Moreover, AR was borderline significantly associated with poor progress free survival (PFS) after adjustment with other variables . Knockdown of ERα inhibited the proliferation, migration and invasion of GC cells possibly via modulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. Downregulation of AR suppressed the migration and invasion of GC cells and inhibited the epithelial-mesenchymal transition (EMT) associated pathways.Despite the mounting studies exploring the role of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ) and androgen receptor (AR) in gastric cancer (GC), there remain controversies in those findings. The present study investigated the expression of ERα, ERβ and AR in Chinese gastric cancer by immunohistochemistry, analyzed their clinical relevance in gastric cancer, and examined the potential mechanisms by which ERα and AR modulated GC progression. The positive rate of ERα, ERβ and AR in GC tissues was 6% (9/150), 93.5% (143/153), and 42.4% (59/139), respectively. The expression of ERα was an independent unfavorable risk factor for overall survival (OS) (hazard ratio [HR] = 3.639, 95% confidence interval [CI] = 1.432-9.246, The present study showed that positive ERα was associated with poor prognosis of Chinese GC patients. ERα might modulate the proliferation, migration and invasion via regulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. ERα could be a valuable prognostic biomarker and promising therapeutic target for Chinese GC patients. Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-related death in the world . A largeIn the past decades, hormonal therapy has been well established in the treatment of hormone-dependent tumors such as prostate cancer and breast cancer , 4. HoweThe relationship between hormone receptors and GC was first reported by Tokunaga et al . Since tThe positive rate of ERα, ERβ and AR in GC tissues was 6.0% 9/150), 93.5% (143/153), and 42.4% (59/139), respectively . Moreover, higher AR expression was observed in patients with better differentiated tumors (p=0.009).Clinicopathological characteristics including patient sex, age, whether received adjuvant chemotherapy, tumor grade, vascular and nerve invasion, TNM classification and stage of the patients enrolled in this study were listed in Table p=0.032; Figure p=0.103; Figure p=0.167; Figure p=0.462; Figure p=0.05; Figure p=0.025; Figure The median follow-up time for the patients was 48.5 months (range 24.7-70.1 months), and nine patients who failed to contact through telephone or email were lost to follow up. Kaplan-Meier survival curves for ERα, ERβ and AR expression were shown in Figure Moreover, univariate analysis of OS demonstrated in Table We next investigated the effects of ERα on the malignant behavior of GC cells. We examined the expression of ERα in GC cell lines, and found that ERα was expressed in low levels in GC cells compared with the normal gastric epithelial cell line (GES-1), which was in accordance with the low positive rate of ERα in GC tissues examined by immunohistochemistry. MKN45 and SNU601 cells with ERα silenced were established by lentivirus infection, as the two cell lines showed relative high basal levels of ERα expression Figure . DownregTo further explore the mechanisms in the decreased proliferation, migration and invasion by ERα knockdown, proliferation associated molecules  as well as the important molecular in adherens junctions of epithelial cells (E-cadherin) were examined by western blot Figure . The expAlthough the present study found that AR was only borderline significantly associated with poor PFS, considering studies on the expression and function of AR in GC was insufficient, we also investigated the effects of AR on the malignant behavior of GC cells. AR was silenced in MGC803 and SGC7901 cells which showed relative high basal levels of AR expression by lentivirus infection . No signin vitro via regulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. And AR suppressed the migration and invasion of GC cells in vitro possibly through inhibiting EMT process.The present study demonstrated 6% (9/150) of GC tissues expressed ERα, 93.5% (143/153) expressed ERβ, and 42.4% (59/139) were AR positive. The correlation coefficients among ERα, ERβ and AR were too small (r < 0.4), indicating that the correlation was too weak to have any clinical significance in GC. The positive expression of ERα was an independent prognostic factor for OS, and AR was borderline associated with PFS for GC patients after adjustment with other variables. In addition, downregulation of ERα might suppress the proliferation, migration and invasion ERα expression patterns varied in different GC patients, for example, the data from TCGA database  showed that among 205 stomach adenocarcinoma samples, 5 samples were detected with ERα amplification, 1 with deep ERα deletion, and 6 with ERα mRNA upregulation. In light of this, high-quality clinical trials that examine the effect of anti-ER therapy on the treatment of ER positive GC patients are needed, and anti-ER therapy might be only effective on ER positive individuals.Treatment of ER positive breast cancer patients with ER antagonist has achieved great success . Howeverp21 [Limited researches have explored the effect of ERα and the underlying mechanisms in GC. Several previous experimental studies has demonstrated that ERα could promote the proliferation of GC cells possibly by interacting with hedgehog pathway , c-Src pp21 . P21 andp21 . E-cadhep21 . Taken tin vitro experiment, and revealed that downregulation of AR suppressed the migratory and invasive ability of GC cells, possibly via inhibition of EMT associated molecules including α-SMA, β-catenin, snail, and slug. In fact, Kominea et al firstly reported that AR was an unfavorable prognostic factor for the OS of GC patients [Since the prognostic role of AR in GC and the potential mechanisms are insufficient, even though the association of AR with PFS only reached borderline significance in the present study, we performed patients . Later Zpatients . Taken tIn summary, the present study showed that positive expression of ERα was significantly associated and positive expression of AR had a tendency to associate with poor prognosis of Chinese GC patients. In addition, knockdown of ERα suppressed the proliferation, migration and invasion of GC cells probably via modulating the expression of p53, p21, p27, cyclin D1 and E-cadherin. And downregulation of AR suppressed the migration and invasion of GC cells possibly via inhibiting EMT process. The results suggested that ERα and AR might serve as prognostic biomarkers and therapeutic targets to improve the survival of Chinese GC patients. In the next period of time, precise approaches are needed to identify the expression patterns of ERα and AR in GC patients, and clinical trials involving highly selected ERα and AR positive patients are required to illustrate the potential of anti-ERα and anti-AR therapy in GC.Formalin-fixed and paraffin-embedded tumor tissues were collected from 155 patients with gastric carcinoma who underwent surgical resection at the Department of Gastric Cancer and Soft Tissue Sarcomas, Shanghai Cancer Center of Fudan University, Shanghai, China, from Oct 2007 to Jan 2010. The samples were used for tissue microarray construction and immunohistochemistry. The patients were followed up every 4 months until death or the end of the study , except those lost to follow up. Overall survival (OS) was defined as the interval between the date of surgery and the date of death or last follow-up visit. Progress free survival (PFS) was defined as the time from the date of surgery to the time of disease progression, death, or last follow-up visit if the disease did still not progress.Informed consents were obtained from all patients, and the research was approved by the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center, and complied with the principles of the Helsinki Accord.ERα, ERβ and AR expression was detected by immunohistochemistry using UltraSensitive™ SP kit  according to the manufacturer's instructions. In brief, the sections were deparaffinized, rehydrated and subjected to antigen retrieval . The sections were then incubated overnight at 4°C with the primary mouse monoclonal antibodies to ERα , ERβ  and AR , respectively. The sections were subsequently washed and incubated with a secondary antibody. Reaction products were visualized with 3, 3’diaminobenzidine tetrahydrochloride and counterstained with hematoxylin and eosin.Cases displaying brown cytoplasmic and/or nuclear stainings were regarded as positive. The staining intensity was graded by the Allred score system . A scoreNine human GC cell lines , one normal gastric epithelial cell line (GES-1) and HEK293T cell line were obtained from the Cell Resource Center, Shanghai Institute of Biochemistry and Cell Bank at the Chinese Academy of Sciences. All cell lines were routinely authenticated by DNA-fingerprinting and isoenzyme analysis and free of contamination by mycoplasma. GC cell lines were cultured in RPMI 1640, MEM, or DMEM containing 10% fetal bovine serum at 37 °C under humidified atmosphere with 5% CO2.