the photothrombotic stroke model.In non-clinical studies, cilostazol has been shown to attenuate brain injury induced by middle cerebral artery (MCA) occlusion All study protocols were reviewed and approved by the Committee on Ethics of Animal Experiments of Kowa Company, Ltd.. All surgery was performed under anesthesia with sodium pentobarbital, ether or urethane, and all efforts were made to minimize suffering.in vitro pharmacological experiments or in methanol for preparing standard solution in pharmacokinetic studies, or suspended in 1% (w/w) hydroxypropylmethyl cellulose aqueous solution  for in vivo experiments. Male Sprague–Dawley (SD) rats and male ICR mice, 5–8 weeks old, were purchased from Japan SLC Inc.  and CLEA Japan, Inc. .PDE3 inhibitors K-134 (molecular weight (MW) = 399.48 g/mol), cilostazol (MW = 369.46 g/mol), OPC-13015 (MW = 367.44 g/mol) and OPC-13213 (MW = 385.46 g/mol) were obtained from Kowa Company Ltd. , and dissolved in dimethylformamide for n = 12), because K-134 reached plasma peak levels at 1–2 h Brain infarction was produced by photothrombotic MCA occlusion essentially as reported previously Twenty-four hours after MCA occlusion, rats were sacrificed by an overdose of pentobarbital and cutting an abdominal vein. The brain was immediately removed, cooled with cold spray , and cut into six slices (2 mm thick) using a slicer  and razor . The cutting base point (0 mm) was set at the position of bregma, and brains were cut at −2, −4, 0, +2, +4, +6, +8 mm from the base point. The brain slices were stained with 1% (w/v) 2,3,5-triphenyl-2H-tetrazolium chloride  in saline solution at 37°C for 20 min. The stained slices were photographed with a digital camera . Whole brain areas and infarcted areas were quantified by image analysis , and infarct volume were calculated with the trapezoidal rule.5/µL with platelet-poor plasma prepared by centrifugation . PRP was incubated with antiplatelet agents at 37°C for 3 min and stimulated with collagen  or adenosine diphosphate (ADP)  (n = 5). Platelet aggregation was quantified by measuring the area under the curve (AUC) during the 5-min period after addition of trigger using a light transmittance aggregometer .Blood was collected via the inferior vena cava under ether anesthesia from SD rats and ICR mice which were fasted overnight, and anticoagulated with a 1/10 volume of sodium citrate (3.0% (w/v) for rats, 3.8% for mice). Platelet-rich plasma (PRP) was prepared by centrifugation  of blood , and the platelet count was adjusted to 5×10n = 12). Ninety minutes after oral administration of a single dose of K-134 or cilostazol, one end of a polyethylene catheter  filled with physiological saline was inserted into the left jugular vein, with the other end of the catheter inserted into the right common carotid artery to create a loop. The end of the catheter was removed from the jugular vein 4 h after creation of the loop. A thrombus was then regarded to have been formed when blood no longer ran from the catheter. In a separate experiment, blood samples were taken from the inferior vena cava in a rat arteriovenous shunt model under the same conditions as in the above method to create an arteriovenous shunt and heparinized at 0.5, 1.5, 3.5, and 5.5 h after oral administration of a single dose of K-134 or cilostazol (n = 6). The serum concentrations of K-134 and cilostazol were determined by high-performance liquid chromatography (HPLC).Immediately after a single oral administration of the test compound, SD rats were anesthetized by subcutaneous injection of 1.2 g/kg of urethane . All statistical analyses were performed using SAS 9.1.3 . In all analyses, 3) than that of control group (131.4±18.9 mm3)  were not significantly different from that of the control group  . In cont8.9 mm3) . On the ol group . No bleein vitro. The half-maximal (50%) inhibitory concentration (IC50) values of K-134 were 2.5 µM and 3.2 µM, respectively, and those of cilostazol were 42 µM and 83 µM, respectively  using data of 1.5–5.5 h and the inhibition rate of arteriovenous shunt thrombus formation indicates that K-134 inhibited arteriovenous shunt thrombus formation at a lower plasma concentrations compared with cilostazol . On the other hand, cilostazol significantly reduced the incidence of occlusive shunt thrombi at doses above 30 mg/kg (ED50 = 18 mg/kg) . The plalostazol .in vitro and ex vivo with greater potency than cilostazol.The present study provided novel data indicating that K-134 blocks brain injury more potently than cilostazol in a photothrombotic