N,N-methylethyl)imidazopyridine-3-acetamide (CLINME) was radiolabelled with iodine-123 and assessed for its sensitivity for the TSPO in rodents. Moreover neuroinflammatory changes on a unilateral excitotoxic lesion rat model were detected using SPECT imaging. [123I]-CLINME was prepared in 70–80% radiochemical yield. The uptake of [123I]-CLINME was evaluated in rats by biodistribution, competition, and metabolite studies. The unilateral excitotoxic lesion was performed by injection of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid unilaterally into the striatum. The striatum lesion was confirmed and correlated with TSPO expression in astrocytes and activated microglia by immunohistochemistry and autoradiography. In vivo studies with [123I]-CLINME indicated a biodistribution pattern consistent with TPSO distribution and the competition studies with PK11195 and Ro 5-4864 showed that [123I]-CLINME is selective for this site. The metabolite study showed that the extractable radioactivity was unchanged [123I]-CLINME in organs which expresses TSPO. SPECT/CT imaging on the unilateral excitotoxic lesion indicated that the mean ratio uptake in striatum (lesion : nonlesion) was 2.2. Moreover, TSPO changes observed by SPECT imaging were confirmed by immunofluorescence, immunochemistry, and autoradiography. These results indicated that [123I]-CLINME is a promising candidate for the quantification and visualization of TPSO expression in activated astroglia using SPECT.The high affinity translocator protein (TSPO) ligand 6-chloro-2-(4′-iodophenyl)-3-( The 18 kDa translocator protein .Most intriguing are the TSPO's relatively low concentrations in the CNS, particularly in microglia and astrocytes , 4 that 11C]-PK11195 [123I]-PK11195 pyridine-3-acetamide or CLINME  from the internal standard tetramethylsilane in the specified deuterated solvents. Low resolution mass spectra were performed using either a VG Quattro triple quadrupole mass spectrometer  in electrospray mode or a Micromass ZMD Quadrupole Mass Spectrometer for Electron Impact.NMR spectra were performed on a Bruker Avance DPX 400 operating at 400 MHz for Melting points were performed on a Gallenkamp melting point apparatus and are uncorrected. Elemental analysis was performed on a Carlo Erba 1106 Elemental Analyser by the Australian National University  and results were within ±0.40% of the theoretical value.1H NMR, 13C NMR, MS, and microanalysis (or HRMS).All reagents and solvents if not specified were purchased from Sigma-Aldrich and were used without further purification. The structures of the synthesised compounds were confirmed by The synthesis of the radiolabelling precursors and standards followed the scheme in  4 pyridin-3-yl)acetic acid 4 , 33  (m.p 65–67°C). 1H NMR (CDCl3): (Rotamers 1 : 1) δ 1.06 and 1.13 , 2.95 , 3.29 and 3.35 , 4.03 and 4.06 , 7.17 and 7.18 , 7.38 and 7.39 , 7.56 , 7.8 , 8.21 and 8.23 . 13C NMR (CDCl3) d: 12.31, 13.52, 29.66, 30.11, 33.15, 35.05, 43.06, 44.74, 93.94, 93.97, 115.35∗, 117.75∗, 120.59, 120.64, 122.48, 122.54, 126.03, 126.08, 130.25∗, 133.69, 133.76, 137.82∗, 143.6, 143.61, 143.92, 144.01, 167.03, 167.1. (Note: doublets in carbon spectrum are due to restricted rotation (Rotamers). The numbers with ∗ are the only carbons not appearing as doublets.) MS: m/z: 456 (32% [M+H]+), 454 . Anal. (C18H17N3OClI) C, H, N.CLINME 5 pyridin-3-yl)acetic acid 5 , 33  (m.p 159-160°C). 1H NMR (CDCl3): (Rotamers 1 : 1) δ 1.07 and 1.12 , 2.95 , 3.28 and 3.44 , 4.03 and 4.07 , 7.17 and 7.18 , 7.51–7.61 , 8.23 . MS (ES+ve) m/z: 410 ([M+H]+ C18H17N3O81Br37Cl), 408 ([M+H]+ C18H17N3O81Br35Cl) and ([M+H]+ C18H17N3O79Br37Cl), 406 ([M+H]+ C18H17N3O79Br35Cl). 13C NMR (DMSO) d: , , , , 117.42, , , , , , 131.60, , , , 167.18 (note: numbers in parentheses indicate Rotamer pair).The bromo analogue2)  in toluene (20 mL) were added hexabutylditin  and a catalytic amount of palladium(0) tetrakis(triphenylphosphine) . The reaction mixture was heated to reflux for 40 h, cooled, and filtered through celite and the filtrate evaporated to dryness. The resulting brown residue was purified on silica gel eluting firstly with 10% petroleum ether/ethyl acetate followed by 40% petroleum ether/ethyl acetate to give the title compound (3).To a solution of . 1H NMR (CDCl3): δ (Rotamers ~ 55 : 45) 0.88−1.60 ; 2.95 , 2.96 ; 3.26 , 3.46 ; 4.10 , 4.13 , 7.30 , 7.61 , 7.83 , 8.32 , 8.36 . MS (ES+ve) m/z: major peak 618 ([M+H]+ C30H44N3O35Cl120Sn).The stannane labelling precursorγ-detector, using a Phenomenex Bondclone C18 column . The analytical HPLC system was equipped with a Varian 9002 pump with a Spectrophysics-Linear UV detector set at 230 nm and 254 nm and an Acemate γ-detector using a Phenomenex Synergi hydro C18 column (4 μm 250 × 4.6 mm).Semipreparative radio-High Pressure Liquid Chromatography (HPLC) was performed on a Berthold LB501 system equipped with a Waters 510 pump, a Spectrophysics-Linear UV detector, set at 254 nm, and an online NaI-Berthold radioactivity μmol) in acetic acid (100 μL) was treated with a solution of Na123I  or Na125I  (0.5–1.1 GBq) followed by peracetic acid . After 5 min the solution was quenched (Na2S2O5) , neutralised with NaHCO3 , and diluted with mobile phase (300 μL). The resulting mixture was purified by semipreparative radio-HPLC, eluting with a mobile phase consisting of acetonitrile : ammonium acetate (0.01 M) (50 : 50) at a flow rate of 4 mL/min. For radiochemical purity and stability measurements, the radioiodinated solution was ascertained on an analytical HPLC system, eluting with acetonitrile : 0.1 M ammonium acetate (60 : 40) at a flow rate of 1 mL/min.The tributyltin precursor  of CLINME for the TPSO and for the central benzodiazepine receptor (CBR) was determined as described previously -CLINME, the rats were sacrificed by CO2 administration followed by cervical fracture. Peripheral organs , olfactory bulbs, and the remainder of the brain were removed. Samples of organs and tissues were weighed and the radioactivity was measured using an automated γ-counter. The percent of injected dose (%ID) was calculated by comparison to a diluted standard solution derived from the initial injected sample. Radioactivity concentrations were expressed as percent of injected dose per gram of wet tissue (% ID/g).No carrier added [123I]-CLINME uptake was studied in competition experiments by injecting CLINME (1 mg/Kg), 5 min prior to [123I]-CLINME , via the tail vein, to 8–10-week-old male SD rats.The saturation effect of [ in vivo specificity of [123I]-CLINME uptake in rats was investigated in competition experiments using flumazenil (CBR selective drug), Ro 5-4864, and PK11195 (TPSO selective drugs) (1 mg/Kg). The drugs were dissolved in dimethyl sulfoxide/saline (1 : 5) and injected intravenously 5 min prior to [123I]-CLINME. Groups of rats (n = 3-4) were sacrificed by CO2 administration followed by cervical fracture 30 min after administration of [123I]-CLINME and peripheral organs , blood, olfactory bulbs, and the remainder of the brain were removed. The radioactivity concentrations were calculated as described previously. Results were analysed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for comparing the tissue radioactivity concentrations in the treated animals (n = 3-4) and in the controls (n = 6). The criterion for significance was P < 0.05.The123I]-CLINME  and sacrificed at 15, 60, and 180 min p.i. Adrenals, kidney, heart, and brain were quickly excised.Groups of male SD rats were injected with -CLINME administration, thyroid blocking was performed with an intraperitoneal injection of 2 mg/Kg potassium iodide. [123I]-CLINME  was injected via the tail vein. One hour p.i., the animals were imaged on a dual-head μ-SPECT system  equipped with medium-energy pinhole collimators , under isoflurane anaesthesia (1-2% in O2). Two acquisitions  were performed sequentially. The energy window was 20%, centred over 159 keV. Following the SPECT, an X-ray Computed Tomography (CT) scan was performed (voltage 70 kVp).Two hours prior to -CLINME was performed on brain tissue. Nonspecific binding was assessed by coincubating the slides with 20 μM of either PK11195 or CLINME. The specificity of [123I]-CLINME for the TSPO was assessed by coincubating slides with flumazenil (20 μM). Sections were incubated at RT for 1 h in 100 mM Phosphate-Buffered Saline  containing 16 nM [125I]-CLINME, either alone or with 20 μM of one of the drugs above. They were then rinsed twice in ice-cold buffer and once in distilled H2O, dried overnight, exposed for 3 h to film , then developed, fixed, and dried.Autoradiography with [125I]-CLINME alone or coincubated with one of the 3 blocking drugs) was compared using the nonparametric Mann-Whitney U test. Differences between the coincubated slides and those incubated with [125I]-CLINME alone were compared separately for lesion and control ROIs using the nonparametric Kruskal-Wallis test and post hoc Mann-Whitney U tests. A P value < 0.05 was considered significant.Images were digitised and analysed using MCID image analysis software. To allow comparison with the SPECT images, a 2D region of interest (ROI) was placed over the dorsal striatum, with corresponding ROI placed on the contralateral side as a control region, the square ROIs having the same area as one facet of the cubic VOIs used to analyse the SPECT images. The mean relative optical density (ROD) was then determined in each region. Mean ROD in the ipsilateral versus the control ROI within each of the 4 incubation conditions  at 10x magnification, acquired with Image-Pro software. Image fusion was performed with MCID image analysis software. in vivo uptake of [123I]-CLINME. The CLINME log P7.5 value was 3.27 in comparison with the log P7.5 of PK11195 that was 3.35.The lipophilicity of CLINME was ascertained to understand theKi values for the TSPO of 1.6 ± 0.2 nM ([3H]-PK11195) and 4.5 ± 0.6 nM ([3H]-Ro 5-4864). The inhibition constant for the CBR was 183 ± 20 nM.Data from the binding assays on membranes from kidney and brain cortex gave 123/125I]-CLINME was prepared by electrophilic substitution of the tributyltin precursor with 123/125I-NaI in acetic acid using peracetic acid as the oxidant followed by HPLC purification. [123/125I]-CLINME was eluted with a retention time k′ = 14 min in a radiochemical yield of 74 ± 5% (n = 8) and 86 ± 8% (n = 4) for [123I]- and [125I]-CLINME, respectively. Radiochemical purity as assessed by analytical HPLC was >99% and the specific activity (SA) for [123I]-CLINME was >185 GBq/μmol based on the limit of detection of the HPLC system. The SA for [125I]-CLINME was measured as 80 GBq/μmol close to the theoretical specific activity of the 125I-iodine. [123I]-CLINME was reconstituted in saline for in vivo pharmacological studies. The radiochemical purity of [123]-CLINME in saline at room temperature, 3 h after synthesis, was >97% whereas [125I]-CLINME was stable (>97%) for up to 6 weeks in ethanol when stored at −80°C.-CLINME to achieve a specific activity of 3.7 GBq/μmol and reconstituted in ethanol.For123I]-CLINME in rats. In the peripheral organs, a plateau was observed in the adrenals after 30 min to 6 h p.i. (≈7.5% ID/g). In liver, spleen, kidney, heart, and lung the activity peaked  at 5 or 15 min p.i. and decreased over time to less than 0.7% ID/g at 6 h. Very low concentrations of [123I]-CLINME were found in blood (<0.18% ID/g) throughout the time of measurement. In the olfactory bulbs (0.38–0.17%), a significantly higher concentration of the radioactivity was found compared to the remainder of the brain (0.2–0.02%), with ratios increasing from 2 at 5 min to 8 at 6 h. The uptake in the thyroid increases from 5 min to 1 h (4–7%) and then a decrease was observed at 3 h to 4% which remained constant till 6 h.P < 0.01), respectively. A nonsignificant reduction of uptake was also observed in the adrenals. A significant increase (P < 0.01) of [123I]-CLINME uptake was observed in the blood (45%).The saturating effect of CLINME (1 mg/Kg) was observed with high significant decreases in the heart, kidney, lungs, and olfactory bulbs by 86, 71, 76, and 38% , respectively, compared to control animals. A significant decrease (P < 0.05) of 36% was also observed in the olfactory bulbs. A nonsignificant reduction was observed in the adrenals. With this drug, the uptake of [123I]-CLINME in blood was significantly increased by 29% compared to controls (P < 0.01). Ro 5-4864 also significantly reduced the uptake of activity in the heart, kidney, lungs, and the remainder of the brain by 77, 68, 62, and 23% (P < 0.01), respectively. A nonsignificant reduction was observed in the olfactory bulbs. The reduction of uptake of [123I]-CLINME in the blood was not significant, while the concentration in the adrenals was significantly increased by 73% compared to controls (P < 0.01). With flumazenil nonsignificant differences of [123I]-CLINME uptake in peripheral organs and brain were observed.Drug competition experiments were carried out to test the ability of TSPO and CBR drugs to inhibit 12I-CLINME  Figures . PK11195123I]-CLINME was studied in adrenals, kidney, brain, heart, and