® HD Transfection Reagent (Promega) to generate lentivirus. MKN45, SNU601, MGC803 and SGC7901 cells were infected with the recombinant lentivirus with 10μg/mL Polybrene® .Lentiviral vector expressing shRNA targeting ERα was purchased from Obio Tech , and lentiviral vector expressing shRNA targeting AR was purchased from Genechem . Those shRNAs were transfected into HEK293T cells using FuGeneMKN45 and SNU601 cells were plated at a density of 2000 cells per well in 96-well plates. MGC803 and SGC7901 cells were plated at a density of 1000 cells per well in 96-well plates. Cell proliferation was determined with Cell Counting Kit 8  for 5 days.in vitro were carried out in chambers of 8-μm transwell inserts  in the presence or absence of Matrigel . Eighty thousand MKN45 and SNU601 cells, 40,000 MGC803 cells and 60,000 SGC7901 cells were seeded in the top chamber in serum-free medium, while medium containing 20% serum was in the lower chamber as the attractant. After 24-48 hours, migrated cells were fixed with 4% paraformaldehydeand then stained with 0.1% crystal violet. The number of migrated cells was counted using an IX71 inverted microscope .Migration and invasion assays Cells were lysed and the protein concentration was measured using the bicinchoninic acid protein assay kit . The lysates were subjected to SDS-PAGE gel and transferred to a PVDF membrane . The membrane were blocked with 5% non-fat milk and incubated with indicated primary antibodies, and then probed with the horseradish peroxidase–conjugated secondary antibody. The primary antibodies against ERα was purchased from Abcam. The primary antibodies against p53 and p27 were purchased from Santa Cruz Biotechnology. The primary antibodies against p21 were purchased from Abways Technology. The primary antibodies against AR, snail, slug, α-SMA, and β-catenin were purchased from Cell Signaling Technology. The antibody against GAPDH was purchased from Proteintech Group. The blots were visualized with Pierce™ enhanced chemiluminescence reagents (Life Technologies).p<0.05) variables remained in multivariate survival analysis. The data of functional experiments were expressed as the mean ± standard deviation from three independent experiments, and analyzed using Student's t-test. All statistical analyses were performed by the SPSS 19.0 for windows . A two-tailed p< 0.05 was considered statistically significant.The associations of ERα, ERβ and AR expression with clinicopathological characteristics were evaluated by Chi-square test or Fisher's exact test. Survival data were analyzed using Kaplan-Meier method (log-rank test). Cox proportional hazards was used for univariate and multivariate survival analysis, clinical factors considered relevant for the prognosis including age, adjuvant therapy, TNM classification and stage according to the REMARK criteria , as well


The structure, morphology, and electrochemical performances of the composites are characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, galvanostatic discharge/charge tests, and electrochemical impedance spectroscopy. It was found that the polytriphenylamine particles were composited with LiV3O8 nanorods which acted as a protective barrier against the side reaction of LiV3O8, as well as a conductive network to reduce the reaction resistance among the LiV3O8 particles. Among the LiV3O8/polytriphenylamine composites, the 17 wt % LVO/PTPAn composite showed the largest d100 spacing. The electrochemical results showed that the 17 wt % LVO/PTPAn composite maintained a discharge capacity of 271 mAh·g−1 at a current density of 60 mA·g−1, as well as maintaining 236 mAh·g−1 at 240 mA·g−1 after 50 cycles, while the bare LiV3O8 sample retained only 169 and 148 mAh·g−1, respectively. Electrochemical impedance spectra (EIS) results implied that the 17 wt % LVO/PTPAn composite demonstrated a decreased charge transfer resistance and increased Li+ ion diffusion ability, therefore manifesting better rate capability and cycling performance compared to the bare LiV3O8 sample.LiV When the solid-liquid rheological body presented its intrinsic appearance, the cylindrical Teflon-lined autoclave was placed in an air oven at 80 °C for eight hours. After cooling to 25 °C, the materials were dried again at 100 °C for 12 h. The precursor materials were then calcined in an aerated muffle furnace at 350 °C for 10 h to obtain the bare LVO sample.The bare LVO sample was synthesized by a rheological phase reaction technique . First, 3) as a solvent and ferric trichloride (FeCl3) as an oxidant. LVO (0.2 g), TPA monomer (0.04 g)  and CHCl3 (50 mL) were added into a flask and stirred for 10 min in N2, and then 0.648 mmol FeCl3 oxidant was divided into four portions and added within 1 h. The mixture was constantly stirred at 50 °C for six hours. Finally, the solution was poured into methanol to precipitate, then washed with deionized water and dried at 80 °C overnight to acquire the LVO/PTPAn composite with a content of 20 wt % PTPAn. The preparation of other LVO/PTPAn composites (30 and 40 wt % PTPAn content) was similar to the above route, except for the amount of TPA monomer.The LVO/PTPAn composites were prepared by in situ chemical polymerization of triphenylamine (TPA) using chloroform  was implemented via a Setaram 92 instrument . Power X-ray diffraction (XRD) measurement was carried out through Rigaku MiniFlex 600 diffractometer  with Cu Kα radiation. The morphological features of the specimen were examined by scanning electron microscopy  and transmission electron microscopy . The Fourier transform infrared (FT-IR) spectra of samples were verified on a Nicolet Avatar 360  with KBr pellets.−3 LiPF6 in ethylene carbonate, ethyl methyl carbonate and dimethyl carbonate (EC/EMC/DMC)  electrolyte and assembled in an argon-filled glove box . The assembled cells were galvanostatically discharged and charged on a Land battery tester . Cyclic voltammetry (CV) and EIS were performed on a CHI 660D electrochemical workstation . The amplitude of EIS signal was ±5 mV and the frequency ranged from 100 kHz to 10 mHz under the open-circuit condition. The recorded specific capacities of LVO/PTPAn composites are rely on the mass of the LVO/PTPAn samples.The cathode film was fabricated by 70 wt % as a prepared composite, with 20 wt % acetylene black and 10 wt % polytetrafluoroethylene (PTFE) microemulsion (60 wt %) added into the paste, then rolled into a sheet and dried in an oven. The sheet was then pressed on an aluminum net. The discharge/charge test was accomplished using 2016 type coin cells. The cells were composed of a cathode, a Li metal anode, a commercial polyethylene separator and 1 mol·dm−1. As shown in To determine the content of PTPAn in the composites, TGA was implemented in an aerated environment with a heating rate of 5 °C·minP21/m) [0.3V2O5, which can be usually be observed in the LVO [d100sinθ100 = nλ, the lattice plane spacing (d100) of the crystal was calculated and recorded in d100 spacing first increased, and then subsequently decreased. A maximum d100 spacing of 6.5727 Å was reached for the 17 wt % LVO/PTPAn composite. It is well known that a larger d100 spacing facilitates Li+ ions mobility and distribution in the framework of electroactive host material, which suggests that the 17 wt % LVO/PTPAn composite may demonstrate the excellent electrochemical performances.XRD patterns of the bare LVO sample and LVO/PTPAn composites are shown in P21/m) ,35, and  the LVO ,37,38. T the LVO ,39. Ther the LVO ,41. Usin−1, which are characteristic of the V=O, the symmetric V-O-V, and the asymmetric V-O-V stretching vibrations, respectively [−1, which are attributed to C=C stretching due to the quinoid structures formed, C–C stretching, C–H bending, and C–H out-of-plane vibrations of the PTPAn [The FT-IR spectra of the bare LVO sample, PTPAn polymer and 17 wt % LVO/PTPAn composite are displayed in ectively ,43,44. Mhe PTPAn . The IR The SEM and TEM micrographs of the bare LVO sample, PTPAn polymer, and the 17 wt % LVO/PTPAn composite are shown in +/Li) at the current density of 60 mA·g−1 are presented in −1. However, the LVO/PTPAn composites showed more noticeable plateaus and a reversible capacity of 260 mAh·g−1 for the 14 wt % LVO/PTPAn composite, 267 mAh·g−1 for the 17 wt % LVO/PTPAn composite, and 198 mAh·g−1 for the 33 wt % LVO/PTPAn composite. It is noteworthy that the bare LVO showed the highest initial discharge capacity, however, the 17 wt % LVO/PTPAn composite showed symmetrical charge and discharge potential plateaus, indicating a lower electrochemical polarization. The results also showed that the specific discharge capacities of the LVO/PTPAn composites increased first and then decreased with increased PTPAn content in the composites, because the 17 wt % LVO/PTPAn composite showed the largest d100 spacing listed in + ion insertion in the tetrahedral sites. The second plateau of discharge curves at around 2.55 V was attributed to the phase transformation of LiV3O8 to Li4V3O8 [4V3O8 [The charge-discharge behaviors for the initial cycle of the bare LVO sample and LVO/PTPAn composites between 1.8 and 4.0 V  at the 50th cycle, respectively. On the other hand, the 17 wt % LVO/PTPAn composite provided initial capacities of 275, 222, 223 and 247 mAh·g−1 at the same current rates, and kept 271, 244, 218, and 236 mA·hg−1  at the 50th cycle, respectively. This data showed that the 17 wt % LVO/PTPAn composite has a favorable rate performance and structural stability, even if operated at a high current rate, and can act as a high-performance cathode material for RLB.The cycling performances of the bare LVO sample and the 17 wt % LVO/PTPAn composite at the different current rates are shown in −1 to 120 mA·g−1, the 17 wt % LVO/PTPAn composite still provided a capacity of around 263 mAh·g−1. Even with a higher current rate of 240 mA·g−1, the 17 wt % LVO/PTPAn composite remained at a capacity of 244 mAh·g−1. Additionally, the rate capability of the 17 wt % LVO/PTPAn composite was much better than the bare LVO sample. Even after 60 cycles, the current rates were restored to 30 mA·g−1. The 17 wt % LVO/PTPAn composite was able to recover to near-primary capacity, showing excellent cycling stability. As stated above, we found that 17 wt % LVO/PTPAn composite exhibited better cycling stability and rate capability, which may be explained by the following: First, in comparison with the bare LVO and other LVO/PTPAn composites, 17 wt % LVO/PTPAn composite has the largest d100-spacing, promoting quicker movement of Li+ ions in the host material. Second, PTPAn coating on the surface of LVO may restrain the dissolution of LVO to improve the cycle stability of the LVO. Third, PTPAn composited with LVO may enhance the electrical conductivity of the LVO.As part of the evaluation of the