ischemic stroke model  and othein vitroK-134 exerts a similar dose-dependent vasodilatory effects on rat femoral arteries contracted by KCl 1/2 = about 2 h) of K-134 when administered to rats at a dose of 10 mg/kg In the stroke model, 30 mg/kg of K-134 inhibited photothrombotic MCA occlusion and reduced cerebral infarct size more potently than 300 mg/kg of cilostazol ,2. On thin vivo because this model permits observation of not only time to occlusion by thrombus formation but also effects on cerebral infarction at a certain period of time after endothelial injury by less-invasive approach without injuring dura mater, thereby enables taking account of effects of rate of drug metabolism and long-term pharmacological effects of drugs. We previously reported that K-134 blocks stable platelet accumulation but not initial platelet adhesion onto Von Willebrand factor (vWF)-coated surface under high shear conditions in vitroin vivo model. Hence, intravital videomicroscopy analysis in vivo.The photothrombotic MCA occlusion model has numerous advantages for the study of antiplatelet inhibitors The photothrombotic stroke model is a suitable model for evaluating the effects of antiplatelet inhibitors because an arterial platelet aggregation is induced through endothelial damage by a photochemical reaction In the photothrombotic cerebral infarction model, photoactivation of rose bengal by illumination with green light results in reactive oxygen intermediates (predominantly singlet oxygen and superoxide). In our electron spin resonance (ESR) experiments, K-134, cilostazol, and OPC-13015 (one of cilostazol metabolites) did not show scavenging activities against both singlet oxygen and superoxide anion radical (data not shown). Therefore, we concluded that the observed inhibitory effects of K-134 on photothrombosis in this model are relevant to its antiplatelet activity but not radical scavenging activity.in vivo experiments, antiplatelet activities of its active metabolites, OPC-13015 and OPC-13213, could also contribute to inhibition of platelet thrombus formation. OPC-13015 has 3 times more potent antiplatelet activity than cilostazol, whereas OPC-13213 has 3 times less potent activity than cilostazol 0–24 h were 30.1, 18.3, and 127.3 µM·h, respectively, after a single oral administration of cilostazol at a dose of 300 mg/kg in non-fasting male SD rats , respectively 0–24 h of K-134 after a single oral administration to non-fasting male SD rats at doses of 10 mg/kg and 30 mg/kg were low  because of its short elimination half-life (T1/2) compared with that of cilostazol in vivo. In the arteriovenous shunt thrombosis model, K-134 showed antithrombotic activity at a dose of 10 mg/kg which gives much lower plasma concentration compared with that of cilostazol at an effective dose of 30 mg/kg  or collagen (B), and inhibitory effects were estimated by measuring the AUC during the 5 or 7 min after addition of ADP or collagen, respectively. Values are means ± SEM .(TIF)Click here for additional data file.Figure S2Inhibitory effects of PDE3 inhibitors on mouse platelet aggregation in vitro. Citrated PRP derived from mice was preincubated with K-134 or cilostazol, and stimulated with collagen (A) or ADP (B). Inhibitory effects of agents on collagen (A) and ADP (B) were estimated by measuring the AUC during the 5 min after stimulation. Values are means ± SEM .(TIF)Click here for additional data file.Figure S3Bleeding time in mice. K-134 and warfarin were administered to mice at 10 min and 24 h before testing of tail bleeding time, respectively. Values are means ± SEM (n = 27–28). **P<0.01 vs. vehicle control by two-tailed t-test. N.S. = not significantly different by two-tailed Dunnett's test.(TIF)Click here for additional data file.Figure S4Vasodilatory effects of cilostazol. Vasodilatory effects of cilostazol were evaluated as previously described (TIF)Click here for additional data file.Table S1Pharmacokinetic parameters of cilostazol and its active metabolites (OPC-13015 and OPC-13213) in rats.(DOC)Click here for additional data file.Table S2Plasma concentration of K-134 in mice.(DOC)Click here for additional data file.Methods S1Description of pharmacokinetic studies, ex vivo antiplatelet effects study, vasodilation study and bleeding time study.(DOC)Click here for additional data file.