olfactory bulbs as well as in plasma. With the homogenisation technique used in this experiment, more than 85% of the activity from tissues and plasma was extracted into acetonitrile and analysed by radio thin layer chromatography. In the adrenals, kidney, and heart, more than 95% of the extractable radioactivity was shown to be unchanged [123I]-CLINME, which remained constant over the three hours studied. Brain tissue, even the low activity present, represents >95% of unchanged radiotracer. In plasma, metabolite analysis showed that 15, 5, and 2% of the extracted activity were unchanged tracer, 15 min and 1 and 3 h p.i., respectively.The time course of unchanged [123I]-CLINME showed a significantly higher signal in the area of the lesion than in the corresponding control striatum at both time points p.i.  study with this same model -CLINME in the olfactory bulbs versus the rest of brain increased from 2- to 8-fold between 5 min and 6 h; this ratio is more than twice that reported for [18F]FE-DAA1106 of 3.5 and comparable with [123I]-CLINDE (6 at 8 h). The uptake in the olfactory bulbs is also always much higher than that of the blood (ratio 6 : 1) indicating that the uptake in the brain is not due to blood flow but to binding to TPSO sites. In addition, metabolite analysis confirmed that the uptake in the brain was due to intact [123I]-CLINME. These results indicated that [123I]-CLINME crosses the brain-blood barrier (BBB), a prerequisite for brain imaging.l glands , in peril glands , 40 and l glands , 42. In ]-CLINDE  and othetracers) , 45. The123I]-CLINME in the peripheral organs that are occupied by the competing drugs. This observation was also found with carbon-11 and fluorine-18-labelled analogues belonging to the dimethylpyrazolopyrimidin-3-yl)acetamide (DPA) series [123I]-PK11195 by 85% in the adrenals of rats [123I]-CLINDE by 51% [123I]-CLINME in peripheral organs or CNS.The pharmacological competition studies confirm that the uptake in the olfactory bulbs and peripheral organs could be blocked by PK11195, Ro 5-4854, and CLINME indicating that the uptake was specific to TPSO binding sites. However, the uptake in the adrenals could not be blocked by CLINME or PK11195 (1 mg/Kg) whereas an increase in uptake was found with Ro 5-4864. These observations could be attributed to the much higher density of TPSO in the adrenals, the amount of competing drugs used , or the decreased availability of TSPO binding sites for [) series  whereas ) series . Gilders of rats  and MattE by 51% . As expe123I]-CLINME, was also investigated in a rat model of neuroinflammation following excitotoxic lesioning of the striatum. In previous PET studies [11C]-CLINME was shown to be superior to [11C]-PK11195 [123I]-CLINME SPECT analogue showed comparable results in the same animal model, which was confirmed with immunohistochemistry  was consistent with that found in the same animal model with [11C]-CLINME PET (2.14 ± 0.09) and considerably higher than [11C]-PK11195 (1.62 ± 0.05) -CLINME in the lesion versus contralateral striatum visualised with SPECT was observed with in vitro autoradiography of [125I]-CLINME alone  or SPECT (123I) imaging as well as for biochemical and pharmacological studies using the 125I-analogue.[


Sexing sperm is an important biotechnical that enables improve animal production, with achievement of maximum animal breeding. However, all sex semen methodologies can promote irreparable damages to sperm cell, which compromise their fertility . Among tpools from four mature goats, collected with artificial vagina. The fresh semen pools (G1) were analyzed and diluted in 10X DMEM to 800 × 106 sperm/mL. Aliquots of 500 µL of the diluted semen were added on Percoll discontinuous gradients, and prepared according Resende et al. [pellet of cell recovered. The spermatic cells recovered from each gradient, with or without resveratrol, were evaluated too. The Sperm parameters accessed were plasma membrane integrity (PMi) by CFDA and PI [To perform this study were used three semen e et al. , withouta; G2 = 67.83 ± 0.76b; G3 = 76.50 ± 4.82ab). No statistics differences (P > 0.05) were observed between all the experimental groups for +MMP , TM  and PM . Nevertheless, sexed semen with resveratrol showed numerically, higher values to the parameters evaluated than without it, fact that consolidates the Sarlós et al. [The percentage of spermatic cells with PMi was higher to G1 (P < 0.05) than to G2 (G1 = 83.00 ± 5.07


IS decreased the cell viability of THP-1 derived macrophages but promoted the production of inflammatory cytokines  and reactive oxygen species. IS reduced macrophage cholesterol efflux (IS 0.5 mM: 30.3% ± 7.3% vs. 0 mM: 43.5% ± 1.6%) and decreased ATP-binding cassette transporter G1 expression. However, lipid uptake into cells was not enhanced. A liver X receptor (LXR) agonist, T0901317, improved IS-induced production of inflammatory cytokines as well as reduced cholesterol efflux. In conclusion, IS induced inflammatory reactions and reduced cholesterol efflux in macrophages. Both effects of IS were improved with activation of LXR. Direct interactions of uremic toxins with macrophages may be a major cause of atherosclerosis acceleration in patients with CKD.One of the possible causes of enhanced atherosclerosis in patients with chronic kidney disease (CKD) is the accumulation of uremic toxins. Since macrophage foam cell formation is a hallmark of atherosclerosis, we examined the direct effect of indoxyl sulfate (IS), a representative uremic toxin, on macrophage function. Macrophages differentiated from THP-1 cells were exposed to IS  Cardiovascular disease (CVD) is a major cause of death in patients with chronic kidney disease (CKD). Cardiovascular (CV) events and mortality are known to increase with decreased glomerular filtration rate ,2, and Cvs. 0.09 ± 0.04 fluorescence/complete cell area without IS, p < 0.05, An MTS assay showed that exposure of THP-1 macrophages to IS at a concentration range of 0 to 1.0 mM did not affect their viability. However, IS at 2.5 and 5.0 mM significantly impaired the viability of macrophages a. IS incvs. 2.51 ± 0.93 mg/g protein without IS; vs. 43.5% ± 1.6% without IS, p < 0.05, vs. 1.31 ± 0.49 ABCG1/β-actin without IS, p < 0.05, vs. 0.98 ± 0.19 ABCA1/β-actin without IS, vs. 1.07 ± 0.08 SRB1/β-actin without IS, To determine the effect of IS on macrophage foam cell formation in atherosclerotic lesions, cellular lipid uptake and efflux reactions were examined. IS did not significantly influence the uptake of acetylated low-density lipoprotein (LDL) by THP-1 macrophages , a member of the nuclear receptor family, regulates macrophage inflammation and cholesterol efflux . The LXRThe results of the present study demonstrate that IS, a protein-bound uremic toxin, can directly promote macrophage inflammation and impair lipid metabolism, and that these effects were ameliorated by treatment with an LXR agonist.Uremic toxins such as IS and p-Cresyl sulfate (PCS) accumulate with increased production induced by CKD-related perturbation of the intestinal microbiome ,17, as win vivo, IS has a high protein binding rate [et al. reported that IS enhanced lipopolysaccharide-induced inflammatory reactions in J774A.1 murine monocyte macrophages [In the present study, IS promoted IL-1β and TNF-α production by macrophages b,c as weing rate . Higher ing rate ,26,27,28in vitro study showed that macrophages from LXR α and β knockout mice exhibited increased inducible nitric oxide synthase activity, whereas LXR agonists reduced this activity in wild type macrophages [The LXR agonist T0901317 modulated IS-induced macrophage inflammatory cytokine expression a. LXRs ain vitro. For example, HDL is less capable of promoting cholesterol efflux in patients who receive maintenance hemodialysis therapy [Macrophage cholesterol efflux is one of the metrics of macrophage and HDL functionality  therapy ,33. Peri therapy . The pre therapy . Further therapy . Express2. THP-1 cells at a density of 1 × 106/mL were differentiated into macrophages by using 50 ng/mL phorbol 12-myristate 13-acetate  for 72 h (THP-1 macrophages) [THP-1 human monocytic leukemia cells  were cultured in RPMI 1640, supplemented with 10% FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 1× MEM vitamin, and 0.5 µM 2-mercaptoethanol . Cells were incubated at 37 °C in humidified air with 5% COophages) . Human mophages) .6 THP-1 macrophages in 80 µL of medium on 96-well plates were exposed to IS (0–5.0 mM) for 24 h. MTS reagent (20 µL) was then added to cells, which were allowed to incubate at 37 °C for 1 h. Absorbance was measured at 492 nm using a Surise™ plate reader .The MTS assay was performed to assess the viability of cells reacted with IS (Sigma-Aldrich), using Cell Titer 96 Aqueous One Solution , according to the manufacturer’s protocol . BrieflyMacrophages were exposed to IS in FBS free medium at concentrations of 0–1.0 mM for 24 h. Cell supernatants were then collected, and the concentrations of IL-1β and TNF-α were measured using the human IL-1β  and TNF-α ELISA Kits , according to the manufacturer’s protocol.Macrophages were exposed to IS at a concentration of 0–1.0 mM for 24 h. Total RNA was extracted from cells by using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) in accordance with the manufacturer’s instructions. Quantitative real-time PCR was performed using the One Step SYBR Plus RT PCR Kit on a Thermal Cycler Dice Real-time System . Primers for human pro-IL-1β , TNF-α (HA072156 Takara), and GAPDH  were used to quantify mRNA expression. GAPDH was used as an internal control.5 THP-1 macrophages on glass slides were reacted with the ROS/RNS 3-plex Detection Mix for 2 h. Cells were then exposed to IS (1.0 mM) for 30 min. Production of ROS was measured by calculating the fluorescent cell area divided by the complete cell area by fluorescence microscopy .THP-1 macrophages were exposed to IS at concentrations of 1.0 mM for 24 h. ROS production was examined using the ROS/RNS Detection Kit , according to the manufacturer’s instructions. Briefly, 2 × 10THP-1 macrophages were exposed to IS at concentrations of 1.0 mM for 24 h. Western blot analysis was performed to detect the expression of ABC transporters, IL-1β, and caspase-1 in cells. Whole-cell lysates were prepared in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 500 mM EDTA, and 1% TritonX-100). The protein (18 µg) was separated on NuPAGE 4%–12% SDS-polyacrylamide gels , and transferred to a PVDF membrane. Membranes were blocked with 5% nonfat dry milk in TBST. To detect ABCA1, ABCG1, SRB1, IL-1β, and caspase-1, membranes were incubated overnight at 4 °C with monoclonal anti-ABCA1 antibodies , polyclonal anti-ABCG1 antibodies , monoclonal anti-SRB1 antibodies (Abcam), polyclonal anti- IL-1β antibodies , and monoclonal anti-caspase-1 antibodies (Santa Cruz Biotechnology Inc.). To detect β-actin, membranes were incubated with anti β-actin antibodies  for 30 min at room temperature. Horseradish peroxidase-linked secondary antibodies were used at a concentration of 1:5000 for ABCA1, 1:1000 for ABCG1, 1:1000 for SRB1, and signals were visualized as chemiluminescence . Chemiluminescence intensity was calculated using ImageJ.versus the cholesterol content after reaction with acetylated LDL.To study lipid uptake, THP-1 macrophages were loaded with acetylated LDL  for 8 h. Cells were washed with D-PBS 3 times and air-dried. Cellular lipid was extracted using 2-propanol overnight, and lipid content was quantified using the Cholesterol/Cholesteryl ester Quantitation Kit  according to the manufacturer’s instructions. Cellular proteins were solubilized by addition of NaOH (0.1 N), and protein content was measured using the Bradford assay . Cholesterol uptake was determined as the increase in cellular cholesterol content at baseline versus the cholesterol content after the reaction with HDL [To study cholesterol efflux, macrophages were reacted with acetylated LDL (50 µg/mL) for 48 h. Lipid-rich macrophages were then reacted with HDL cholesterol (50 µg/mL) isolated from the plasma of healthy subjects using density-gradient ultracentrifugation for 24 h . Cholestwith HDL .THP-1 macrophages were exposed to the LXR agonist, T0901317 (Sigma-Aldrich) to intervene with IS induced disruption of macrophage function. THP-1 macrophages were exposed to IS (1 mM) and T0901317 (2 µM), and inflammatory cytokine production and cholesterol efflux were evaluated.t-test or a single-factor analysis of variance followed by Bonferroni correction. p < 0.05 was considered significant.Results are expressed as mean ± standard deviation. Statistical differences were assessed using the unpaired Student IS directly promoted inflammatory reactions and impaired lipid homeostasis in macrophages, both of which were modulated by LXR agonists. These findings emphasize the importance of the interaction between uremic toxins and macrophages, which may be, at least in part, the underlying mechanism driving the acceleration of atherosclerosis in patients with CKD. In addition, this may also serve as a therapeutic target to ameliorate this condition.