electrochemical performance of the 17 wt % LVO/PTPAn composite, the symmetric stepped rate capability at the various current rates are given in + ions. The CV of the bare LVO and 17 wt % LVO/PTPAn composite at the second cycle is shown in + ion insertion/extraction reactions and the phase transformation in + ions intercalation/deintercalation for LVO/PTPAn composite was decreased, and accordingly, the electrochemical reaction become more reversible due to the suppressed phase transition, thus improving the electrochemical performances of LVO/PTPAn composite. In order to further prove the structural stability of 17 wt % LVO/PTPAn composite, the differential capacity curves on the 10th, 50th, and 90th cycle were compared (The CV features of the 17 wt % LVO/PTPAn composite at various scan rates are shown in compared c. It wasRct) and the Li+ diffusion resistance in the electrodes (Zw), respectively . It was evident that the PTPAn coating provided both an electronic and Li+ ion pathway among the LVO particles, which significantly improved the charge and ionic transfer, consequently elevating the electrochemical performance of LVO sample. Zre vs. ω−1/2, from which the slope  could be attained, and the diffusion coefficient value (D) was calculated by the following equation [R is the gas constant (R = 8.314 J·K−1·mol−1), T is the temperature (T = 298 K), A is the effective contact area between the electrode and the electrolyte (A = π × 1 cm2/4 = 0.7854 cm2), n is the number of electrons transferred per mole of the active material involved in the electrode reaction (n = 3), F is Faraday’s constant (F = 96500 C·mol−1), and C is the concentration of the Li+ ion in the cathode calculated based on the crystallographic cell parameter of LVO (nLi+ = (3/6.02 × 1023) mol, V = 275.5 Å3, C = nLi+/V = 1.808 × 104 mol·m−3). The DLi+ value of the bare LVO (σω = 91.5 Ω·s−1/2) and the 17 wt % LVO/PTPAn composite (σω = 36.2 Ω·s−1/2) was calculated to be 2.58 × 10−16 and 1.65 × 10−15 cm2·s−1, respectively, signifying the quicker Li+ ions diffusion ability of LVO/PTPAn composite.To better understand the reasons for the electrochemical performance of the 17 wt % LVO/PTPAn composite, EIS of the bare LVO sample and the 17 wt % LVO/PTPAn composite were measured and compared for electrochemical resistance, with the results shown in ectively a. The fiequation :D = 0.5d100 spacing and exhibited the highest rechargeable capacity, better cycling behavior, and appropriate rate performance. The results revealed that the 17 wt % LVO/PTPAn composite provided a second reversible capacity of 293 mAh·g−1 and remained as high as 242 mAh·g−1 at the 100th cycle, at the current rate of 60 mA·g−1. Even at the high current density of 240 mA·g−1, the 17 wt % LVO/PTPAn composite gave an initial capacity of 247 mAh·g−1 and maintained 236 mAh·g−1 up to 50 cycles, which was attributed to low charge transfer resistance, quick Li+ ions diffusion ability, and stable structural characteristics. It was suggested that PTPAn can act both as a conductor to promote the transport of electrons and Li+ ions among LVO particles, and as a coating to improve structural stability. These results confirm that LVO composited with proper polymer could be an attractive means for building effective structures for high-performance RLB.LVO/PTPAn composites were prepared via an in situ oxidative polymerization route. Compared to the bare LVO sample, the 17 wt % LVO/PTPAn composite had the largest


Doryteuthis opalescens, is an important forage species for the inshore ecosystems of the California Current System. Due to increased upwelling and expansion of the oxygen minimum zone in the California Current Ecosystem, the inshore environment is expected to experience lower pH and [O2] conditions in the future, potentially impacting the development of seafloor-attached encapsulated embryos. To understand the consequences of this co-occurring environmental pH and [O2] stress for D. opalescens encapsulated embryos, we performed two laboratory experiments. In Experiment 1, embryo capsules were chronically exposed to a treatment of higher  pH (7.93) and [O2] (242 μM) or a treatment of low pH (7.57) and [O2] (80 μM), characteristic of upwelling events and/or La Niña conditions. The low pH and low [O2] treatment extended embryo development duration by 5–7 days; embryos remained at less developed stages more often and had 54.7% smaller statolith area at a given embryo size. Importantly, the embryos that did develop to mature embryonic stages grew to sizes that were similar (non-distinct) to those exposed to the high pH and high [O2] treatment. In Experiment 2, we exposed encapsulated embryos to a single stressor, low pH (7.56) or low [O2] (85 μM), to understand the importance of environmental pH and [O2] rising and falling together for squid embryogenesis. Embryos in the low pH only treatment had smaller yolk reserves and bigger statoliths compared to those in low [O2] only treatment. These results suggest that D. opalescens developmental duration and statolith size are impacted by exposure to environmental [O2] and pH (pCO2) and provide insight into embryo resilience to these effects.The market squid,  However century –6. In ad century . These c0–240 μM . By 20602]  and low pH waters for the entire embryogenesis process (i.e. 3–5 weeks) potentially causing additional energy costs and reducing the scope for aerobic performance  –12. Devec wastes –15. We dc wastes . Under elular pH , 18. D. formance . In cont demands .D. opalescens statoliths are aragonite structures  conditions on D. opalescens embryos, we analyzed the same laboratory experiments and embryo capsules as described in Navarro et al. 2014. Briefly, the first experiment assessed exposure to combined low [O2] and low pH, while the second experiment tested the effects of low [O2] as compared with low pH [a priori that embryos exposed to low [O2] and low pH would have longer developmental duration and be smaller in size  at the same embryonic stage . For the second experiment, we hypothesized that D. opalescens would have a morphological response to decoupled pH and [O2] treatments. Embryos exposed to the low [O2] treatment were expected to have a longer developmental duration, while embryos exposed to the low pH treatment were expected to be smaller in size [D. opalescens is coupled to and can be affected by environmental pH and [O2].To assess the effects of chronic exposure to realistic low environmental pH and , pH, and alkalinity were measured discretely (2] and Durafet (pH) sensors among tanks. Samples of embryos from this study were taken from unique aliquots from the same capsules described in Navarro et al. 2014  of 80.4 μM (± 18.7), a pH level of 7.57 (± 0.066) and an alkalinity of 2,215.1 μM (± 6.0). Treatment values in Experiment 1 fell within the range characteristic of upwelling (low pHOx) and relaxation (high pHOx) events in the region at ~30 m depth [D. opalescens are exposed to a range of [O2] and pH conditions because they can spawn at all times of the year in the Southern California Bight [2] and pH exposure. This type of exposure could reasonably occur during conditions with a persistent isopycnal uplift, such as occurring during a La Niña and/or a strong upwelling season [2] and pH each as a single stressor (i.e. low [O2] only or low pH only). This experiment was designed to identify if squid develop differently when [O2] and pH levels are de-coupled from one another, to assess whether or not [O2] and pH coupling impacts development. The “low pH” only treatment pH level was 7.56 (± 0.028) with alkalinity of 2,242.7 μM (± 6.5) and [O2] at 241.4 μM (± 8.4). Whereas the “low [O2]” only treatment had an [O2] of 84.7 μM (± 10.6), with a pH level of 7.92 (± 0.054) and alkalinity of 2,240.5 μM (± 5.0). In both experiments, pH was controlled by manipulating pCO2, and alkalinity remained relatively constant   were different between low pHOx and high pHOx tanks; post hoc Dunn’s pairwise joint ranking tests were performed for temperature and alkalinity. Among all tanks, temperature was distinct only between tanks in the low [O2] treatment; alkalinity was not distinct between tanks  , 29. Treeatments . A Krusken tanks . Abioticen tanks ; S2 Fig.2] only) developed to the same stages over a similar duration of time as Experiment 1. Upon removal, embryo capsules were immediately photographed, and the total number of embryos per capsules was quantified. Then embryos were either preserved for morphological analyses or frozen for statolith analyses. A single capsule contains hundreds of embryo-filled chorions , with each embryo having two statocysts and statoliths. Per capsule, approximately 50 embryos and their chorions, from the middle position of the capsule  (pHOx) affect D. opalescens embryo development stage, yolk and statolith size. Embryos exposed to low pHOx had 149.9% larger YV, were 39.6% shorter in DML and had 13.0% wider HWs than those, of the same age exposed to high pHOx (Chronic exposure (≥ 24 d) to varied levels of pH and  for 32 d were 19.1% shorter in DML and had 379.2% bigger yolks by volume compared to those, of the same age, from the low pH, 32-d treatment. Nearly all the outer yolk was absorbed by embryos from the low pH treatment. The embryos removed after 28 d of exposure from the low [O2] treatment had a 20.4% shorter dorsal mantle length and 85.9% larger yolk volume than those exposed to low pH for the same time duration , reduced YV  and longer TL . Exposure duration did not affect the HW between groups. For low pH treatment, the YV of the 32-d exposure group (0.201 mm3) approached zero and was reduced by 74.9% compared to the 28-d exposure group (0.801 mm3). For the low [O2] treatment 32-d exposure group, there was a 35.2% reduction of the YV (0.965 mm3) compared to embryos in the 28-d exposure group (1.490 mm3).Older embryos (32-d exposure) had longer DML , YV  and HW , largely driven by differences between tanks in the pH treatment at 28 days of exposure. At 