To analyze the clinical and dosimetric risk factors of acute esophagitis (AE) in non-small-cell lung cancer (NSCLC) patients treated with concomitant chemoradiotherapy.Seventy-six NSCLC patients treated with concomitant chemoradiotherapy were retrospectively analyzed. Forty-one patients received concomitant chemoradiotherapy with vinorelbine/cisplatin (VC), 35 with docetaxel/cisplatin (DC). AE was graded according to criteria of the Radiation Therapy Oncology Group (RTOG). The following clinical and dosimetric parameters were analyzed: gender, age, clinical stage, Karnofsky performance status (KPS), pretreatment weight loss, concomitant chemotherapy agents (CCA) (VC vs. DC), percentage of esophagus volume treated to ≥20 (V20), ≥30 (V30), ≥40 (V40), ≥50 (V50) and ≥60 Gy (V60), and the maximum (Dmax) and mean doses (Dmean) delivered to esophagus. Univariate and multivariate logistic regression analysis were used to test the association between the different factors and AE.Seventy patients developed AE . By multivariate logistic regression analysis, V40 was the only statistically significant factor associated with Grade ≥2 AE . A V40 of <23% had a 33.3% (10/30) risk of Grade ≥2 AE, which increased to 89.1% (41/46) with a V40 of ≥23% (p<0.001). CCA  and V50  were most significantly correlated with grade 3 AE. A V50 of <26.5% had a 6.7% (3/45) risk of Grade 3 AE, which increased to 38.7% (12/31) with a V50 of ≥26.5% (p = 0.001). On the linear regression analysis, V50 and CCA were significant independent factors affecting AE duration. Patients who received concomitant chemotherapy with VC had a decreased risk of grade 3 AE and shorter duration compared with DC.Concomitant chemotherapy agents have potential influence on AE. Concomitant chemotherapy with VC led to lower risk of AE compared with that using DC. V40 and V50 of esophagus can predict grade ≥2 and ≥3 AE, respectively. Lung cancer is currently the most common malignancy and also the leading cause of cancer-related mortality in the world . Non-smaBetween October 2006 and March 2009, 76 patients diagnosed with NSCLC were enrolled in this study. All patients had histologically or cytological proven NSCLC . There were 59 men and 17 women. The median age was 59 years . All patients were staged according to the AJCC 2002 classification. Three patients had stage II, 32 stage IIIa, 37 stage IIIb, and 4 stage IV. Of these patients, 11 patients had undergone surgical intervention before chemoradiotherapy.All patients received concomitant chemoradiotherapy, which were administered by medical oncologists according to our institutional standards. Of these patients, 41 received concomitant chemotherapy with VC, 35 with DC. Concomitant chemotherapy regimens were summarized in the Table 20 < 33% , and spinal cord Dmax < 48 Gy. No dose limits were used for the esophagus, trachea, and heart. Dose distribution was calculated with tissue heterogeneity correction. A series of dose volume parameters including Dmean, Dmax, V20, V30, V40, V50 and V60 were recorded for normal structures. Radiotherapy was delivered with linear accelerators using 6 or 15-MV X-rays. Treatment techniques typically included initial anterior-posterior beams followed by oblique off-cord beams. Most of plans employed 4 to 6 fields. Respiration motion was managed by using the Active Breathing Coordinator device (Elekta Inc.) for 18 patients (23.7%). Electronic portal images acquired with large orthogonal fields (18 cm × 18 cm) with 18 monitor units were used to guide daily patient positioning for 32 patients (42.1%), while cone-beam CTs were used to position patient at the first three treatments and once a week subsequently for 44 patients (57.9%).Seventy-one patients were treated with three-dimensional conformal radiotherapy (3D-CRT), 5 with intensity modulated radiation therapy (IMRT). Patients were set up in treatment position and immobilized using vacuum bag to improve the reproducibility during daily treatments. Computed Tomography (CT) scans with a slice thickness of 5 mm thick was obtained from the lower end of the cricoid cartilage to the lower edge of the liver. The obtained CT images were directly transmitted to a 3D planning system . Gross tumor volume (GTV) was delineated to encompass all detectable tumors, including primary mass and metastatic regional lymph nodes observed on CT. Planning target volume (PTV) included GTV and margins of 5 to 12 mm for lymph nodes, 8 to 20 mm for primary tumor. Fifty-eight patients (76.3%) were treated with involved-field irradiation, 18 (23.7%) with elective nodal irradiation. The external surface of the esophagus was contoured uniformly on each of the axial images of the planning CT by the same physician. The normal esophagus was defined from the inferior