Cr), a newly proposed index that is a surrogate for nephron load, and CKD progression in patients with CKD.Whether phosphate itself has nephrotoxicity in patients with chronic kidney disease (CKD) is controversial, although phosphate excretion into urine may cause tubular damage in rat models. To evaluate actual phosphate load on each nephron, we examined the association between 24-h urinary phosphorus excretion per creatinine clearance (24-h U-P/CCr levels as Quartiles 1–4. The association was examined in three models: unadjusted (Model 1), adjusted for risk factors for CKD progression (Model 2), and factors that affect renal phosphate handling (Model 3).We conducted a single-center, retrospective cohort study. To avoid potential confounders for protein intake, only patients on our educational program for CKD with a fixed diet regimen and aged 20 years or older were included. The observation period was 3 years. Primary outcomes were CKD progression defined as a composite of end-stage kidney disease (ESKD) or 50 % reduction of estimated glomerular filtration rate. Patients were stratified by quartiles of 24-h U-P/CCr showed a higher risk for the composite outcomes. The hazard ratios [95 % confidence interval] for 24-h U-P/CCr levels in Quartile 2, 3, and 4, respectively, versus Quartile 1 were 2.56 (1.15–6.24), 7.53 (3.63–17.62), and 12.17 (5.82–28.64) in Model 1; 1.66 (0.63–4.97), 3.57 (1.25–11.71), and 5.34 (1.41–22.32) in Model 2; and 3.07 (0.97–11.85), 7.52 (2.13–32.69), and 7.89 (1.74–44.33) in Model 3.A total of 191 patients met the eligibility criteria. Patients with higher 24-h U-P/COur study showed that higher phosphorus excretion per creatinine clearance was associated with CKD progression. Cr), as a prognostic factor for CKD progression. In animal studies, renal biopsy and magnetic resonance imaging were used to estimate glomerular number. A previous study reported a positive correlation between glomerular filtration rate (GFR) and glomerular number in stable renal transplants [Cr as a marker for nephron number. To avoid potential confounders for protein intake, only patients on the educational program for CKD with a fixed diet regimen were included. The association between 24-h U-P/CCr and CKD progression in these patients was examined.Serum phosphorus level is a risk factor for cardiovascular disease in patients with chronic kidney disease (CKD) –3. Severnsplants . Thus, wCr and CKD progression.We conducted a single-center, retrospective cohort study in a Japanese population to examine the association between 24-h U-P/CPatients aged 20 years and older who were admitted to the hospital and who were in the educational program for CKD between January 2001 and December 2006 were enrolled. A fixed amount of protein  was served to each patient during the whole admission period. The proportion of animal protein in the diet regimen was 50–60 % of the total. Therefore, the phosphate intake of each patient was also fixed at about 0.8 to 1.0 g/kg of standard body weight. Patients with acute kidney injury or primary hyperparathyroidism were excluded. Although the observation period was 3 years, patients who were lost to follow-up were censored at the date of the last contact with an attending physician of the hospital.Cr (ml/min), 24-h urea nitrogen excretion (g/day), 24-h urinary protein (g/day) and 24-h urinary phosphorus (mg/day). Serum calcium concentration was corrected for serum albumin concentration using the following formula: Corrected calcium = measured calcium −  [−1.094 × age−0.287  [Data on age, weight, and sex of the patients, and comorbid conditions, including history of diabetes mellitus and laboratory data, were obtained by review of medical records. Laboratory data included serum creatinine (mg/dL), urea nitrogen (mg/dL), albumin (g/dL), calcium (mg/dL), phosphorus (mg/dL), intact parathormone (PTH) (pg/mL), 1,25(OH)VitD (pg/mL), Calbumin) . Estimat× 0.739) . Daily p× 0.739) . BecauseCr levels: ≤ 11.15 mg/day/CCr (Quartile 1), 11.16 to 17.07 mg/day/CCr (Quartile 2), 17.08 to 29.61 mg/day/CCr (Quartile 3), and ≥ 29.62 mg/day/CCr (Quartile 4). We also examined the association between 24-h U-P/CCr and serum phosphorus. Associations were examined in models with incremental multivariable adjustments: unadjusted (Model 1); comorbid conditions, and risk factors for CKD progression such as age, male sex, diabetes, CCr, protein intake, urinary protein, and corrected calcium (Model 2); and factors that influence phosphate reabsorption such as age, male sex, CCr, protein intake, corrected calcium, 1,25(OH)VitD, and PTH (Model 3). Unadjusted and multivariable-adjusted relative risks for progressive CKD in relation to 24-h U-P/CCr were calculated using Cox proportional hazard models. The assumption of proportional hazards in the Cox regression models was assessed and found to be acceptable  or 50 % reduction in eGFR. We compared subgroups stratified by quartile of 24-h U-P/CCr with common markers of urinary phosphorus excretion, we examined for the association of fractional excretion of phosphorus (FeP), tubular reabsorption of phosphate (TRP), and maximum tubular reabsorption of phosphorus per unit volume of glomerular filtration rate (TmP/GFR) with primary outcomes. TRP was calculated using the following formula: TRP = 1 − (urinary phosphate × serum creatinine)/(serum phosphate × urinary creatinine). We also analyzed an alternate model, where 24-h U-P/eGFR, instead of 24-h U-P/CCr, was applied to a variable, because this is more commonly used by Japanese physicians.To compare 24-h U-P/Cp < 0.05 was considered statistically significant.A total of 191 patients met the eligibility criteria. Of these, one (0.5 %) patient had primary hyperparathyroidism, eight (4 %) patients had evidence of acute kidney injury and seven (3 %) patients had missing values, excluding them from analyses. 1,25(OH)VitD or PTH were missing in 24 (15 %) patients, excluding them from analyses only in Model 3. The final analyses were carried out in 175 patients in Model 1 and Model 2, and 151 patients in Model 3. Statistical analyses were performed using JMP, version 11.2, , and Excel-Toukei 2012 , where This study was approved by the ethics committee of JA Toride Medical Center, and the research was performed in accordance with the ethical principles of the Declaration of Helsinki. According to the ethics committee of JA Toride Medical Center, there was no need for patient consent for this anonymous retrospective study.Cr quartile. Mean age of the studied population was 65 years, and 69 % were male. Prevalence of diabetes mellitus was 34 %. Mean eGFR was 19.1 ml/min/1.73 m2. The patients with CKD stage G5 represented the largest population and there was no patient with CKD stage G1. Patients with Quartile 3 had lower protein intake, but when 24-h U-P/CCr was included as a continuous variable, a significant statistical correlation was not observed between 24-h U-P/CCr and protein intake . In contrast, a highly significant statistical correlation between urinary phosphorus and protein intake was observed   Fig. . Fig. 3 Cr was 20.9 ± 12.7 mg/day/CCr and the median value was 17.07 mg/day/CCr. The maximum and minimum values were 53.97 mg/day/CCr and 3.95 mg/day/CCr, respectively. The first quartile value was 11.15 mg/day/CCr, and the third quartile was 29.61 mg/day/CCr. A significant correlation was observed between serum phosphorus and U-P/CCr in patients with CKD stage G5 , but not in patients with CKD stage G3b  as shown in Fig. The mean 24-h U-P/CMedian follow-up period was 1.56 year. Ninety (51 %) patients reached the composite outcomes of ESKD or 50 % reduction of eGFR over 3 years, of whom 50 29 %) reached ESKD and 40 (23 %) had a greater than 50 % reduction. More patients in Quartile 4 reached the composite outcomes , 7.53 (3.63–17.62), and 12.17 (5.82–28.64). The corresponding values were 1.66 (0.63–4.97), 3.57 (1.25–11.71), and 5.34 (1.41–22.32) in Model 2, and 3.07 (0.97–11.85), 7.52 (2.13–32.69), and 7.89 (1.74–44.3) in Model 3. The alternate model, where 24-h U-P/eGFR, instead of 24-h U-P/CCr, was applied to a variable, showed similar results . However, neither TRP nor TmP/GFR were associated with the composite outcomes (data not shown). Moreover, the association between FeP and the outcome was observed in Model 1, while in Models 2 and 3, higher FeP was not associated with a higher risk for the outcomes.The hazard ratio (HR) and 95 % confidence interval (CI) of the composite outcomes in the unadjusted Cox proportional hazard model and after adjustment are shown in Table Our study showed that higher phosphorus excretion per creatinine clearance was associated with CKD progression. This association remained after adjustment for other possible risk factors related to CKD progression.Cr would be a confounding factor for FGF-23. FGF23 cannot be measured in clinical use in Japan so U-P/24 h-CCr may be a suitable surrogate marker.In a previous animal experiment, phosphate excretion per nephron over 1 μg/day caused tubular damage  while inCr would be a confounding factor for FGF23. A significant correlation was observed between serum phosphorus and U-P/24-h-CCr in patients with CKD stage G5, but not those with CKD stage G3b. In early stage CKD, serum phosphorus was reported to remain in the normal range due to an increase in phosphorus excretion by FGF-23 [U-P/24-h-Cy FGF-23 , 22. In y FGF-23 , 24, 25.Cr as a marker for nephron numbers, but eGFR is more commonly used by physicians. Therefore, we also examined U-P/eGFR, instead of U-P/24-h-CCr, as a marker for phosphorus burden to nephrons, and found similar reliability.We applied 24-h CCr may be superior to 24-h U-P/eGFR for the following reason. It is difficult to collect 24-h urine accurately because of residual urine. The units of U-P/24-h CCr and U-P/eGFR are presented as follows: (mg/24 h)/(ml/min/24 h) and (mg/24 h)/(ml/min). If collected urine is sampled only over 23 h, the value of U-P/24-h CCr does not change greatly, because U-P and 24-h CCr were collected during the same time. In this case, the unit of U-P/24-h CCr is (mg/23 h)/(ml/min/23 h). However, when normalizing eGFR, each urine sample must be collected accurately over 24 h.24-h U-P/CCr, which showed that 24-h U-P/CCr is a risk factor independent of protein intake.The strength of our study is that we included protein intake as a potential confounding factor. As a direct effect of phosphate on the kidney, oral phosphate overload damaged the kidney while oral phosphate restriction prevented CKD progression in animals , 27. HowAnother strength of our study was using 24-h collected urine samples to measure urinary phosphorus excretion. Compared with spot urine, measuring 24-h collected urine samples takes much time and effort. In CKD patients, the circadian rhythm of urinary phosphorus excretion along with U-P/Cr/eGFR remains unclear, although urinary phosphorus excretion increases from morning to the middle of the night in healthy subjects .Using 24-h collected urine can ensure more accurate measurement of phosphorus excretion than spot urine. However, spot urine could be an alternative prognostic marker if the circadian change of U-P/Cr was resolved.Our study has several limitations. First, it is a single-center, retrospective observational study, and not an interventional study, in a Japanese population. Second, we mitigated the effect of confounding factors by using multivariate Cox proportional hazard analysis. We applied PTH and 1,25(OH)VitD to the confounding variables in Model 3, but the sample size in each group may have been insufficient for full evaluation.Cr, but the number of nephrons was not fully proportionate to creatinine clearance. However, a reliable marker for residual nephrons is not available at present. Finally, we measured daily urinary phosphorus excretion only once even under the fixed menu in admitted patients. Day to day variability in urinary phosphorus excretion was not studied.Third, we assumed the number of nephrons was proportionate to 24-h CCr was associated with more rapid CKD progression. Because an increase of urinary phosphate excretion is reported to be earlier than an elevation in serum phosphorus levels, 24-h U-P/CCr may be a useful marker for suggesting phosphate burden to each nephron unit, thus enabling earlier interventions. Further study is needed to resolve the mechanism of 24-h U-P/CCr as a risk factor for CKD progression.Our study showed that higher 24-h U-P/C