32 days, DML and HW were similar, and only YV was statistically distinct. Further, these biological tank effects did not mirror abiotic tank effects , HW , YV, and TL . These differences are biologically significant and are likely caused by the capsular and chorion membranes and maternal effects [Capsules effects were observed for DML ( effects .pCO2 = 1440 μatm) and 90 μM O2 affected embryos by slowing their development by on average 2.8 stages compared to all those exposed to the high pHOx treatment  at stages 25–28 were similar between low pHOx and high pHOx treatments in Experiment 1. Thus we infer that differences in size at age  are due 2] group. DML and HW were similar between embryos from the low pH and low [O2] treatments.In Experiment 2, for most stages  the embryos from the low pH treatment had a smaller yolk volume . Also, s2] treatment compared to those from the same stage (29) in the low pH treatment . These embryos did not significantly differ from control embryos in size or yolk volume, suggesting that some squid embryos may be resilient to low pHOx conditions.We observed clear effects of exposure to chronic low pHOx on perature . Small eth rates –42. In o2] is important for embryo development. Embryos exposed to low pH had smaller external yolk sacs at each stage  compared to those from the low [O2] treatment, possibly indicating that these embryos were absorbing more yolk. This difference in YV suggests these embryos may be allocating more energy towards maintaining their internal pH balance relative to embryos exposed to low [O2]. Loliginid embryos exposed to acidified conditions have been shown to upregulate ATP-dependent ion pumps on the yolk epithelium to mediate extracellular pH regulation, suggesting a potential mechanism for elevated energy utilization in response to low pH [2] could represent an extreme environmental change for the D. opalescens at the embryo life stage and pose serious challenge to their physiological limits. As such, future investigations not only should assess the magnitude for change of pH and [O2] under future climate change scenarios but should also assess whether or not the relationship between pH and [O2] will change from historic values.The coupling of pH and ). During embryonic development, some cephalopods experience increased metabolic costs caused by additive effects of combined pH and pCO2 with another environmental stressor [2] over the course of development. As a result, cephalopods such as cuttlefish  may be within the upper-shelf waters < 40 m depth, with harsher habitat occurring deeper. At shallow depths, semi-diurnal tidal currents usually bathe embryos attached to the seafloor with high [O2] and high pH/ lowpCO2 waters [2] and pH levels in water masses are cues driving spawning site selection, D. opalescens might attach embryo capsules at different depths on the shelf at different times throughout the year [2] and pH that are predicted to shrink due to human-induced sources of hypoxia and hypercapnia  treatment. * = tank effect . Bars = ± 1 standard error.Seawater properties per tank. Box plot distribution of daily averages and variability for total alkalinity, temperature, pH and dissolved oxygen. A Kruskal-Wallis revealed tanks effects were statistically significant for temperature and total alkalinity (TA) in Experiment 2. (TIF)Click here for additional data file.S2 Fig2] tank replicates 1 and 2. Y-axis = Biological response variable. (a) Dorsal mantle length (mm) = DML. (b) Yolk volume (mm3) = YV. (c) Head width (mm) = HW. (d) Total length (mm) = TL. Bars = ± 1 standard error.X-axis = . (b1) ANOVA results. (b2) post hoc TUKEY results. DML = dorsal mantle length, YV = external yolk sac volume, HW = head width, TL = total length of the embryo and external yolk sac. All results were Bonferroni corrected (ɑ = 0.125). Bold and italicized font = significant.(a) Experiment 1, treatments = low pHOx, high pHOx. (a1) ANOVA results. (a2) (DOCX)Click here for additional data file.S3 Tablepost hoc Tukey test results. (b) Experiment 2: Results of the Wilcoxon test. DML = dorsal mantle length, YV = external yolk sac volume, HW = head width, TL = total length of the embryo and external yolk sac. All results were Bonferroni corrected (ɑ = 0.125). Bold and italicized font = significant.(a1) Experiment 1: Results of the hierarchal ANOVA. (a2) Experiment 1: (DOCX)Click here for additional data file.


Salmonella compared to birds fed control diet, 87 ± 17% versus 74 ± 13%, respectively. However, no impact of DON was observed on the Salmonella induced disease signs, organ lesions, faecal and organ Salmonella counts. The presented risk assessment indicates that pigeons are frequently exposed to mycotoxins such as DON, which can affect the outcome of a Salmonella infection. The increasing number of pigeons shedding Salmonella suggests that DON can promote the spread of the bacterium within pigeon populations.Seed-based pigeon diets could be expected to result in exposure of pigeons to mycotoxins such as deoxynivalenol (DON). Ingestion of low to moderate contamination levels of DON may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host-pathogen interactions and thus different outcome of infections. Here we demonstrate that DON was one of the most frequently detected mycotoxins in seed-based racing pigeons feed, contaminating 5 out of 10 samples . Subsequently, a toxicokinetic analysis revealed a low absolute oral bioavailability (F) of DON in pigeons (30.4%), which is comparable to other avian species. Furthermore, semi-quantitative analysis using high-resolution mass spectrometry revealed that DON-3α-sulphate is the major metabolite of DON in pigeons after intravenous as well as oral administration. Following ingestion of DON contaminated feed, the intestinal epithelial cells are exposed to significant DON concentrations which eventually may affect intestinal translocation and colonization of bacteria. Feeding pigeons a DON contaminated diet resulted in an increased percentage of pigeons shedding  Fusarium is one of the most important toxigenic fungal genera in small grain cereals. Although members of this genus can cause grain yield losses, the interest in Fusarium head blight is primarily fuelled by the ability of the majority of the Fusarium species to produce mycotoxins. These mycotoxins exhibit an intrinsic toxicity and pose a potential acute or chronic health threat to humans and animals upon ingestion of contaminated food or feed [F. graminearum and F. culmorum [ or feed , 2. The  or feed . DON is culmorum . DON hasculmorum . Differeculmorum , 6. Afteculmorum , 8. Thisculmorum . Furtherculmorum . HoweverIt is well known that DON acts as an inhibitor of the protein synthesis at the ribosomal level , 11. TheSalmonella Typhimurium infection by enhancing Salmonella invasion and translocation across the intestinal epithelium, as well as by potentiating the early intestinal immune response induced by the infection [Salmonella Typhimurium by macrophages, through modulation of the cytoskeleton due to extracellular signal-regulated kinase 1/2 (ERK1/2) activation [Salmonella Typhimurium-related organ lesions or mortality was seen [Salmonella Typhimurium variant Copenhagen strains [Since the intestinal tract is also a major portal of entry to many pathogens, DON exposure could affect the animal susceptibility to infectious diseases, such as salmonellosis , 20. Feenfection , 20. Furtivation . In broiwas seen –24. Salm strains , 26. The strains .Columba livia) feed, (2) to assess the toxicokinetic characteristics of DON in racing pigeons, and (3) to evaluate the impact of DON on the susceptibility and course of an experimental Salmonella Typhimurium variant Copenhagen infection in racing pigeons.The consumption of seed-based diets could result in high exposure to mycotoxins such as DON in pigeons, which in turn may affect the animal's susceptibility to infectious diseases . Therefo1, B2, G1 and G2, altenuene, alternariol (AOH), alternariol methylether (AME), DON, 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivelenol (15ADON), diacetoxyscirpenol, enniatin B (ENNB), fumonisin B1 (FB1), B2 (FB2) and B3 (FB3), fusarenon-X, neosolaniol, nivalenol, ochratoxin A (OTA), roquefortine-C, sterigmatocystin, T-2 toxin, HT-2 toxin and zearalenone (ZEN). The decision limit (CCα) was defined as the concentration at “the y intercept plus 2.33 times the standard deviation of the within laboratory reproducibility” (α = 1%) in the case of substances for which no permitted limit has been established. In the case of substances with a maximum limit such as aflatoxin B1, CCα was established by spiking blank material around the maximum limit. The corresponding concentration at the permitted limit plus 1.64 times the standard deviation of the within laboratory reproducibility equals the decision limit (α = 5%) [Ten different commercial racing pigeon feeds, all seed mixtures, were bought from local stores in Flanders, Belgium. On average, the major commodities of these seed mixtures were corn (34 ± 6.9%), peas (17 ± 8.0%), wheat (15 ± 6.9%), sorghum (15 ± 8.4%), rice (7 ± 6.6%), barley (6 ± 3.8%) and safflower seed (6 ± 4.0%). Minor commodities in some of the seed mixtures were oats (3 ± 2.4%), hemp seed (2 ± 2%), sunflower seed (2 ± 0.6%), buckwheat (2 ± 1.4%), rape seed (2 ± 1.8%), linseed (1 ± 0.9%), milk thistle seed (1 ± 1%), millet (1 ± 0.4%) and canary seed (1 ± 0.5%). Samples of 500 g were taken at three different locations in one bag of each of the 10 feeds (20 kg), subsequently pooled and analyzed for mycotoxin contamination. Analysis was performed by a validated multi-mycotoxin liquid chromatography-tandem mass spectrometry method (LC-MS/MS) . The fol(α = 5%) .ad libitum. Animals were deprived of feed 8h before the administration of DON until 2 h post-administration (p.a.). Pigeons were administered a single bolus of DON (0.3 mg/kg BW), either by oral gavage (PO)  or by intravenous injection (IV) in the vena cutanea