border of the cricoid cartilage to the gastroesophagea junction. All patients were treated with conventional fractionation scheme, 5 days per week, with fractional dose of 2.0 Gy for 70 patients and 1.8 Gy for 6 patients. The median of fraction number was 30, ranging 28–35. At least 90% of a PTV was required to be covered by the prescription dose. Dose constraints for normal tissue used in the planning process were as follows: VAll patients were hospitalized during the course of radiotherapy, and were seen everyday as routine evaluation of the treatment. After completion of therapy, patients were followed up every month for the first 3 months and every 3 or 4 months thereafter. If necessary, telephone follow-up was conducted to acquire more information on radiation induced toxicity. AE (measured within 3 months of initiating radiotherapy) were evaluated according to the RTOG and European Organization for Research and Treatment of Cancer toxicity grading scale . These dt test. Mann–Whitney U test was used to verify the difference of occurring time. Patients experiencing grade 2 or worse, grade 3 or worse, were counted as positive events. The following factors: gender , age (continuous), clinical stage (≤IIIa vs. ≥IIIb), KPS (<90 vs. ≥90), pretreatment weight loss (<5% vs. ≥5%), CCA (VC vs. DC), Dmean, Dmax, V20, V30, V40, V50 and V60, were included in the statistical analysis. Univariate logistic regression analysis was used to test the association between the different factors and positive events . Pearson correlation analysis was used to correlate these parameters with duration (continuous). For multivariate analysis, the forward Wald procedure was performed using a logistic regression model containing all statistically significant variables in univariate analysis (p<0.10). Receiver operating characteristic (ROC) curves have been used to identify discriminate threshold. Linear regression model was used to evaluate their importance in duration (continuous). All statistical tests were 2-sided. Statistically significant differences are reported for p<0.05. Statistical analyses were performed using SPSS (version 15.0 for Windows).The maximal grade and the AE duration for each patient were used in the statistical analysis. A descriptive statistical analysis was performed and results are reported as mean ± Standard deviation (SD) for continuous variable. The correlations among dosimetric parameters were assessed with Pearson Correlation coefficient test. The difference of dosimetric parameters between VC group and DC group were evaluated by Of all patients studied, fifteen patients experienced interruption in radiotherapy delivery, 5 due to AE , 7 due to neutropenia, and 3 due to machine problems. For patients with radiotherapy interruptions, the median delay was 3 days . After completion of radiotherapy, fifteen patients experienced delay of consolidation chemotherapy, 6 for neutropenia, 6 for AE , 1 for transaminases, and 2 for refusal of patient. The median delay was 5 days .The mean ± SD of radiation dose to PTV was 60.7 ± 2.6 Gy . VC group had slightly numerically higher dosimetric parameters of esophagus than DC group, but there were no statistical difference by t test (p:0.293-0.616). Most of dosimetric parameters were closely correlated with one another when assessed by Pearson correlation coefficient test  except the correlation between Dmax and V20.A total of 70 patients  developed grade ≥1 AE. Among them, 19 patients experienced grade 1 AE , 36 grade 2  and 15 grade 3 . No grade 4 or 5 AE was observed. In the VC group, 5 patients experienced grade 0 , 11 grade 1 , 19 grade 2  and 6 grade 3 . In the DC group, 1 patients experienced grade 0 , 8 grade 1 , 17 grade 2  and 9 grade 3 .Of the clinical factors investigated, such as gender, age, clinical stage, KPS, pretreatment weight loss and CCA, none was found to be significant for predicting grade ≥2 or grade 3 AE in univariate analysis (p<0.05). The CCA (VC vs. DC) was weakly related to grade 3 AE . All dosimetric parameters investigated, including Dmax, Dmean, V20, V30, V40, V50 and V60 of esophagus, were found to be significantly associated with the risk of grade ≥2 or grade 3 AE (p<0.05) in univariate analysis. These results were summarized in Table The predictive power of V40 for grade ≥2 AE was tested by ROC analysis. The areas under the ROC curve for grade ≥2 AE were 0.888 . These data had been used to identify discrete points on ROC curves. In this curve, V40 ≈ 23% (22.75%) represents the cut point with appropriate sensitivity (80.4%) and