Proteobacteria, are well known for characterized social behaviors and large genomes of more than 9 Mb in size. Myxococcus fulvus is a typical species of the genus Myxococcus in the family Myxococcaceae. M. fulvus 124B02, originally isolated from a soil sample collected in Northeast China, is the one and only presently known myxobacterial strain that harbors an endogenous autonomously replicating plasmid, named pMF1. The endogenous plasmid is of importance for understanding the genome evolution of myxobacteria, as well as for the development of genetic engineering tools in myxobacteria. Here we describe the complete genome sequence of this organism. M. fulvus 124B02 consists of a circular chromosome with a total length of 11,048,835 bp and a circular plasmid of 18,634 bp. Comparative genomic analyses suggest that pMF1 has a longstanding sustention within myxobacteria, and probably contributes to the genome expansion of myxobacteria.Myxobacteria, phylogenetically located in the delta division of the The online version of this article (doi:10.1186/s40793-015-0121-y) contains supplementary material, which is available to authorized users. In ag bodies –5. Myxob 9.14 Mb  in size, So ce56  and 14.7M. fulvus 124B02 [M. fulvus 124B02 is thus meaningful for understanding the evolution of myxobacterial genomes, and providing clues for the presence of pMF1 in strain 124B02. Here we report the complete genome sequence and analyses of M. fulvus 124B02.Extrachromosomal autonomously replicating genetic materials are normally absent from myxobacterial cells. Up to now, pMF1, originally discovered from s 124B02 , is stilM. fulvus 124B02. The optimal growth pH for strain 124B02 is in the range of 6.8–7.6, and the optimal growth temperature ranges between 26 °C and 32 °C. The predominant fatty acids of M.fulvus 124B02 cells were determined as iso-C15:0 (33.18 %), C16:1 ω5c (20.19 %), iso-C14:0 3-OH (6.27 %), C16:0 (5.79 %) and C14:0 (5.65 %). 2-hydroxy and 3-hydroxy fatty acids are the major hydroxyl fatty acid components of strain 124B02.Strain 124B02 was isolated from a soil sample collected in Northeast China . VegetatM. fulvus 124B02 in the Cystobacterineae suborder of myxobacteria (the GenBank accession number of the 16S rRNA gene sequence of strain 124B02 is EU137665). All three 16S rRNA gene copies in the genome of strain 124B02 are identical, but differ by two nucleotides from the previously published 16S rRNA sequence generated from M.fulvus 124B02 (EU137665). According to the morphological and phylogenetic characteristics, M. fulvus 124B02 was determined as a typical strain of Myxococcus fulvus . Then the Phred/Phrap/Consed software package –15 was uThe genome was annotated automatically in GenBank. In addition, we predicted Cluster Regularly Interspaced Short Palindromic Repeats (CRISPRS) with PILER-CR . We anal.M. fulvus 124B02 consists of a circular chromosome with a total length of 11,048,835 bp and a circular plasmid of 18,634 bp. The G+C contents of the chromosome and the plasmid are 69.96 % and 68.7 %, respectively. There were 8,515 predicted coding sequences (CDSs) in the genome, including 9 rRNAs and 80 tRNAs. The protein coding sequences occupied 86.13 % of the whole genome sequence. The majority of the protein-coding genes  were assigned putative functions in categories of orthologous group (COG), while the remaining ones were annotated as hypothetical proteins. The distribution of genes in COGs functional categories is presented in Table The genome statistics are provided in Table 3M. fulvus HW-1 to 14.78 Mb of S. cellulosum So0157-2. Compared with the other sequenced Myxococcus genomes, i.e. 9.14 Mb of M. xanthus DK1622 [M. fulvus HW-1 [M. stipitatusDSM 14675 [M. fulvus 124B02 is rather large. It is known that horizontal gene transfer (HGT) [Myxococcus genomes revealed 576 strain-specific duplications in the strain 124B02 genome  were probably derived from plasmids , transcription , secondary metabolites biosynthesis, transport and catabolism  and replication, recombination and repair . M. fulvus 124B02 had approximately 1,230 kb strain-specific fragments, which scattered throughout the whole genome  , 26 and er (HGT) , 28 are W-1 Fig. , which wW-1 Fig. . We founpMF1.13-pMF1.16) and the partitioning system (pMF1.21-pMF1.23), both of which were determined by narrowing-down of sequence fragments [pMF1.19 and pMF1.20 genes were on the lagging strand, the others were located on the leading strand  to 74 % (MFUL124B02_18095 of M. fulvus 124B02) for pMF1.19 and from 30 %  to 88.0 % (MFUL124B02_18100 of M. fulvus 124B02) for pMF1.20   Fig. . It is atransfer .Fig. 5ThM. fulvus 124B02 is a typical strain of Myxococcus fulvus. The complete genome sequence of M. fulvus 124B02 is much larger than the other sequenced genomes of Myxococcus strains. The phylogenomic analysis of total genome sequence indicates that M. fulvus 124B02 is closer to M. stipitatusDSM 14675, rather than M. xanthus DK1622 or M. fulvus HW-1. Multiple copies of the pMF1.19 and pMF1.20 homologues in different myxobacterial strains suggest that myxobacterial genomes are open, not only to being subject to integrate foreign DNA sequences but also to being duplicated by self, in which the pMF1 plasmid played important roles. The bioinformatics analyses, together with the similar G+C contents of pMF1 and myxobacterial genomes, suggested that pMF1 had a longstanding co-adaption with myxobacteria, probably involving in the expansion of myxobacterial genomes.


Haloarcula californiae icosahedral virus 1 (HCIV-1), a halophilic euryarchaeal virus originating from salt crystals. HCIV-1 also retains its infectivity under low-salinity conditions, showing that it is able to adapt to environmental changes. The release of progeny virions resulting from cell lysis was evidenced by reduced cellular oxygen consumption, leakage of intracellular ATP, and binding of an indicator ion to ruptured cell membranes. The virion contains at least 12 different protein species, lipids selectively acquired from the host cell membrane, and a 31,314-bp-long linear double-stranded DNA (dsDNA). The overall genome organization and sequence show high similarity to the genomes of archaeal viruses in the Sphaerolipoviridae family. Phylogenetic analysis based on the major conserved components needed for virion assembly—the major capsid proteins and the packaging ATPase—placed HCIV-1 along with the alphasphaerolipoviruses in a distinct, well-supported clade. On the basis of its virion morphology and sequence similarities, most notably, those of its core virion components, we propose that HCIV-1 is a member of the PRD1-adenovirus structure-based lineage together with other sphaerolipoviruses. This addition to the lineage reinforces the notion of the ancient evolutionary links observed between the viruses and further highlights the limits of the choices found in nature for formation of a virion.Despite their high genomic diversity, all known viruses are structurally constrained to a limited number of virion morphotypes. One morphotype of viruses infecting bacteria, archaea, and eukaryotes is the tailless icosahedral morphotype with an internal membrane. Although it is considered an abundant morphotype in extreme environments, only seven such archaeal viruses are known. Here, we introduce  Under conditions of extreme salinity, the majority of the organisms present are archaea, which encounter substantial selective pressure, being constantly attacked by viruses. Regardless of the enormous viral sequence diversity, all known viruses can be clustered into a few structure-based viral lineages based on their core virion components. Our description of a new halophilic virus-host system adds significant insights into the largely unstudied field of archaeal viruses and, in general, of life under extreme conditions. Comprehensive molecular characterization of HCIV-1 shows that this icosahedral internal membrane-containing virus exhibits conserved elements responsible for virion organization. This places the virus neatly in the PRD1-adenovirus structure-based lineage. HCIV-1 further highlights the limited diversity of virus morphotypes despite the astronomical number of viruses in the biosphere. The observed high conservation in the core virion elements should be considered in addressing such fundamental issues as the origin and evolution of viruses and their interplay with their hosts. Archaea dominate in extreme environments; consequently, ~140 known archaeal viruses have been isolated, mainly from geothermal springs and high-salinity environments     (ronments . At presl virus)  and STIVvirus 2) –7, SNJ1 irus 2) –, HHIV-2 virus 2) , PH1 (10virus 1) .Paramecium bursaria chlorella virus 1)  (T) of T=28   as well virus 1) , IN93 (2virus 1) , and SSIphage 1)  and sevephage 1) , 22, 23 phage 1) , SNJ1 (8phage 1) , and PH1phage 1) . High-rephage 1) . VP16 ho of T=28 . Two oth of T=28 , 24.SH1 is now the best studied tailless icosahedral virus with an internal membrane that infects halophilic archaea –7. The S–Haloarcula hispanica viruses HHIV-2 and PH1 (Sphaerolipoviridae family (Alphasphaerolipovirus genus). The SNJ1 virus of Natrinema also belongs to the Sphaerolipoviridae family. It is a temperate virus with a plasmid prophage  were stable for at least 4 weeks at 4°C. Although HCIV-1 originated from high-salinity conditions, it tolerates high ionic strength  as well as low-ionic-strength environments. Its infectivity decreased only at the lowest salinities tested  were infected using a multiplicity of infection (MOI) of 25. The highest virus production (~1 × 1010 PFU/ml in culture supernatants) was obtained when cells were infected during mid-exponential-phase growth. Varying the MOI from 3 to 30 had no significant effect on virus production (8 × 109 to 1 × 1010 PFU/ml). Consequently, virus production and life cycle studies were carried out using cells infected at a MOI of 10 in mid-exponential-phase growth.To optimize HCIV-1 production in liquid culture, Haloarcula californiae was efficient but rather slow, with an adsorption rate constant of 5.7 × 10−11 ml/min . At 4 to 5 h p.i., ~80% of the particles were adsorbed to cells (see 8) demonstrated efficient infection. Transmission electron microscopy (TEM) of infected cells showed viruses adsorbed on the host cells at 2.5 h p.i.  and also by monitoring cellular oxygen consumption and the release of ATP from the cells . The GC content is high (~68%), which is typical for halophilic organisms, and there are short inverted terminal repeats in the genome ends . Comparison of the levels of mobility during agarose gel electrophoresis of HCIV-1 nucleic acids extracted with and without protease treatment suggested the possible attachment of terminal proteins to the DNA ends (data not shown) , 29.A total of 47 ORFs, possibly coding for peptides greater than 38 amino acids in length, were identified. The ORFs are tightly packed in the genome 1.5 ORFs/kb) . Ten ORF ORFs/kb Halorubrum viruses: HSTV-2, HRTV-7, HRTV-5, HRTV-8, and HF2 (see A BLAST search found that 43 of the 47 HCIV-1 (putative) proteins had matches to haloarchaeal viral sequences see , it is u HF2 see . Interes24 (encoding VP2), and at the right end of the genome (upstream of ORF 47)  see , and PH1 47) see . The hep 47) see . The onl 47) see .Haloarcula californiae and Haloarcula hispanica. However, we were not able to identify any ORF with canonical DNA polymerase motifs, suggesting that HCIV-1 uses its host cell replication machinery. Putative protein 44 was predicted to have an RNA polymerase sigma subunit domain similar to the transcriptional regulators of archaea and bacteria. ORF 13 is predicted to encode a packaging