ulnaris superficialis (wing vein) , using a two-way crossover design. This administered dosage was based on the average daily feed intake (30 g per pigeon) and maximum European guidance level of DON in poultry feed . The DON bolus was prepared immediately prior administration by dissolving DON analytical standard  in ethanol (10 mg/mL) and further diluting with water (PO bolus) or saline solution (IV bolus) up to a concentration of 0.3 mg/mL. Blood samples (0.2 mL) were collected in heparinized tubes from the vena metatarsalis plantaris superficialis (leg vein) before administration (t = 0) and at 5, 10, 20, 30, 40, 50, 60, 90 and 120 min p.a. Blood samples were centrifuged  and plasma was stored at ≤-15°C until further analysis. After a two weeks wash-out and recovery period, the protocol was repeated. The birds that previously received an IV injection of the mycotoxin then received a PO bolus and vice versa. The dosing, blood collection, and sample storage was performed in the same way as the first administration. The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine and the Faculty of Bioscience Engineering of Ghent University (EC 2014/67).Ten racing pigeons : 430 ± 34 g) were obtained from a commercial breeder. A one week acclimatization period was respected and animals were group housed applying a 14 h light/10 h dark cycle. A seed-based diet , 25 μg/mL) stock solution in ACN were purchased from Sigma-Aldrich . All the above mentioned stock solutions were stored at ≤−15°C. Individual working standard solutions of 1 μg/mL were prepared by diluting the above stock solutions with HPLC-grade ACN and were stored at ≤−15°C. ACN solutions of DON have been reported stable for 24 months at room temperature, for DOM-1 no published stability data are available [DON analytical standard was purchased from Fermentek and was dissolved in acetonitrile (ACN) yielding a stock solution of 1 mg/mL , time to maximal plasma concentration (Tmax), area under the plasma concentration-time curve from time 0 to 90 min (AUC0-t), elimination rate constant (kel), elimination half-life (T1/2el), mean residence time (MRT), volume of distribution (Vd), and clearance (Cl). The absolute oral bioavailability (F) was calculated according to the following formula:Toxicokinetic analysis was performed using a non-compartmental model . Plasma levels below LOQ were not included in the toxicokinetic analysis. The most important toxicokinetic parameters of DON were calculated: maximal plasma concentration HPLC coupled to high resolution mass spectrometry (HR-MS) analysis for identification and semi-quantification of the phase II biotransformation metabolites of DON. Analysis was based on a method for turkeys and broiler chickens, as previously described by Devreese et al. .1 (42 ± 5 μg/kg) and FB2 (80 ± 8 μg/kg) were detected in the control feed. The DON level in the mycotoxin contaminated diet was 3,538 ± 1,000 μg/kg feed.DON contaminated feed was produced by adding DON to a control diet, a commercial extruded pellet feed . DON was kindly provided by dr. M. Lemmens . Purity of the provided stock was > 99%. DON was dissolved in ethanol and further diluted in a fructose-glucose syrup . Subsequently, this syrup was sprayed homogeneously over the pellets to a final level of 2% (v/m). Similarly, a blank syrup was sprayed over the control diet. Mycotoxin contamination of both the control and DON contaminated diet was analyzed by the validated multi-mycotoxin LC-MS/MS method as mentioned in section 2.1 . Low levSalmonella and were negative for the presence of Salmonella bacteria in their feces at multiple sampling points as well as for serum agglutinating antibodies and the presence of anti-Salmonella IgG antibodies as tested by an in-house enzyme-linked immuno-sorbent assay (ELISA). Feed and water were given ad libitum throughout the trial and a 14 h light/ 10 h dark cycle was applied. During a two week acclimatization period all animals were fed the control diet and group housed in an aviary of 6 m3.Twenty racing pigeons were divided into two groups , one group was fed the control diet and the other group was fed the DON contaminated diet. The animals were obtained from a breeding colony free of Salmonella Typhimurium variant Copenhagen DAB69 strain [8 colony forming units (CFU) of the strain. Post inoculation, Salmonella fecal shedding was assessed daily by counting the numbers of Salmonella CFU per gram excreta. This was done by plating ten-fold serial dilutions on Brilliant Green Agar (BGA) plates . If negative after direct plating, the samples were pre-enriched in buffered peptone water  at 37°C and then enriched in tetrathionate brilliant green broth  at 37°C. The animals were examined daily for the presence of disease signs such as apathy, diarrhea, and polyuria. Faecal consistency was scored daily as described by Pasmans et al. [Salmonella lesions by the same person as follow: 0, no macroscopic lesions; 1, visual organ enlargement; 2, presence of granuloma’s (< 3 mm); 3, presence of granuloma’s (3–6 mm); 4, presence of granuloma’s (7–9 mm); 5, presence of granuloma‘s (> 9 mm). The small intestines were scored exclusionary using the following system: 0, no macroscopic lesions; 1, serosal congestion; 2, abnormal content; 3, haemorrhagic content; 4, presence of granuloma’s (<3 mm); 5, presence of granuloma‘s (3–6 mm). From each animal, the left lung, liver, spleen, kidney, gonads, brain, ileal mucosa, ileal content were homogenized, and the number of CFU of Salmonella per g tissue was determined as described above. Tissue samples were fixed in 10% buffered formalin. After fixation, the samples were processed for histological examination. Paraffin sections were stained with haematoxylin-eosin. Ten serial histologic sections of three randomly selected lung samples per group were submitted to further histologic examination. The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University (EC 2014/108).Next, experimental diets, i.e. control or DON contaminated feed, were fed from day 1 of the experiment onwards until the end of the experiment (day 28). From day 1 until day 7 birds were housed per experimental group in an aviary. From day 8 onwards each animal was housed individually in a cage of 50 x 70 x 40 cm (width x depth x height) with a wire mesh floor. The BW of animals was measured on day 7, 14, 21 and 28. A pigeon-adapted 9 strain  was grows et al. : 0, normSalmonella growth: 0, 0.2, 2 and 20 μg DON/mL. A stationary phase culture of Salmonella Typhimurium variant Copenhagen strain DAB69 was 1:2500 diluted in LB broth medium, and incubated aerobically at 37°C. Tenfold dilutions were prepared in phosphate buffered saline (PBS), and subsequently plated on BGA at 0, 1, 2, 3, 4, 5, 6, 7, 8, and 24h after incubation to determine the number of CFU/mL and establish a growth curve. After aerobic incubation overnight at 37°C, the number of CFU per mL was determined. All tests were performed in triplicate.Following concentrations of DON were tested for their effect on Salmonella CFU were tested for normality using the Shapiro-Wilk test. Normally distributed data were analyzed using the (un)paired two-sided Student’s t-test to address the significance of difference between mean values with significance set at p ≤ 0.05. Categorical dependent variables faecal consistency score and Salmonella lesion score were converted to a binary dependent variables, with a score of two or more considered as poor faecal consistency and positive for Salmonella-related organ lesions, respectively. Subsequently, faecal consistency, Salmonella lesion score, and percentage of pigeons shedding Salmonella per day were analyzed by logistic regression.Statistical analysis was done using SPSS 23 software . BW and number of 1/kg, 181 μg FB2/kg, and 169 μg FB3/kg)  and OTA (16 ± 1.9 μg/kg) (CCα = 3 μg/kg). None of the observed contamination levels of ZEN, fumonisins, and OTA exceeded the European maximum guidance level for poultry feed [Alternaria mycotoxins AOH and AME. No levels above the CCα were detected of the following mycotoxins: aflatoxin B1 (CCα = 7 μg/kg), B2 (CCα = 2 μg/kg), G1 (CCα = 2 μg/kg) and G2 (CCα = 2 μg/kg), altenuene (CCα = 5 μg/kg), 15ADON (CCα = 3 μg/kg), diacetoxyscirpenol (CCα = 1 μg/kg), fusarenon-X (CCα = 17 μg/kg), neosolaniol (CCα = 7 μg/kg), nivalenol (CCα = 36 μg/kg), roquefortine-C (CCα = 1 μg/kg), sterigmatocystin (CCα = 5 μg/kg), T-2 toxin (CCα = 9 μg/kg), and HT-2 toxin (CCα = 9 μg/kg).DON was detected in 5 out of 10 commercial pigeon feed samples (CCα = 61 μg/kg), with an average contamination level of 515 ± 537 μg/kg  . Howevertry feed . Current1/2el = 0.3 ± 0.09 h), as well as after PO (T1/2el = 0.6 ± 0.19 h) administration. DOM-1 was not detected in any sample. The HR-MS analysis revealed the presence of the deoxynivalenol-3a-sulphate (DON3S) metabolite in plasma , respectively . Anorexia was only observed in one bird in the DON group and two birds in the control group. Polyuria was only observed in two pigeons in the DON group and not in the control group. Following the oral inoculation with Salmonella, a decrease in the bird’s BW was observed, although no difference was observed between both experimental groups (P> 0.1) , kidneys (60%–70%) and small intestines (50%–60%), in both, control and DON group, respectively. In only one animal in the control group and none in the DON group a Salmonella-related lesion was observed in the spleen. No significant differences were observed between both experimental groups. In contrast, a significant lower mean number of Salmonella CFU (P = 0.01) was observed in the lung of pigeons fed a DON contaminated diet, compared to pigeons fed a control diet  compared to the control group (90%), and only in one pigeon in the control group and none in the DON group organ lesions were observed. Histology of the lung revealed typical lesions seen in Salmonella infections. Within the DON group lymphocyte aggregates were found throughout the lung parenchyma and