specificity (80.0%). If V40 is <23% there is a 33.3% (10/30) risk of grade ≥2 AE, which increases to 89.1% (41/46) if V40 is ≥23% (p<0.001).The predictive ability of V50 to for grade 3 AE was also tested by ROC analysis. The areas under the ROC curve for grade 3 AE were 0.841 . In these curves, V50 = 26.5% represents the cut point with sensitivity (80.0%) and specificity (68.9%). If V50 is <26.5% there is a 6.7% (3/45) risk of Grade 3 AE, which increases to 38.7% (12/31) if V50 is ≥26.5% (p = 0.001).All 70 patients experienced grade ≥1 AE were included in the analysis, 36 in VC group, and 34 in DC group. The median occurring time was 22 day (ranging 8-41 day) for all 70 patients, 23 day (ranging 9-41 day) for the VC group (n = 36) and 21.5 day (ranging 8-29 day) for the DC group (n = 34). There were significantly statistical difference between VC and DC groups by Mann–Whitney U Test . Grade ≥1 AE occur later in patients treated with VC than those with DC. Distribution of VC and DC group patients according to the occurring time were depicted in Figure All 70 patients with grade ≥1 AE were included in the duration analysis. Dosimetric parameters of VC and DC groups were summarized in Table The mean ± SD durations of 70 patients suffered grade ≥1 AE were of 41.2 ± 11.1 days. There were significant differences in AE duration between VC group and DC group by t test . A longer duration (about one week) was found in DC group (45.0 ± 10.9 days) in comparison to the VC group (37.6 ± 10.3 days).2 = 0.643. Patients who received concomitant chemotherapy with VC show a shorter duration compared with DC. The predictive equation of AE duration was as follows: Y(days) = 12.195 + 0.512 × V50 (%) + 10.865 × CCA, where V50 (%) = relative esophageal volume for radiation dose ≥50 Gy and CCA = 1 or 2 for concomitant chemotherapy with VC or DC, respectively.By Pearson correlation analysis, CCA , V20 , V30 , V40 , V50 , V60 , Dmax  and Dmean  were all significantly correlated with grade ≥1 AE duration. Figure AE is one of the main complications of radiotherapy for NSCLC. Meta-analysis  have shoThere were few prior reports about the effect of CCA on developing AE. The CALGB study 9431 , showed Esophagus is lined by nonkeratinized epithelium with a lamina propria and muscularis mucosa. AE is one of the gastrointestinal mucositis (GIM) caused by cancer therapies. Chemotherapy and radiotherapy damage the dividing and differentiating cells and limit the proliferative ability of the epithelium, so that it becomes thin or ulcerated ,10. ChemSeveral studies reported that docetaxel showed heavy GIM compared with vinorelbine in NSCLC patients treated with chemotherapy. In a randomized phase II study of docetaxel or vinorelbine in combination with cisplatin against NSCLC , more paIn the QUANTEC study, Werner-Wasik et al. reviewed many studies on analyzing dosimetric risk factors of AE, the results were not consistent . But thePatients who had received concomitant chemotherapy with VC show statistically lower AE grade, later occurring time and shorter AE duration compared with the patients treated with DC in the present study. This indicates that the CCA may be a critical influencing factor of AE in these patients. The analysis of subgroup with different CCA will be needed in order to explore the most valuable predictor of AE. Dosimetric parameters V40 and V50 were important predictors of grade ≥2 or ≥3 AE, respectively, for NSCLC patients treated with concomitant chemoradiotherapy.AE: Acute esophagitis; NSCLC: Non-small-cell lung cancer; VC: Vinorelbine/cisplatin; DC: Docetaxel/cisplatin; RTOG: Radiation Therapy Oncology Group; KPS: Karnofsky performance status; CCA: Concomitant chemotherapy agents; V20,30,40,50,60: Percentage of esophagus volume treated to ≥20, ≥30, ≥40, ≥50 and ≥60 Gy; Dmax: The maximum doses delivered to esophagus; Dmean: The mean doses delivered to esophagus; 3D-CRT: Three-dimensional conformal radiotherapy; IMRT: Intensity modulated radiation therapy; CT: Computed Tomography; GTV: Gross tumor volume; PTV: Planning target volume; ABC: Active breathing coordinator; SD: Standard deviation; ROC: Receiver operating characteristic; CALGB: Cancer and leukemia group B; GIM: Gastrointestinal mucositis.The authors declare that they have no competing interests.ZCZ and JX carried out the manuscript writing; ZCZ, YY and HSL participated in statistical analysis; ZCZ, JX, TZ, HFS, WH and DQW helped to collect data, BSL and GGY conceived and designed this study. All authors read and approved the final manuscript.