ATPase based on sequence similarity to the putative packaging ATPases of haloviruses SH1, HHIV-2, and PH1  and here designated it provirus HalaCibP1 , and Haloterrigena thermotolerans genomes , 28 (VP3), and 29 (VP6) had homologues only in the SH1 and PH1 genomes and not in HHIV-2. In SH1, proteins VP9, VP3, and VP6 are known to locate in the virion vertices  showed ~50% overall similarity to VP2 proteins of SH1, PH1, and HHIV-2 (see 9) showed only ~30% overall similarity to the SH1 VP1 protein  and the VP12 membrane protein   (HCIV-1 isolated on  genera) . A narro genera) , 35. In  genera) . HCIV-1  genera) , which s genera)  but are  genera) . Even th genera) , 36. HCI genera) , by whicEarly in the infection, TEM revealed tube-like structures between attached HCIV-1 particles and host cells see  and ɸX17− binding to the ruptured cell membranes . On the basis of the model, the building blocks used for the formation of the HCIV-1 capsid lattice are homodimers of VP4 proteins and heterodimers of VP4 and VP7. The single β-barrels of VP7 and VP4 (with only one single β-barrel) form the lattice, whereas the second β-barrel of VP4 resides on top of the lattice-forming β-barrel and makes the turret on the capsomer surface. This arrangement was previously shown for HHIV-2  among the alphasphaerolipoviruses, including HCIV-1. At the same time, the least conserved structural elements in this virus group are the proteins of the spike complex. This observation is in line with the idea that the hallmark of any viral group is their specific virion structure , 14, 44.Tectiviridae, the temperate Bam35-like and the virulent PRD1-like viruses, do not have detectable sequence similarity, although their virion architectural principles are the same -, middle -, and late -exponential-growth phases were infected with the virus stock at a MOI of 25. For optimization, cells  were infected at different MOIs (3 to 30).To optimize virus production in liquid, 2+, the virus stock was diluted in 23% SW devoid of NaCl or Mg2+ and then incubated as described above. For determinations of Ca2+ requirements, the virus stock was diluted 1,000-fold in 23% SW devoid of CaCl2 and then incubated 24 h at 4°C. For determinations of stability at 4°C, HCIV-1 was monitored for 2 weeks at 4°C in the HCIV-1 buffer . For determinations of temperature sensitivity, virus stock was incubated for 30 min at different temperatures (4°C to 90°C). For determinations of pH sensitivity, virus stock was diluted 1,000-fold in modified 23% SW containing 61 mM potassium phosphate  or 61 mM bis-Tris (pH 6.4 or 7.1) or 61 mM Tris-HCl (pH 8.0 or 9.0) and then incubated for 3 h and 24 h at 4°C.The infectivity of HCIV-1 samples was determined by plaque assay following the specified treatments. For determinations of sensitivity to lowered ionic strength, the virus stock was diluted 1,000-fold in 0.7% to 23% SW buffers and then incubated for 3 h and 24 h at 4°C. For determinations of the effects of NaCl and MgHaloarcula californiae cells  were infected at a MOI of ~2 × 10−3 and then incubated aerobically at 37°C as previously described  were infected at a MOI of 10 and incubated aerobically at 37°C. A similarly treated but noninfected culture was used as a control. The numbers of infective centers and viable cells were determined during the first 5 h p.i. Unadsorbed viruses were removed at 5 h p.i. by washing the cultures three times. The incubation was continued in vessels, and the turbidity of the cultures (OD550) was monitored. To determine the number of infectious progeny viruses in the medium, samples withdrawn from the infected culture starting at 6 h p.i. were centrifuged  and the supernatant was analyzed by plaque assay. Light microscopy analysis of the noninfected and infected cells (nondiluted samples) was performed using ×400 and ×1,000 magnification, oil immersion, and an Olympus BX50F-3 optical microscope equipped with a SensiCam cooled digital 12-bit charge-coupled-device (CCD) camera.To determine the adsorption rate constant of HCIV-1, escribed , 55. HCIescribed . Haloarc− was monitored in the medium by selective electrodes using Ag/AgCl reference electrodes (Sigma-Aldrich) as described previously  were prepared by incubating the cells  in boiling water for 10 min. The dissolved oxygen level in the medium was monitored by the use of selective electrodes , and the amount of extracellular ATP in the medium was measured as described previously  were infected at a MOI of 10. At ~50 h p.i., the cell debris was removed . Particles were precipitated from the supernatant using 10% (wt/vol) polyethylene glycol (PEG) 6000, collected , and resuspended in 18% SW (100-fold concentration). Aggregates were removed , and the specimen was subjected to rate-zonal centrifugation in a linear 5% to 20% (wt/vol) sucrose gradient–18% SW . The viral zone (once-purified HCIV-1) was collected and further purified by equilibrium centrifugation in a CsCl gradient in 18% SW (mean density of 1.3 g/ml) . The virus zone (twice-purified HCIV-1) was diluted 2-fold with 18% SW without NaCl, concentrated by differential centrifugation , and resuspended in HCIV-1 buffer. For further analyses, CsCl equilibration centrifugation was used .Haloarcula californiae cells  were infected by HCIV-1 (MOI of 10 or 60). Collected cells  were resuspended in the original volume of MGM buffered with 61 mM 4-morpholineethane-sulfonic acid (pH 6.7), fixed with 3% (wt/vol) glutaraldehyde , and washed three times with the same medium. Thin sections were prepared as previously described , eluting the samples to 0.1% TFA–50% acetonitrile. The dried peptides were reconstituted in 0.1% TFA–1% acetonitrile (buffer A). Mass spectrometry analysis was carried out with an Orbitrap Elite ETD mass spectrometer  coupled with Proxeon Easy-nLC . The separation gradient included 5% buffer B (0.1% TFA–98% acetonitrile) processed for 5 min, 35% buffer B processed for 60 min, 80% buffer B processed for 5 min, and 100% buffer B processed for 10 min. Data were acquired with LTQ Tune software. Protein identification was carried at the Proteomics Unit, Institute of Biotechnology, University of Helsinki.For N-terminal sequencing by Edman degradation , proteins were separated in gels, transferred onto a polyvinyl difluoride membrane , and stained with Coomassie brilliant blue R 350 . For mass spectrometry, protein bands from the gels stained with Coomassie brilliant blue R 250 (Serva) were subjected to in-gel digestion . CysteinHaloarcula californiae and Haloarcula hispanica early-stationary-phase cells as previously described  (0.5 µg/ml) and 2% (wt/vol) SDS . Phenol-ether-extracted nucleic acid was precipitated with NaCl and ethanol. To obtain the genome with possible terminal proteins, proteinase K was omitted from the reaction mixture. Purified nucleic acid was treated with RQ1 DNase (Promega), RNase A (Fermentas), Exonuclease III (Fermentas), Mung Bean nuclease (Promega), and restriction enzymes AseI, MseI, SspI, SalI, and HincII (New England Biolabs), as well as with SmaI and NotI (Fermentas). The genome was sequenced via the use of a shotgun library and subsequent Sanger sequencing . After quality clipping (PHRED20) and vector clipping of the raw data, the program gap4  was used for data assembly. The 192 reads assembled into 4 contigs that provided 5.4× average coverage of the entire genome. Gaps were closed by PCR. In addition, terminal genome regions were sequenced directly on the genomic DNA toward the ends using a protocol optimized for large constructs (99 sequencing cycles) with several primers. The cycle sequencing was performed with a BigDye Terminator v3.1 cycle sequencing kit. Sequencing runs were executed using an ABI3730 XL sequencer and POP7 polymer.http://www.sciencebuddies.org/science-fair-projects/project_ideas/Genom_GC_Calculator.shtml). Isoelectric points and molecular masses of putative ORF products were calculated using the Compute pI/MW tool , HaloMukP1  Stability of infectivity for the virus stock (open circles) and virus in HCIV-1 buffer (closed circles). The infectivity scales (left y axis) are the same for panels A to D. (E) Infectivity after 30 min of incubation at different temperatures. (F) Infectivity of the virus after 30 min (closed circles) and 24 h (open circles) of incubation in modified 23% SW of different pHs. The infectivity scales (left y axis) are the same for panels E and F. Download Stability of HCIV-1 virions. (A) Infectivity in SW with decreasing ionic strength  determined after 3 h (closed circles) and 24 h (open circles). (B) Infectivity in SW adjusted to the specified NaCl concentrations. (C) Infectivity in SW adjusted to the specified MgFigure S1, TIF file, 0.6 MBFigure S2 Haloarcula californiae cells at 37°C. Error bars represent standard deviations. (B) Light microscopy of uninfected and HCIV-1-infected Haloarcula californiae cells (MOI of 10) cultured at 37°C with aeration in the reaction vessels. In both cultures, cells were washed at 5 h p.i., which removed the unadsorbed virus particles from the infected culture. Cell debris is circled. Undiluted samples, ×1,000 magnification (oil immersion). Download (A) Adsorption of HCIV-1 to Figure S2, PDF file, 1.6 MBFigure S3 HCIV-1 structural proteins analyzed in polyacrylamide-Tricine-SDS gel stained with pro-Q Emerald 300 (for glycoproteins only) (A) and SYPRO-Ruby (for proteins) (B). CandyCane molecular mass standards  , including both glycosylated and nonglycosylated proteins, are indicated. Major HRPV-1 and HCIV-1 virion proteins (right), including glycosylated HRPV-1VP4, are shown. Download Figure S3, TIF file, 0.1 MBFigure S4 HCIV-1 protein VP2 heptapeptide repeat . The HCIV-1 VP2 repeat region (with numbered amino acids residues 225 to 456) is represented by the aligned repeat units. Download Figure S4, TIF file, 1.9 MBFigure S5 Significant (>55%) amino acid similarity between HCIV-1 ORFs/gene products and those of SH1, PH1, and HHIV-2. Similar ORF/gene products in different viruses are connected. ORFs/genes, light grey; noncoding regions, dark grey; genome reading directions, arrows see . ORFs orFigure S5, PDF file, 0.2 MBTable S1 Recovery of infectious particles during HCIV-1 purification.Table S1, PDF file, 0.01 MBTable S2 HCIV-1 ORFs and genes.Table S2, PDF file, 0.2 MBTable S3 Repeats in HCIV-1 genome.Table S3, PDF file, 0.02 MBTable S4 Conserved domains in predicted HCIV-1 proteins.Table S4, PDF file, 0.04 MBTable S5 HCIV-1-related elements in archaeal genomes.Table S5, PDF file, 0.03 MB