granuloma’s consisting of central necrosis surrounded by giant cells. The control group showed lymphocyte aggregates in the lung parenchyma, but in lower numbers and smaller in size compared to the ones in the DON group. Granuloma’s  were not present within the control group. The results of the in vitro Salmonella growth assay showed no influence of 0, 0.2, 2 or 20 μg DON/mL on the bacterial growth curve (Feeding pigeons a diet contaminated with DON significantly increased the percentage of pigeons shedding ectively . However samples . No sign(P> 0.1) . None ofrol diet . Salmoneth curve .For the first time, to our knowledge, a risk assessment was made for the mycotoxin DON in pigeons. The occurrence of different mycotoxins was clearly demonstrated in commercially available racing pigeons feed samples. The percentage of pigeon feed samples contaminated with DON and the average contamination level were in accordance with a long-term global mycotoxin survey program in feed and feed raw materials . In thisAlternaria mycotoxins. However, the impact of Alternaria mycotoxins on animal health is still largely unknown [It is important to emphasize the frequent co-occurrence of different mycotoxins in feed and feed raw materials. Indeed, different fungi are able to simultaneously produce a variety of mycotoxins, and on the other hand, any given commodity is likely to be infected with different types of fungi . Moreove unknown , 37.Differential sensitivity to the toxic effects of DON might be related to differences in toxicokinetic properties among animal species , 6. The Fusarium mycotoxins and Salmonella infection is difficult to predict, and can be influenced by the animal host species, the bacterial strain and the effect of the mycotoxin on the bacterium, the host cells and the host-pathogen interaction. In the present study, it was demonstrated for the first time that feeding pigeons a diet contaminated with DON increased the percentage of pigeons shedding Salmonella compared to pigeons fed a control diet. This increasing number of pigeons shedding Salmonella might suggest that DON may promote the spread of the bacterium within or to other pigeon populations. As horizontal transmission of enteric pathogens is important in a population, the pathogen will benefit from an increasing number of individuals able to spread the pathogen. For pathogens which can induce a chronic carrier state and need a stable pathogen reservoir, a higher number of pathogen excreting animals is beneficial. A rapid and efficient intra- and inter-flock spread is primordial for horizontal transmission, especially in the case of pigeon salmonellosis. The difference in percentage of animals shedding the bacterium was not reflected in differences in the faecal and organ Salmonella counts between both groups in the present experimental setting. Besides, no impact of DON was observed on the occurrence of Salmonella-related clinical symptoms and organ lesions. Similarly, no obvious impact on Salmonella Typhimurium translocation could be demonstrated in pigs fed a fumonisins contaminated feed [Salmonella Typhimurium invasion and translocation [et al. [Salmonella counts correlated to the direct toxic effect of T-2 toxin on the bacterium. Although, no such direct toxic effect of DON was observed on the pigeon-adapted Salmonella Typhimurium variant Copenhagen strain DAB69, it remains unclear why the DON-exposed pigeons had lower lung Salmonella counts. Contradictorily, histologic examination of randomly chosen lungs showed more and more severe lesions in the DON group compared to the control group. It is important to emphasize that Salmonella organ count is not positively correlated with the number and severity of lesions. The fact that more microscopical lesions were noticed in the DON group might be related to the immunomodulating effect of DON [As salmonellosis is the main bacterial disease in pigeons , 26, it ted feed . In contlocation , 40, altt of DON .Salmonella infection in a pigeon flock by increasing the number of pigeons shedding the bacterium and a tendency towards more and extensive paratyphoid lesions. Future work looking at dose response, the effect of chronic exposure and the effect of DON in chronic Salmonella infections should be able to clarify the complex interplay between host, mycotoxin and pathogen.In conclusion, our study clearly demonstrated that DON frequently contaminates feed for racing pigeons. Toxicokinetic analysis revealed that the absolute oral bioavailability of DON in pigeons was low, and comparable to other avian species. DON showed a high clearance, which results in a short elimination half-life. In accordance to other avian species, DON is predominantly metabolized to DON3S in pigeons. Feeding a DON contaminated diet may modulate the spread of a S1 Table8 colony forming units of Salmonella Typhimurium variant Copenhagen DAB69 strain.Pigeons were fed either a control diet or a DON contaminated diet for 28 days. BW was measured at day 7 , day 14, 21 and 28. At day 15, each pigeon was inoculated with 10(TIFF)Click here for additional data file.S1 FigSalmonella Typhimurium variant Copenhagen strain DAB69 was 1:2500 diluted in LB broth medium with 0, 0.2, 2 and 20 μg DON/mL, and incubated aerobically at 37°C. Samples were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24h after incubation following inoculation with Salmonella. The number of colony forming units (cfu) per mL was determined by bacterial plating of 10-fold dilutions. Results are presented as the mean cfu/mL. There is no significant difference between the different test conditions.A stationary phase culture of (TIFF)Click here for additional data file.


Non-replicating vaccines, such as those based on recombinant proteins, require adjuvants and delivery systems, which have thus far depended on mimicking pathogen danger signals and strong pro-inflammatory responses. In search of a safer and more efficacious alternative, we tested whether vaccinations with influenza recombinant hemagglutinin (HA) mixed with a novel vegetable oil in water emulsion adjuvant , based on the immune-enhancing synergy of vitamins A and E and a catechin, could protect against intra-nasal challenge with live influenza virus. Vaccinations of inbred Brag Albino strain c (BALB/c) mice, with HA mixed with NIDS compared to other adjuvants, i.e., a squalene oil in water emulsion (Sq. oil), and the Toll Like Receptor 3 (TLR3) agonist Poly (I:C), induced significantly lower select innate pro-inflammatory responses in serum, but induced significantly higher adaptive antibody and splenic T Helper 1 (TH1) or TH2, but not TH17, responses. Vaccinations with NIDS protected against infection, as measured by clinical scores, lung viral loads, and serum hemagglutination inhibition titers. The NIDS exhibited a strong dose sparing effect and the adjuvant action of NIDS was intact in the outbred CD1 mice. Importantly, vaccinations with the Sq. oil, but not NIDS, induced a significantly higher Serum Amyloid P component, an acute phase reactant secreted by hepatocytes, and total serum IgE. Thus, the NIDS may be used as a clinically safer and more efficacious vaccine adjuvant against influenza, and potentially other infectious diseases. Most non-replicating vaccines require adjuvants and delivery systems. Several adjuvants and delivery systems for protein-, DNA-, and RNA-based vaccines have been devised and used in animal and early clinical studies . CommercIn search of safer and more efficacious vaccine adjuvants and delivery systems, we focused on select vitamins and flavonoids. We previously demonstrated the ability of NIDS to enhance humoral and cellular immune responses against co-administered HIV-1 envelope antigens ,9. We haFor prophylactic vaccines, the safety bar is rightfully set high, as the vaccinees are otherwise healthy with regards to the disease they are vaccinated against. Therefore, it is important to find biomarkers that define safety in preclinical studies. Serum Acute Phase Reactants (APR) could serve assuch safety biomarkers. APR refers to a group of proteins that change their concentration significantly in response to an inflammatory response such as an infection or a wound . One reaIn the present study, we compared the adjuvanticity of NIDS with that of two licensed adjuvants and delivery systems, i.e., aluminum hydroxide and the squalene oil based oil in water emulsion, which are known to preferentially induce T Helper 2 (TH2)-type responses . As a cov/v Span80 and Tween80 (Sigma Aldrich) and was homogenized at high speed with a Silverson homogenizer to yield an emulsion. The amount used for each vaccine mouse dose was 30 µg for Vit A, 2 mg Vit E, 240 µg for EGCG, and 35% v/v for seed oil. Recombinant HA protein (H1N1sw 2009) corresponding to 0.25 µg and 0.025 µg per dose was used as indicated in the immunogenicity studies. For the protective efficacy study, the rHA  dose was 0.4 µg. The total volume for each Intra-muscular (IM) thigh dose was 100 µL, adjusted with Dulbecco’s phosphate buffered saline (Cat# 21-030-CV). All vaccines were prepared with endotoxin free reagents and in endotoxin free 2.0 mL tubes (Eppendorf biopur safe-lock microcentrifuge tubes).Vitamin A palmitate (Vit A) and alpha-tocopherol (Vit E) , were mixed into mustard seed oil  to form the oil phase. EGCG, (Sigma/Aldrich)) was mixed in 0.1× PBS and then added to the oil phase in the presence of 2% each ® that has been licensed in Europe for adjuvanted flu vaccines, at 1:1 v/v for antigen:Addavax), was used as a well-known and licensed systemic adjuvant. Serum was collected 21 days after the first immunization or 10 days after the second immunization. For the measurement of T cell responses in systemic tissues, 10 days after the final immunization, mice were euthanized and spleens (SP) were harvested for the detection of antigen-specific T cells as measured by cytokine production. The mice were maintained at the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved vivarium of the University of Connecticut, Storrs. The investigators adhered to the “Guide for the Care and Use of Laboratory Animals” prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council.All animal studies were approved by the IACUC of the University of Connecticut  and the Trudeau Institute before the onset of studies. Female inbred Brag Albino strain c (BALB/c) or outbred CD1 Swiss Albino-derived mice  (5 per group for immunizations studies and six per group for the protective efficacy study) were immunized once or twice intra-muscularly in the thigh (IM) at a 3 week interval. All immunizations were performed on unanesthetized mice. As a comparison, HA mixed with AddaVax™  was performed on serum from individual mice collected at 3 weeks after the 1st or 10 days after the second immunization. Maxisorp 96-well flat-bottom plates  were coated overnight at 4 °C with 0.6 μg/mL HA in phosphate-buffered saline pH 7.4 (PBS). The coated wells were blocked for 1 h at room temperature with 300 μL of PBS pH 7.4, 0.1% BSA, and 0.05% Tween-20 with 1% goat serum. The plates were washed with PBS pH 7.4, 0.1% BSA, and 0.05% Tween-20, tapped. Serum samples were initially diluted 1:300 with the dilution buffer , then transferred into coated-blocked plates in which the samples were serially diluted three-fold with the same buffer. Antigen specific IgG1, IgG2b, IgG2a, and IgA titers were revealed with HRP-conjugated goat anti-mouse IgG1, IgG2b, IgG2a, and IgA . Antibody titers are expressed as the logarithm of the enzyme-linked immunosorbent assay titers that give an optical density (OD) higher than the mean plus five times the standard deviation (SD) of the average OD obtained in the pre-immune or naïve sera.6 per mL in the presence or absence of 0.5 μg/mL HA from the 2009 H1N1 swine strain. Supernatants were collected and stored at −80 °C. Innate cytokines and chemokines and adaptive cytokines as stated above, were measured by the Multiplex Luminex Assay using an in-house MAGPIX  instrument and using Millipore kits as per the manufacturer’s protocol.For the detection of serum innate responses, sera were collected at 16 hours following the first IM vaccination and analyzed for concentrations of G-CSF, IL-5, MCP-1, MIP1, MIP-1β, IL-6, IP10, KC, MIP-2, RANTES, and TNFα by the Luminex multiplex assay using a Millipore MAGPIX instrument. For the detection of adaptive TH1 , TH2 , TH17 (IL-17A/F), and Treg (IL-10) cytokine responses and all chemokine responses, single-cell suspensions from SP were cultured overnight in 24 well plates at a concentration of 5 × 10Mouse Serum Amyloid P (SAP) protein was measured by endpoint enzyme linked immunosorbent assay (ELISA). The assay was performed using the Mouse Serum Amyloid P (SAP) ELISA Test Kit  according to the manufacturer’s protocols. Briefly, microtiter wells were coated with a peptide-specific antibody that recognizes different epitopes on mouse SAP. Another peptide-specific antibody is conjugated to Horseradish peroxidase (HRP) and used for detection. Serum samples collected from mice at 16 hours after a single IM vaccination with HA alone, or HA mixed with NIDS, Sq. oil, Poly (I:C) or Imject Alum were diluted 1:20 and 100 µL of each sample were added to the pre-coated wells and incubated with 100 µL of HRP-conjugate for an hour on a micro-plate shaker at 150 rpm at room temperature. 100 µL SAP standards were diluted at the following concentrations, 500, 250, 125, 62.5, 31.25, 15.63, and 7.81 ng/mL and incubated with the HRP-conjugated secondary antibody for an hour on the shaker. Unbound HRP-conjugate were washed away with washing buffer included in the kit. Next, 100 µL of TMB Reagent were added to the wells and the plates were incubated for 20 min at room temperature. Color development was stopped using the stop reagents included in the kit. Using a plate reader, the optical density was measured at 450 nm and the serum SAP concentrations were calculated from the standard curve and expressed as ng per mL serum.Isolation of Serum: Mice were bled through the mandibular vein one day prior to the beginning of the challenge study. In brief, mice were scruffed by the neck. The grip was tight enough so that the cheek was taut, but did not restrict respiration of the mouse. The slight depression in the fur near the middle/rear of the mandible was located. Gently, in an upward motion toward the mandible, an 18 gauge needle penetrated the skin not further than 1 mm. Blood was collected into the BD Microtainer with serum separator tubes. Blood was left at room temperature for 30 min. Tubes containing blood was spun at 10,000 rpm for 10 min. Serum was isolated and placed into a fresh Eppendorf tube and stored at −20 °C.Hemagglutination Inhibition Assay: The Hemagglutination Inhibition assay detects serum antibodies to the viral hemagglutinin by measuring the ability of antibodies to inhibit virus-mediated agglutination of erythrocytes. In brief, chicken red blood cells (RBCs) (Charles River) were washed 3–5 times in PBS and resuspended at a final concentration of 1%. The influenza virus was thawed and adjusted to appropriate hemagglutination units in 0.05 mL of PBS. Serum was initially diluted in PBS 1 to 10 and serially diluted in five-fold increments resulting in dilutions from 1:10 to 1:781,250. Then 0.025 mL of diluted serum was added to the 0.05 mL of the virus and incubated for 30 min at room temperature. After this incubation, 0.05 mL of 1% RBCs was added to every well and incubated for one hour at room temperature. Plates were observed for agglutination, with nonagglutinating cells being defined as a button appearance of cells at the bottom of the well. The Hemagglutination Inhibition Assay titer of the serum samples was determined to be the inverse of the last dilution where cells were not agglutinated.Viral Stock Preparation for Intranasal Administration: The virus stock of the H1N1 2009 swine influenza virus strain and clade was retrieved from the −80 °C freezer and quickly thawed. The EID50 stock was serially diluted to a concentration of 1.0 × 105 EID50 in PBS without Ca2+/Mg2+.Anesthesia: Animals were anesthetized using an Isoflurane special circuit system ; working setup: 3% Isoflurane in O2 at 1.75 L/min O2 at 14.7 PSIA flow rate. Animals were subjected to anesthesia prior to viral infection and prior to each IN instillation.Viral Infection: Anesthetized mice were infected intranasally with the LD50  of the H1N1 2009 swine Influenza virus in 50 µL PBS without Ca2+/Mg2+.Monitoring: Following IN instillations, and during the postoperative recovery period, mice were observed for possible respiratory disorders . Mice were observed as they recovered from anesthesia and again one day later for signs of illness and to be sure they were eating and drinking as approved and reviewed by the IACUC committee. Follow up: Survival, weight, and clinical scoring were performed daily starting prior to infection on day 0 (baseline), until death or scheduled study termination, whichever came first. Clinical scoring was performed by a person blinded to the study design and animal’s identity, based on the following scale:0 = no visible signs of disease;1 = Slight ruffling of fur;2 = ruffled fur, reduced mobility;3 = ruffled fur, reduced mobility, rapid breathing;4 = ruffled fur, minimal mobility, huddled appearance, rapid and/or laboured breathing;5 = death/euthanize.Unscheduled Euthanasia/Animal Death: If animals displayed moribund signs (see below), they were humanely euthanized and recorded as dead on the particular day. Mice, once infected, were monitored daily for weight changes and euthanized humanely immediately if they became recumbent, failed to move upon stimulation, exhibited an inability to eat or drink, or if they lost > 30% of their initial body weight as stipulated and approved by the Institutional Animal Care and Use Committee (IACUC).Scheduled Euthanasia and Lung Harvest: Animals from the challenge study were euthanized on day 6 after the viral infection, through intaperitoneal injection of tribromoethanol followed by exsanguination. Lungs were harvested into a 2 mL Sample Tube RB (Qiagen) with 1 mL of PBS without Ca++ or Mg++. Tissue will be homogenized using a TissueLyser II (Qiagen). Samples were spun down at 12,000 rpm for 10 min at 4 °C. The lung homogenate was then split into two 2.0 mL cryovials and stored at −80 °C until a viral plaque assay was performed to determine the viral titers within each lung sample.Viral Load Determination: Viral titers were determined using a viral foci assay using Nitro Blue Tetrazolium. In brief, Madin-Darby Canine Kidney Epithelial Cells (MDCK) cells were seeded into a 96 well plate and grown to confluence. Media was replaced with Trypsin/Zero-Serum media while viral dilutions were made. Lung homogenate was serially diluted using aerosol barrier tips in a 1:10 initial dilution followed by 1:5 serial dilutions. Trypsin/Zero-Serum media were then removed from the 96 well plate and diluted lung homogenate was added. Plates were spun at 700× g for 1.5 h. Sample dilutions were then aseptically aspirated and Trypsin/Zero-Serum media were added to the cells. Plates were incubated for 24 h at 32 °C, 5% CO2. After 24 h, plates were aspirated and fixed with acetone for 30 min at −20 °C. Next, acetone was removed and plates were allowed to dry completely. Once dry, plates were washed in staining buffer and then incubated with mouse anti-influenza A-biotin monoclonal antibody for 1 h at 37 °C. After primary antibody incubation, plates were washed with staining buffer and then stained using streptavidin-alkaline phosphatase for 1 h at 21 °C. After the incubation, the plates were washed with staining buffer and developed using nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate (BCIP/NBT) substrate for 0.5 to 1.0 h at 21 °C. Once maximal spot development occurred, the substrate was removed via aspiration and the plates dried at room temperature overnight. Viral foci were