Fusarium fungi were the main contaminants found. Some deoxynivalenol (DON) was found in 100% of the tested samples of wheat , triticale, winter barley and oats, while the maximum permissible DON level (as defined in the EU Commission Regulation No. 1881/2006) was exceeded in 10 samples. Zearalenone (ZEN), DON metabolites and enniatins were also commonly found. The presence of mycotoxins in grain reflected the prevailing weather conditions during the plant flowering/earing stages, which were favorable for the development of blight. Among all investigated wheat genotypes, cv. Fidelius was the least contaminated, while Bamberka, Forkida and Kampana were the most contaminated. However, the single-factor ANOVA analysis of variance did not reveal  any differences between levels of mycotoxins in individual genotypes. Triticale was the most contaminated grain among all of the tested varieties. ZEN, DON and the sum of 3-acetyldexynivalenol and 15-acetyldeoxynivalenol (3- and 15-ADON) were found in 100% of the tested triticale samples at concentrations within the 4–86, 196–1326 and 36–374 µg·kg−1 range, respectively. Of particular concern was the fact that some “emerging mycotoxins” (enniatins)  were also found in the tested triticale samples (enniatin B (Enn-B), enniatin B1 (Enn-B1), enniatin A-1 (Enn-A1), 100% of samples, and enniatin A (Enn-A), 70% of samples). Depending on the toxin, they were found at levels between 8 and 3328 µg·kg−1.The levels of 26 mycotoxins were determined in 147 samples of the grain of cereals cultivated in five regions of Poland during the 2014 growing season. The HPLC-HRMS (time-of-flight) analytical technique was used. An analytical procedure to simultaneously determine 26 mycotoxins in grain was developed, tested and verified. Samples from eastern and southern Poland were more contaminated with mycotoxins than the samples from northern and western Poland. Toxins produced by  Generally, mycotoxins are toxic metabolites of various fungi found in various plant-origin food around the world. Several toxins may be found in a single foodstuff. Mycotoxins may negatively impact consumer health and are therefore regarded as a serious threat to food safety . The ecoFood safety concerns are increasing due to the potential consequences of climate changes that might influence crop yield and the degree to which the crops are contaminated with mycotoxins. Climate changes might have an enormous impact on landscapes around the world. A significant increase in the average temperature and a decrease in rainfall during the summer season are predicted for some regions. Weather conditions may be favorable for the development of fungi that have not yet been observed within a given area ,4.Agriculture contributes significantly to the value of the national economy in Poland. Plant production accounts for approximately 43% of total agricultural production, while cereals make up approximately 37% of the plant production. In 2013, Polish farmers produced approximately 9.5 million tons of wheat, 3.4 million tons of rye, 2.9 million tons of barley, 1.2 million tons of oats and 4.3 million tons of triticale. Poland is ranked second (after France) in the EU with respect to arable land and third (after France and Germany) with respect to crop volume. Even though crop yield and total production of cereals in Poland fluctuate on an annual basis, reflecting changes in weather conditions, we may observe a clear upward long-term trend in crop yield, indicating a clear increase [1, the sum of B1/B2/G1/G2 aflatoxins, zearalenone, deoxynivalenol, ochratoxin A (OTA) and the sum of B1/B2 fumonisins [1 [1/B2 fumonisins [Alternaria fungal, ergot alkaloids, patulin, citrinin, sterigmatocystin, moniliformin (MON) and phomopsin [Mycotoxins, for which maximum levels in grain for food have been regulated, include aflatoxin Bmonisins ,7,8. Mormonisins . In graiisins [1 , and limmonisins . Howeverhomopsin .Even if analyses of mycotoxins in food are typically difficult due to low concentrations of the analytes and the high complexity of the matrices , a largeThe aims of this work were: (i) to develop/test/verify an HPLC-HRMS analytical procedure capable of simultaneously determining many various mycotoxins in grain; (ii) to apply the developed method to determine the levels of mycotoxins in various grains (including various genotypes of winter wheat) cultivated in different regions of Poland; and (iii) to correlate the measured mycotoxin levels in various wheat genotypes and with weather conditions during the grain growing season. This is one of the first scientific reports on the natural occurrence of certain mycotoxins  in cereals grown in Poland.A total of 147 samples of cereals cultivated in five regions of Poland were analyzed . The sami.e., until the end of the grain ripening period. In The communes of Osiny, Borusowa and Werbkowice (situated relatively close to one another) are located in the southeastern part of Poland, with Żelisławki in the north and Wielichowo in the west. The air temperatures and rainfall were monitored at each location from March to July 2014, Fusarium fungi, hence mycotoxin biosynthesis. Czaban et al. [F. graminearum (r = 0.976/0.996), F. culmorum (r = 0.998/0.981) and F. tricinctum (r = 0.879/0.933), respectively. Franz et al. [During the wheat flowering period, rainfall was lower in Żelisławki than in Osiny, Werbkowice and Borusowa by 11.2 mm, 7.9 and 6.1 mm, respectively. The rainfall was also lower in Wielichowo by 5.9, 2.6 and 0.8 mm, respectively. Average temperatures were lower in Żelisławki than in the three other locations by 2.1, 2.3 and 2.4 °C, respectively. The temperatures were also lower in Wielichowo by 1.2, 1.4 and 1.5 °C, respectively. Lower temperatures and lower air humidity must have limited the growth of n et al.  reportedz et al.  found thFusarium toxins were found most frequently in the former samples: DON, ZEN, Enn-B, Enn-B1 , Enn-A1 (found in 89%–100% of samples), T-2 (78%–100%), Enn-A (56%–100%), β-ZOL (44%–100%), 3-/15-ADON (67%–83%), DON-3G (44%–78%), HT-2 (17%–67%), and FB1 (6%–22%). Neosolaniol (NEO) was only found in a single sample from Osiny and in 15 samples from Borusowa. The highest concentrations (in µg·kg−1) found in the samples cultivated in Osiny, Borusowa and Werbkowice included: DON 209–2,975 (average 960); Enn-B 20–1,981 (average 275); DON-3G 40–356 (average 118); DON-3G 43–166 (average 106); Enn-B1 8–368 (average 89); 3-/15-ADON 32–98 (average 59); and ZEN 7–100 (average 43). Wheat cultivated in Żelisławki and Wielichowo was contaminated with mycotoxins to a much lesser extent. Among the Wielichowo samples, ZEN was found in two cases (3–4 µg·kg−1), DON in only a single sample (25 µg·kg−1), Enn-B in 30 samples (1–156 µg·kg−1) and Enn-B1 in 23 samples (1–22 µg·kg−1). Among the Żelisławki samples, Enn-B was found in 18 cases and Enn-B1 in 15 samples; however, the measured mycotoxin levels were low (1–7 µg·kg−1). The maximum acceptable levels of mycotoxins in grain (as defined in the EU Commission Regulation No. 1881/2006 [−1 DON level (for unprocessed grain) was exceeded in seven samples , while the 100 µg·kg−1 ZEN (level for unprocessed grain) level was exceeded in a single sample from Osiny.As observed in , Enn-A 5%–100%, βFusarium fungi [F. poae, F. tricinctum, F. avenaceum, F. culmorum and F. graminearum [F. avenaceum, F. culmorum and F. graminearum are the main pathogens of wheat in Poland [F. langsethiae from winter wheat. Fusarium spores in wheat grain are rather ubiquitous. Factors influencing the development of fusariosis include other crops cultivated in close vicinity and/or residues of maize left on the soil surface after harvesting [It is possible that local weather conditions prevailing during the wheat blossoming period are responsible for the fact that the wheat cultivated in Żelisławki and Wielichowo was clearly less contaminated with mycotoxins than that cultivated in Osiny, Borusowa and Werbkowice. Cereal plants cultivated in a temperate climate are frequently infected with um fungi . The varminearum . F. avenn Poland . Lukanown Poland  have alsrvesting . Pathogervesting . Rainfalrvesting ,24,25.Fusarium fungi) was observed in wheat cultivated in Osiny, Borusowa and Werbkowice. The single-factor ANOVA analysis of variance did not reveal significant differences between levels of majority of mycotoxins in the tested genotypes of wheat. DON is an exception: significant differences were found between the Fidelius and Kampana/Forkida/Bamberka varieties, as well as between the Kepler/Dacanto and Bamberka ones; see −1)  to the most contaminated cv. Bamberka .The same genotypes of winter wheat were cultivated in all five regions. Significant contamination with mycotoxins (mostly produced by QTL and Fhb1 genes), is commonly regarded as the most resistant genotype and is widely applied in breeding programs around the world [F. culmorum, most cultivars prove susceptible or very susceptible to ear fusariosis compared to commonly-known resistant cultivars, e.g., Arina [et al. [et al. [The selection of genotypes resistant to fusariosis (FHB) is one of the methods used in wheat cultivation to restrict the incidence of mycotoxins in wheat grain. However, none of the tested genotypes have proved fully immune ,27. FHB he world ,30. Stud., Arina . Data co., Arina  reported [et al.  reported [et al.  suggeste [et al.  reported [et al. .Fusarium toxins, mostly DON, ZEN, 3-/15-ADON, Enn-B, Enn-B1, Enn-A , Enn-A1 (89%–100% of samples) and Enn-A1 (70%). The highest concentrations (in µg·kg−1) included: Enn-B 473–3328 (average 1360); DON 196–1326 (average 573); Enn-B1 149–1347 (average 552); Enn-A1 67–882 (average 357); 3-/15-ADON 36–374 (average 162); Enn-A 8–135 (average 32); and ZEN 4–86 (average 29). Maximum acceptable levels were exceeded in two samples: an excessive concentration of DON was found in a single sample, and another single sample contained OTA at a concentration of 8 µg·kg−1, i.e., above the 5 µg·kg−1 level specified in the EU Commission Regulation No. 1881/2006 [−1) were as follows: DON 54–1,602 (average 602); Enn-B 49–253 (average 150); DON-3G 43–277 (average 146); and Enn-B1 8–81 (average 46). Maximum acceptable DON levels were exceeded in two samples. Spring barley and oats were contaminated to a much lesser extent. The significance of the differences between the levels of the individual compounds in the tested samples is shown in Grain sampled at Osiny also included eight samples of spring barley, 16 samples of winter barley, four samples of oats and 20 samples of triticale. As observed in Fusarium fungi are the main threat to food/feed safety. Studies on the AF, OTA, DON, NIV, HT-2, T-2, ZEN and FB mycotoxins in fodder based on grain (including maize) cultivated between 2006 and 2009 in various regions of Poland identified DON, ZEN, OTA, T-2 and HT-2 as the most frequently-occurring toxins [−1; ZEN 45%–74%, 1.20–6.22 µg·kg−1; OTA 26%–88%, 1.99–33 µg·kg−1; T-2 23%–52%, 3.9–5.5 µg·kg−1; and HT-2 30%–67%, 9.6–11.8 µg·kg−1 (depending on the grain vegetation season).Mycotoxins are commonly found in cereals around the world. In Poland, mycotoxins produced by g toxins . The perFusarium toxins  were also identified in Triticum aestivum wheat cultivated in Lithuania during the 2010–2012 seasons [−1 in 2010), moderate  or high . These findings correlate well with the fact that intense rainfall combined with high average air temperatures prevailed in the third decade of July 2012. The ZEN concentrations were 11.0–12.7 µg·kg−1 (2010), 11.6 µg·kg−1 (only a single ZEN-positive sample in 2011) and 17.2–302.5 µg·kg−1 (2012), and the HT-2 + T-2 toxin concentrations were 10.2–24.6 µg·kg−1 (2010), 10.0–115 µg·kg−1 (2011) and 10–23.7 µg·kg−1 (2012). seasons . Some gret al. [−1). Similar results were reported by Pettersson et al. [et al. [F. langsethiae.Edwards et al.  reportedn et al.  for oats [et al.  .The so-called “masked” mycotoxins (including DON/ZEN metabolites) are becoming increasingly important. However, there are very few reports on their occurrence in Polish cereal grains. Globally, DON-3G is the most frequently identified among masked mycotoxins in wheat grain . Conseque to DON ,43,44. Wet al. [−1 for Enn-A/A1/B/B1, respectively). Enniatins were also found in oats/barley/wheat cultivated in Norway between 2000 and 2002 [−1, Enn-A1 22 µg·kg−1, Enn-B 790 µg·kg−1 and Enn-B1 180 µg·kg−1 (for wheat), Enn-A <3 µg·kg−1, Enn-A1 6 µg·kg−1, Enn-B 47 µg·kg−1 and Enn-B1 20 µg·kg−1 (for oats) and Enn-A <4.5 µg·kg−1, Enn-A1 35 µg·kg−1, Enn-B 490 µg·kg−1 and Enn-B1 170 µg·kg−1 (for barley). Alkadri et al. [et al. [−1; Enn-A1 44%, 8–851 µg·kg−1; Enn-B 91%, 13–941 µg·kg−1; and Enn-B1 80%, 8–785 µg·kg−1.Reports on the occurrence of enniatins in Polish cereal grains are equally sparse. Globally, the levels seem to depend on the particular climatic zone. Oueslati et al.  analyzedand 2002 . The per [et al.  reportedF. verticillioides and F. proliferatum to infect maize [1 at levels between 15 and 150 μg kg−1 in six wheat samples, 101 μg kg−1 in one barley sample and 342 μg kg−1 in one triticale sample. The same triticale sample contained 151 and 113 μg kg−1 of FB2 and FB3, respectively. Even if the levels are relatively low, fumonisins in those grains are of concern, since so far, they contaminated mainly maize and rice [1 + FB2 at levels between 10.5 and 1245.7 μg kg−1 [1 + FB2 fumonisins demonstrated in cultivated Spain cereals  were attributed to F. proliferatum [1/FB2/FB/3 was found in 68%/43%/32% of wheat samples at levels of 15–155/12–86/below 100 μg kg−1, respectively. Fusarium strains responsible for the biosynthesis of fumonisins were isolated from the grain [The risk of fumonisin contamination of wheat, barley, oat and/or triticale is rather low due to the known inclination of ct maize . Our anaand rice . As muchiferatum . Fumonishe grain .−1). Regardless of the climatic zone, the presence of OTA in grain reflects an inappropriate method of grain storage. In tropical zones and/or warm climates, OTA may be synthesized by Aspergillus fungi, compared to Penicilliumverrucosum in moderate/cold zones. Aflatoxins are not the most significant contaminants of grain in Poland, although inappropriate grain storage may result in such contamination [−1). However, the analyzed grain was not sampled from heaps or storage bins, and the fact that no AFs were found may merely reflect that no AFs-producing fungi developed in the field.OTA and OTB were found in only one of our triticale samples (the same sample). However, the OTA/OTB LOQ in our method was fairly high (4 µg·kgmination . We founThe investigated samples were often contaminated with more than one mycotoxin. As observed in A high performance liquid chromatography coupled with a (time-of-flight) mass spectrometer was employed to analyze levels of mycotoxins in wheat/barley/oat/triticale grain. An analytical procedure to simultaneously determine 26 mycotoxins in grains was developed, tested and verified.Fusarium fungi were the major contaminants identified in the studied grain samples. The wheat cultivated in the southeastern part of Poland was more highly contaminated with mycotoxins than that cultivated in the northern/western regions of Poland, most likely as a result of the unfavorable weather conditions  prevailing during the plant flowering/earing stages in southeastern Poland. Among the grain of cereals other than winter wheat, triticale was the grain most often contaminated with Fusarium toxins. Spring barley and oats were contaminated to a much lesser extent. Among all of the investigated wheat genotypes, cv. Fidelius was the least contaminated, while Bamberka, Forkida and Kampana were the most contaminated.The levels of 26 mycotoxins were determined in 99 samples of nine various genotypes of winter wheat  cultivated in five regions of Poland during the 2014 growing season and in 48 samples of three other cereals  cultivated in the southeastern part of Poland . Mycotoxins produced by −1) was exceeded in 10 samples , while the maximum acceptable OTA level (5 µg·kg−1) was exceeded in a single sample. The fact that some enniatins  were also found in all tested triticale samples  is cause for concern. The investigated samples were often contaminated with more than one single mycotoxin. As much as 25% of all positive samples (including winter wheat samples) were contaminated with two (out of 26) of the investigated toxins; only 6% of the samples were contaminated with a single toxin. At least three toxins were found in each of the remaining 69% of samples, among which the mycotoxin count peaked at eight. Little is known about the possible synergy of various mycotoxins acting in combination, and it is difficult to assess their real threat to health. However, such combinations may potentially be a serious toxicological problem.The maximum acceptable DON level  were established using a randomized blocks method with 3 replications. Wheat was sown in October 2013 , in soil classified as a good wheat complex. The previous crop was winter rape. The seeding density was 450 sprouted seeds per sqm. Before sowing, the soil was sampled to determine the contents of available forms of macro-elements. According to the test results, NPK fertilizer (8-20-30) was used in the following amounts: 20 kg·N, 70 kg·P and 100 kg·K. Fertilizer (120 kg·N·ha−1) was applied during the growing season . The land plots were protected against pathogens. A Pilmet 100 sprayer was used to perform the spraying operations. In autumn, 1 L·ha−1 of an Alister Granade preparation was used to control weeds; 2 L·ha−1 of Adexar Plus were used to control fungi; and 0.1 L·ha−1 of Furry 100 EW was used to control pests. Crops were harvested during the full ripeness stage using a small harvester.Experimental plots  were collected from various points in the heap using a pipe sampler . The samples were ground using an MLU-202 laboratory mill by Bühler GmbH , and stored in a dry and cold location. Each analysis was performed in 3 independent replicates.The reagents used in this study included: HPLC-grade and LC-MS-grade methanol; HPLC-grade and LC-MS-grade acetonitrile ; LC-MS-grade water ; HPLC-grade and LC-MS-grade formic acid; sodium citrate dibasic sesquihydrate; sodium citrate dihydrate; PSA bonded on spherical silica; C-18 silica gel; LC-MS-grade ammonium formate ; analytical grade magnesium sulfate ; and neutral alumina . HPLC-grade water used to extract the analytes was produced in-house using a Hydrolab demineralizer .1, AFB2, AFG1, AFG2), 1 µg·mL−1 each; α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), ochratoxin A (OTA), ochratoxin B (OTB). 10 µg·mL−1 each; Mix 3 fumonisins , deoxynivalenol-3-glucoside (DON-3G), hydrolysed fumonisin B1 (HFB1), fumonisin B3 (FB3), 50 µg·mL−1 each; HT-2, T-2, deoxynivalenol (DON), fusarenon X (FUS-X), neosolaniol (NEO), diacetoxyscirpenol (DAS), zearalenone (ZEN), 100 µg·mL−1 each; as well as 13C-labelled internal standards of 13C ZEN, DAS, HT-2, FB1: 25 µg·mL−1 each; and FB2, FB3: 10 µg·mL−1 each. Depending on solubility, the standards were dissolved in acetonitrile or in an acetonitrile/water mixture. Analytical standards purchased from Sigma Aldrich  and dissolved in-house in acetonitrile (according to the recommendations provided in standard certificates) included 3- and 15-acetyl-deoxynivalenol (3-ADON and 15-ADON), enniatin A (Enn-A), enniatin A1 (Enn-A1), enniatin B (Enn-B) and enniatin B (Enn-B1). The initial concentration of each of the standards was 100 µg·mL−1. All standards were stored in amber glass vials maintained at approximately minus 20 °C. A mixture of all of the standards necessary for a particular analytical run was prepared immediately prior to the run. To prepare a mixture, specific volumes of individual standard solutions were transferred to a 5-mL volumetric flask. The flask was filled up to the mark with LC-MS-grade acetonitrile and thoroughly shaken. The contents were then transferred to an amber glass vial and stored at approximately minus 20 °C. The mixtures were used both to validate the method and to analyze the samples. The final concentrations (in μg·mL−1) of individual mycotoxin standards were as follows: AFB1, AFB2, AFG1 and AFG2: 0.08; Enn-A, Enn-A1, Enn-B, Enn-B1, OTA and OTB: 0.2; HFB1: 0.25; α-/β-ZOL and DAS: 0.4; HT-2, T-2 and NEO: 1; and FB1, FB2, FB3, DON-3G, 3-/15-ADON, ZEN, DON and FUS-X: 2. Some 13C-labelled internal standards were also used .Analytical standards purchased in the form of ready-to-use solutions from Romer Labs  included: Mix 9 aflatoxins , 2 mL of water and 10 mL of 10% formic acid in acetonitrile. The tube contents were homogenized in a homogenizer for 3 min. Then, 2 g of anhydrous magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of sodium citrate dihydrate and 0.25 g of sodium citrate dibasic sesquihydrate were added, after which the tubes were immediately energetically shaken and centrifuged . The extract in acetonitrile was transferred to another 15-mL falcon test tube and extracted with hexane (5 mL) to remove the lipid fraction. The defatted extract was deep-frozen (at −30 °C) for approximately 24 h. Three hours prior to the end of the freezing period, a 5-mL cartridge with some glass wool in the bottom was also placed in the same freezer. The frozen supernatant was filtered into a 15-mL falcon centrifuge vial. The extract was supplemented with 1 g of anhydrous magnesium sulfate, 0.25 g of C-18 silica gel, 0.25 g of neutral alumina and 0.4 g of PSA. The samples were shaken and centrifuged for 5 min . Then, 2.5 mL of the supernatant were transferred to a reaction vial and evaporated in a stream of nitrogen flowing through a heater. The extract was dissolved in 300 µL of methanol and subsequently supplemented with 200 µL of HPLC-grade water. The solution was filtered through a nylon syringe filter with a 0.2-µm mesh size.The samples were generally prepared in line with the QuEChERS  procedure proposed by Koesukwiwat et al. . However−1. Gradient of Phase A: from 1%–50% for 0–8 minutes, from 50%–95% for 8–11 min, constant at 95% for 11–22 min, from 95% down to 1% for 22–22.6 min and constant at 1% for 22.6–27 min. Sample volume injected into the column: 2.5 μL. Ion source: ESI-type at both positive and negative polarization. Ion source temperature: 150 °C, de-solvation temperature: 350 °C. Gaseous nitrogen flow rate: 750 L·min−1, cone flow rate: 20 L·min−1. Capillary bias: 3000 V/2800 V in positive and negative ion modes, respectively. The ion optics were operated in the V mode. The mass spectrometer was calibrated using solutions of the leucine encephalin solution. The precursor ions for each of the 26 analyzed mycotoxins are specified in An Acquity H-Class ultra-high performance liquid chromatograph coupled to an LCQ Premiere XE time-of-flight high-resolution mass spectrometer equipped with a UPLC C18 Cortecs 2.1 × 100 mm 1.6-µm column and a pre-column was used  as follows. Mobile Phase A: methanol and 0.2% formic acid; Phase B: water, 0.2% formic acid, 2 mM ammonium formate. Flow rate: 0.4 mL·min−1): DON, at 25–670; DON-3G, at 20–840; FUS-X, at 13–1020; NEO, at 2–402; 3- and 15-ADON, at 20–1,700; DAS, at 1–402; AFB1; AFB2; AFG1 and AFG2, at 4–70; HT-2 and T-2 toxin, at 1–670; FB1; FB2 and FB3, at 25–330; OTA and OTB, at 4–180; ZEN, at 2–670; α- and ß-ZOL, at 2–200; and Enn-A; Enn-A1; Enn-B and Enn-B1, at 1–13.To correct the matrix effects (which may reduce sensitivity), matrix matched calibration standards were used for quantification. Calibration standards were dissolved in the sample matrix prepared in the same manner as other samples. The material used for standard calibration preparation was mycotoxin-free. A diluted mixture of the standards and internal standards was added before the matrix samples were finally dissolved in methanol (300 μL) and water (200 μL). The concentrations of the internal standards in the matrix samples corresponded to the concentration of the standards in the samples waiting to be analyzed. The linearity of the individual compounds was evaluated in the following a concentration ranges (in µg·kgThe limits of quantification (LOQs) of the individual mycotoxins were determined as the concentrations at which the signal-to-noise ratio decreased to 10:1. The wheat and triticale matrices were similar in terms of practically identical LOQs for a given mycotoxin . Similarly, the barley and oat matrices were similar in terms of the practically identical LOQs for a given mycotoxin . An overwhelming majority of 100 coefficients of correlation  was above 0.99 with a few exceptions, where values ranged between 0.98 and 0.99 (the worst case: 0.9774). The LOQs and correlation coefficients obtained for the individual mycotoxins in wheat, triticale, barley and oats are shown in 1 ; FB2 ; and FB3 . Indeed, the measured recovery rates were used to calculate the unknown concentrations of the analytes.Matrix samples (2 g) spiked with certain mycotoxin standards at three levels were used to measure the recovery rates (R%) and method repeatability . The results are shown in z-score values fell within the –1.3–+1.2 range depending on the analyte and the matrix and were considered satisfactory in all cases.The developed method was also assessed within the framework of certain FAPAS  and Romer Labs inter-laboratory proficiency tests. As observed in Only samples contaminated above the LOQ level were included in calculations of average and/or median values. The experimental data were statistically evaluated using the Statgraphics 4.1 software package . A one-way ANOVA was used to assess the significance of the differences between the determined mycotoxin concentrations. Tukey's test at α = 0.05 was used for the paired tests.