counted the following day using an inverted dissecting light microscope with a 4× objective lens.Survival of Animals: Daily recording was performed until scheduled study termination.Lung Viral Loads of the Animals: Both raw data of individual animals and average/mean data were analyzed by appropriate statistical methods as described below.p ≤ 0.05, ** p < 0.01 and *** p < 0.001 as shown in the respective figures. Statistical significance was determined by Student’s t test, using Microsoft Office software.Results shown are from one representative study with 5 mice per group for the immunogenicity studies. Each study was repeated twice, i.e., a total of 3 independent studies, and very similar results were obtained. For the protective efficacy study, twelve mice per vaccination group were challenged following two IM vaccinations, divided into two subgroups of 6 mice each. Following challenge, subgroups with 6 mice each were monitored for survival and weight loss, and the other subgroups of 6 mice each were sacrificed on day 6 post challenge to measure lung viral loads. The data are presented as mean values from 5 or 6 mice per group ± SEM (error bars). Statistical significance is represented as * p < 2.78 × 10−8–p < 0.02). Furthermore, vaccination with Sq. oil significantly enhanced the same cytokines and chemokines as NIDS, compared to the antigen alone (p < 3.51 × 10−9–p < 0.01).In initial efforts to develop a safer and more efficacious vaccine against pandemic influenza, we measured several serum innate cytokines and chemokines, i.e., G-CSF, IL-5, MCP-1, MIP1, MIP-1β, IL-6, IP10, KC, MIP-2, RANTES, and TNFα. To this end, serum was collected from mice at 16 h after a single IM injection with HA alone or mixed with NIDS, Sq. oil, or Poly (I:C). Serum concentrations of G-CSF, IL-5, IL-6, KC, MCP-1, and MIP-1β were significantly enhanced following IM vaccination with NIDS compared to the antigen alone  (p < 2.7p < 0.002–p < 0.016). Furthermore, Poly (I:C) induced significantly higher serum IP-10, MIP-1β, and RANTES compared to vaccination with the antigen alone (p < 1.26 × 10−8–p < 8.96 × 10−6), or vaccination with NIDS (p < 2.02 × 10−7–p < 2.95 × 10−6). In addition, vaccination with NIDS significantly enhanced G-CSF, IL-5, IL-6, KC, and MIP-2 compared to Poly (I:C) (p < 3.12 × 10−7–p < 0.033). While TNFα levels showed no significant change in mice vaccinated with NIDS and Sq. oil, vaccination with Poly (I:C) significantly increased TNFα levels compared to the antigen alone (p < 0.034)   (p < 3.1< 0.034) .These results demonstrate that an IM injection with Sq. oil compared to NIDS resulted in significantly higher concentrations of serum innate IL-5, and the pro-inflammatory chemokines G-CSF, MCP-1, and MIP-1β. Furthermore, an IM injection with Poly (I:C) compared to NIDS induced significantly higher serum innate RANTES, MIP-1, and IP-10. Hence, compared to Sq. oil or Poly (I:C), vaccinations with NIDS resulted in significantly lower select innate pro-inflammatory cytokines or chemokines.We next determined whether NIDS would exert any adaptive immune enhancing effects when used as an adjuvant compared to other licensed or in development adjuvants. Groups of mice were vaccinated twice IM with HA alone, or HA mixed with NIDS, Sq. oil, or Poly (I:C).p < 0.001), or HA mixed with Sq. oil (p < 0.01) or Poly (I:C) (p < 0.001) (p < 0.001) and HA mixed with Sq. oil (p < 0.05) or Poly (I:C) (p < 0.001) (p < 0.001), or HA mixed with Sq. oil (p < 0.05) and Poly (I:C) (p < 0.001) (p < 0.01) and HA mixed with Sq. oil (p < 0.05), but not compared to HA mixed with Poly (I:C) (v/v), but the responses were poor and comparable to no adjuvant results . These data demonstrate that the NIDS adjuvant was superior for the induction of serum antibody responses compared to the other adjuvants.Vaccination with HA mixed with NIDS significantly enhanced serum anti-HA IgG1 responses at three weeks following a single IM vaccination compared to vaccination with HA alone (< 0.001) A. Simila< 0.001) B. In add< 0.001) B. Interely (I:C) B. Of notp < 0.001) or HA mixed with Poly (I:C) (p < 0.001)  . Further< 0.001) . Compare< 0.001) . In cont< 0.001) . Interes< 0.001) , and in Based on the promising results of the innate and adaptive antibody responses from the immunogenicity studies, we further examined the protective efficacy of NIDS in BALB/c mice. The protective efficacy study was performed in a single blind fashion, in that the personnel involved with this study were withheld information on the adjuvant used in each vaccination group. The mice were vaccinated twice IM with recombinant HA antigen or inactivated A/Cal/04/09 influenza virus alone or mixed with NIDS or Sq. oil. In addition to measuring the serum anti-HA neutralizing activity by the Hemagglutination Inhibition (HI) assay following two IM vaccinations, clinical signs and lung virus pfu titers were also measured.p < 0.01), but were not compared with vaccinations with HA mixed with Sq. oil (p < 0.001) (p < 0.001). Importantly, vaccinations with HA alone resulted in significantly higher lung pfu titers compared to vaccinations with HA mixed with NIDS (p < 0.05)  A. Intra- < 0.05) B.As a further measure of the protective efficacy of NIDS, we also compared the clinical scores of the mice, vaccinated with HA mixed with NIDS compared to vaccinations with HA alone and no vaccinations (naïve). We found significantly higher clinical scores in unvaccinated mice as well as in mice vaccinated with HA alone, compared to mice vaccinated with HA mixed with NIDS C.p < 0.01) compared to the vaccinations with HA alone or with HA mixed with NIDS . We found that two IM vaccinations with Sq. oil induced significantly higher serum IgE titers compared to vaccinations with HA alone  and 10× lower dose (0.025 µg) following two IM vaccinations with HA alone or HA mixed with NIDS, or Sq. oil. Sera collected at ten days post 2nd vaccination were used to measure antibody responses using ELISA. Vaccination with HA mixed with NIDS resulted in significantly higher serum anti-HA IgG1 responses compared to vaccinations with HA alone with both the standard dose A and 10×p < 0.05) or HA adsorbed to Alum (p < 0.01)  B. Thus, This is the first study demonstrating that a natural vaccine adjuvant, i.e., NIDS, comprising a combination of nutritive compounds, i.e., Vit A, Vit E, and a catechin (EGCG) in a vegetable oil in water emulsion conferred complete protective efficacy against intra-nasally induced infection with live influenza virus. The immunomodulatory properties of each of the components of NIDS have been discussed extensively in two recent reviews ,11. MoreIt is important to note that the NIDS can be prepared as an all natural vaccine adjuvant, and all the components are available as Good Manufacturing Practice (GMP) grade material. Moreover, since it can be prepared as a nano-emulsion with droplet sizes of 200 nm, it can be readily sterile filtered. Thus, this novel adjuvant has immediate industrial applications to be used against existing or emerging pathogens.In these studies we established that NIDS enhanced some systemic innate responses, but not others that may be related to adverse clinical events. Specifically, innate G-CSF, IL-5, MCP-1, MIP-1α, and MIP-1β responses induced following injection with NIDS were significantly lower than the responses induced by Sq. oil. Moreover, innate IP-10, RNATES, and TNFα responses induced following injection with NIDS were significantly lower compared to the responses induced by Poly (I:C).As safety biomarkers, besides early serum innate inflammatory responses, we also measured SAP and total IgE. Normally, serum levels of APR go back to normal levels 1–2 days after the inflammatory response. However chronic inflammation can cause APR levels to remain abnormal which can be detrimental and can cause complications and toxicity issues . APR levThere are several lines of evidence that strongly suggest that influenza infection exacerbates allergic reactions in sensitized individuals in which IgE may play an important role ,21. InfeIn a recent publication, we reported that local, site of injection, pro-inflammatory cytokines and chemokines were significantly decreased in a murine air pouch model following the injection of NIDS compared to Poly (I:C) or imject Alum . MoreoveImportantly, the NIDS significantly enhanced serum IgG1, IgG2b, and IgA antibody responses as well as adaptive splenic TH1 and TH2 but not TH17 responses compared to vaccination with other adjuvants such as Sq. oil or Alum. The Alum data from the BALB/c mice  demonstrated a lack of enhanced adaptive responses, which was similar to the data shown in the CD1 mice. In addition, vaccinations with HA mixed with NIDS induced significantly higher serum HI titers compared to vaccinations with no adjuvant, regardless of whether the antigen was purified recombinant HA or inactivated influenza virus. Most importantly, vaccinations with NIDS followed by IN challenge with live influenza virus, induced significant protection compared to vaccination with HA alone, as demonstrated by significantly lower lung pfu virus titers and clinical scores. Of note, the adjuvant effect of NIDS, was intact in the outbred CD1 mice, significantly increasing the potential of NIDS exerting its adjuvant effect in the outbred human population.Studies have shown that higher doses of some pre-pandemic influenza vaccines were required to achieve 50% protection in healthy adults ,27,28,29Although TH17 responses have been suggested to play a role in the clearance of infection of certain pathogens, their role in protective efficacy following vaccinations may be less clear. In addition, TH17 responses have been implicated in certain autoimmune diseases ,33,34,35The influenza virus gains access to the host through mucosal membranes of the upper respiratory tract. Whether long-term mucosal memory immune responses are required for protection against mucosal pathogens has been subject to debate . Factors