KCB14S006 derived from a saltern. The structures of the isolated compounds were established by 1D-, 2D-NMR and HR-ESIMS, and their absolute configurations were determined by applying advanced Marfey’s method and CD spectroscopy. All isolates exhibited significant antifungal activities against various pathogenic fungi and moderate cytotoxic activities toward HeLa and srcts-NRK cell lines. Moreover, in an in vitro enzymatic assay, compound 4 showed a significant inhibitory activity against indoleamine 2,3-dioxygenase.Salterns, one of the most extreme natural hypersaline environments, are a rich source of halophilic and halotolerant microorganisms, but they remain largely underexplored ecological niches in the discovery of bioactive secondary metabolites. In continued efforts to investigate the metabolic potential of microbial populations from chemically underexplored sites, three new lipopeptides named iturin F Bacillus lipopeptides, represented by three classes, namely the iturins, surfactins and fengycins, have been widely studied for their effective antibacterial or antifungal activities . Thi. Thi2 (29 (3) was isolated as a white amorphous powder. Its molecular formula was assigned as C51H80N12O14 on the basis of HR-ESIMS in combination with the 1D NMR spectroscopic data , thus indicating the presence of Pro in 3 rather than 4-OH-Pro. The peptide backbone of 3 was identical to that of iturin A isomers; however, it possessed an iso-C17 type of β-amino fatty acid chain, which was not previously reported.Iturin Apic data . A 16 am1–3 was defined by MS-detected chromatographic comparison of the derivatives of the acid hydrolysate + .Iturin F2 (2): white amorphous powder; c 0.05, MeOH); UV (MeOH) λmax (log ε) 204 (4.3), 226 (3.9) nm; for 1H and 13C NMR spectroscopic data, see m/z 1123.5758 [M + Na]+ .Iturin F9 (3): white amorphous powder; max (log ε) 205 (4.3), 227 (3.9) nm; for 1H and 13C NMR spectroscopic data, see m/z 1107.5808 [M + Na]+ .Iturin A8 (4): white amorphous powder; c 0.05, MeOH); UV (MeOH) λmax (log ε) 204 (4.3), 226 (3.9) nm; HR-ESIMS m/z 1107.5807 [M + Na]+ .Iturin AStaphylococcus aureus 209 and Escherichia coli HO141. Antimicrobial activities of compounds against these microorganisms were conducted by a standard microdilution method. Briefly, 100 μL of cell suspension containing 0.1% of a 0.5 McFarland standard suspension was plated into 96-well plate. Test compounds were added to the culture medium, and the plates were incubated at 28 °C (S. aureus) or 37 °C (E. coli) for 24 h. The growths of these microorganisms were measured by absorbance at 600 nm.The following microorganisms were used as test strains in the assay; Aspergillusflavus KCTC 6984, Neurospora crassa KCTC 6079, Candida tropicalis KCTC 7830, Candida albicans KCTC 7678, Fusarium oxysporum f. sp. cucumerinum KCTC 6084, Alternaria brassicicola ATCC 96836 and Penicillium griseofulvum KCTC 6435. Potato dextrose broth (Difco Laboratories) was used for a test medium. For C. albicans and C. tropicalis, 100 μL of inoculum suspension of 105 spores/mL was plated into 96-well plates containing diluted test compounds, and the plates were incubated at 28 °C for 48 h. For A. flavus, N. crassa, F. oxysporum, A. brassicicola and P. griseofulvum, 100 μL of cell suspension containing 2% of overnight cultures in 96-well were incubated at 28 °C for 72 h. The MIC values were determined as the lowest concentration of each compound that prevented visible growth.The following microorganisms were used as test strains in the assay; srcts-NRK cells, rat kidney cells that were infected with ts25, a T-class mutant of Rous sarcoma virus Prague strain, were cultured at permissive temperature (32 °C) in MEM (Sigma-Aldrich), supplemented with 10% calf serum . Each cell line was seeded into a 96-well plate  and then exposed to test compounds. After 48 h incubation, cell proliferation was determined using a Cell Count Reagent SF . Briefly, a 1/10 volume of WST-8 solution was added to each well, and the plates were incubated at 37 °C for 1 h. Then, the absorbance at 450 nm was measured in a microplate reader.The human cervix epidermoid carcinoma cell line HeLa was cultured at 37 °C in DMEM , supplemented with 10% fetal bovine serum . E. coli according to previously described method with some modifications [g for 15 min. Finally, 125 μL of supernatant is added to 125 μL of p-dimethylaminobenzaldehyde (pDMAB)  in acetic acid, and the absorbance at 480 nm was measured.The enzymatic inhibition assay was performed with human recombinant IDO expressed in ications . The rea1–4 (each 0.1 mg) were each dissolved in 0.5 mL of 6 N HCl and shaken at 100 °C for 1 h. After excess HCl was removed under vacuum, the reaction product was dissolved in 100 μL of H2O and separated into two portions. The residue was then dried and dissolved in 100 μL of 1 N NaHCO3. Either 100 μL of 1% l- or d-FDLA in acetone was added to each hydrolysate, and the resulting mixture was vortexed and heated at 40 °C for 1 h. The reaction was quenched by the addition of 20 μL of 2 N HCl and 20 μL of the reaction mixture was diluted with 20 μL of CH3CN. The solution was analyzed by LC-MS. Standard amino acids were also derivatized with FDLA in the same manner and were subjected to LC-MS to compare retention times of reaction products.Compounds Bacillus sp. KCB14S006 obtained from salterns has led to the isolation of three new iturin class lipopeptides (1–3), along with one known metabolite, iturin A8 (4). The absolute configurations including a newly identified 4-OH-Pro residue were determined by Marfey’s analysis and CD spectroscopy. All isolates showed antifungal activities with slight differences due to the presence or absence of the hydroxy group in Pro and cytotoxic activities. In particular, compound 4 was found to show IDO-inhibitory activity with an IC50 value of 5.5 μM. Structurally diverse secondary metabolites, including lipopeptides, polypeptides, macrolactones and polyketides, have been continuously discovered from marine-derived Bacillus strains [Bacillus sp. inhabiting hypersaline environments, such as saline lakes and salterns where the salt concentration is much higher than that of ocean [Chemical investigation of  strains ,29,30,31of ocean ,33. The


Arenimonas donghaensis is the type species of genus Arenimonas which belongs to family Xanthomonadaceae within Gammaproteobacteria. In this study, a total of five type strains of Arenimonas were sequenced. The draft genomic information of A. donghaensis DSM 18148T is described and compared with other four genomes of Arenimonas. The genome size of A. donghaensis DSM 18148T is 2,977,056 bp distributed in 51 contigs, containing 2685 protein-coding genes and 49 RNA genes. Arenimonas donghaensisTDSM 18148 (= HO3-R19T = TKACC 11381) was isolated from seashore sand [Xanthomonadaceae. So far, the genus Arenimonas contained seven species, Arenimonas donghaensis (type species) [Arenimonas malthae [Arenimonas oryziterrae [Arenimonas composti [Arenimonas metalli [Arenimonas daejeonensis [Arenimonas daechungensis [A. composti [Aspromonas composti [ malthae , Arenimoeonensis  and Arenungensis . These bungensis , rice rhungensis  and sediungensis , respectcomposti .Arenimonas strains are Gram-staining-negative, aerobic, rod-shaped, non-spore-forming, oxidase-positive, non-indole-producing, non-nitrate-reducing, containing iso-C16:0 and iso-C15:0 as the major fatty acids, phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids, Q-8 as the major respiratory quinone, and possessing relatively high DNA G + C content (63.9–70.8 mol %) [The common characteristics of the ent 63.9–0.8 mol %Arenimonas species, we performed genome sequencing of strains A. donghaensisTDSM 18148, A. compostiTKCTC 12666, A. malthaeTCCUG 53596, A. metalli CF5-1T and A. oryziterraeTKCTC 22247. In this study, we report the genomic features of A. donghaensisTDSM 18148 and compare it to the close relatives.In order to provide genome information and determine genomic differences of A. donghaensisTDSM 18148 shares 93.1–95.7 % 16S rRNA gene identities with the other six type strains of Arenimonas species, A. malthae CC-JY-1T (DQ239766) (95.7 %), A. daejeonensis T7-07T (AM229325) (95.7 %), A. metalli CF5-1T (HQ698842) (94.6 %), A. oryziterrae YC6267T (EU376961) (94.3 %), A. composti TR7-09T (AM229324) (94.3 %) and A. daechungensis CH15-1T (JN033774) (93.1 %). A 16S rRNA gene based neighbor-joining phylogenetic tree of the related strains was obtained using MEGA 5.05 software [Strain A. donghaensisTDSM 18148 are Gram-negative, aerobic, non-spore-forming, straight or slightly curved rods, motile by means of a single polar flagellum. Colonies are yellowish white, translucent and convex on R2A agar after 3 d cultivation .A. donghaensisTDSM 18148 was determined using the Illumina Hiseq2000 [The whole-genome sequence of iseq2000  with theiseq2000  , amino acid transport and metabolism (8.7 %), coenzyme transport and metabolism (5.8 %), lipid transport and metabolism (5.1 %), translation, ribosomal structure and biogenesis (12.4 %), replication, recombination and repair (5.2 %), cell wall/membrane/envelope biogenesis (5.9 %), posttranslational modification, protein turnover, chaperones (6.3 %), general function prediction only (8.4 %), function unknown (7.3 %) and signal transduction mechanisms (5.3 %) , general function prediction only (8.5 %), function unknown (7.3 %) and signal transduction mechanisms (9.0 %). The remaining 242 unique genes (40.3 %) are not classified into any COG categories  value of 0.18, which means an approximate genus-wide nucleotide sequence homology of 82 %.There are 601 strain-specific genes for  unknown .3 % and n-genome  size of n-genome  and DnaSArenimonas genomes using CRISPRfinder program online [A. compostiTKCTC 12666. This CRISPR length is 5331 bp, with 29 bp direct repeat (DR) sequences be separated by 87 spacers.The clustered regularly interspaced short palindromic repeats (CRISPRs) mediate resistance to foreign genetic material and thus inhibit horizontal gene transfer . Screenim online  found thXanthomonadaceae were chosen for genomic based phylogenetic analysis, including the five Arenimonas genomes that were sequenced by us. In total, 1014 core protein sequences were extracted using the cluster algorithm tool OrthoMCL with default parameters [Arenimonas species clustered into the same branch (Fig. Fifteen available genome sequences of the family rameters . The neinch Fig. , which iA. donghaensisTDSM 18148, the TCA cycle is complete and hexokinase is absent in all the five Arenimonas strains. The proteins responsible for the oxidative phase of pentose phosphate pathway are also incomplete in five Arenimonas strains, this may be part of the reasons that the five Arenimonas strains can only use several single carbon sources.Similar to Arenimonas. The genomic based phylogeny is in agreement with the 16S rRNA gene based one indicating the usefulness of genomic information for bacterial taxonomic classification. Analysis of the genome shows certain correlation between the genotypes and the phenotypes especially on utilization of sole carbon sources.To the best of our knowledge, this report provides the first genomic information of the genus