Iron is an essential cofactor in almost all biological systems. The lactic acid bacteria (LAB), frequently employed as probiotics, are unusual in having little or no requirement for iron. Iron in the human body is sequestered by transferrins and lactoferrin, limiting bacterial growth. An increase in the availability of iron in the intestine by bleeding, surgery, or under stress leads to an increase in the growth and virulence of many pathogens. Under these high iron conditions, LAB are rapidly out-competed; for the levels of probiotic bacteria to be maintained under high iron conditions they must be able to respond by increasing growth rate to compete with the normal flora. Despite this, iron-responsive genera are poorly characterised as probiotics.Streptococcus thermophilus that can increase growth rate in response to increased iron availability. The isolate of S. thermophilus selected was able to reduce epithelial cell death as well as NF-κB signalling and IL-8 production triggered by pathogens. It was capable of crossing an epithelial cell barrier in conjunction with E. coli and downregulating Th1 and Th17 responses in primary human intestinal leukocytes.Here, we show that a panel of probiotics are not able to respond to increased iron availability, and identify an isolate of We propose that an inability to compete with potential pathogens under conditions of high iron availability such as stress and trauma may contribute to the lack of efficacy of many LAB-based probiotics in treating disease. Therefore, we offer an alternative paradigm which considers that probiotics should be able to be competitive during periods of intestinal bleeding, trauma or stress. Lactobacillus delbruickii subspecies bulgaricus) isolated from a fermented milk product was of particular interest. Metchnikoff proposed it was the optimal strain to consume because of its ability to produce large amounts of lactic acid with little succinic or acetic acid; its ability to coagulate milk rapidly; and the lack of alcohol and acetone produced Lactobacillus spp., have been trialled in the treatment of a number of diseases.The benefit of consuming specific strains of bacteria was first proposed by Elie Metchnikoff. He suggested that since lactic acid bacteria can prevent putrefaction of stored food, they may also benefit the gastrointestinal tract; Bulgarian bacillus , L. reuteri (ATCC 55730) and L. paracasei (strain F19) have been shown to decrease the incidence of eczema in cohorts of children Some Lactobacillus spp. have also been successful in the treatment of acute infectious diarrhoea in children Lactobacillus spp., VSL#3 and Escherichia coli Nissle 1917, have shown promise in the treatment of inflammatory bowel disease (IBD) E. coli (AIEC) The lactic acid bacteria (LAB) are unusual organisms in that they do not appear to have a requirement for iron S. thermophilus with probiotic potential, based on functionality in in vitro models. We propose an alternative paradigm to the traditional LAB spp. probiotics, which considers that probiotics should be able to be active and functional during periods of intestinal bleeding, trauma or stress.Here, we show that the majority of LAB do not respond to noradrenaline-mediated iron availability by increasing growth rate. We have identified species of bacteria that can increase growth rate under these conditions, and using a rational selection process we have found an isolate of L. acidophilus ASF360 increased its growth more than 7-fold at 48 hours and S. thermophilus NCIMB 41856 increased growth at all time points studied, with a maximum of an almost 5-fold increase at 48 hours ; AIEC HM615 reduced proliferation by 80% (p = 0.0001); and E. coli Nissle 1917 by 82% (p = 0.001) (E. coli treatment: E. coli K12 induced a reduction of 77% compared to untreated cells (p = 0.003); AIEC HM615 induced a reduction of 76% (p = 0.003); and E. coli Nissle 1917 of 78% (p = 0.004) (E. coli K12 induced a 254% increase in cell death in Caco-2 cells (p = 0.0005); AIEC HM615 induced a 498% increase in death in T84 cells (p = 0.0003) and a 254% increase in Caco-2 cell death (p = 0.001); E. coli Nissle 1917 induced a 498% increase in T84 cell death (p<0.0001) and a 218% increase in Caco-2 cell death (p = 0.001) (S. thermophilus NCIMB 41856 reduced the proliferation of Caco-2 cells treated with E. coli K12 or AIEC HM615 by 35% (p = 0.003) and 31% (p = 0.05), respectively; while E. coli Nissle 1917 reduced proliferation induced by E. coli K12 by 32% (p = 0.007) and 37% in response to AIEC HM615 treatment (p = 0.03) (L. acidophilus ASF360 further reduced proliferation of Caco-2 cells treated with AIEC HM615 by 22% (p = 0.02) (S. thermophilus NCIMB 41856 reduced proliferation of T84 cells treated with AIEC HM615 and E. coli Nissle 1917 by 33% (p = 0.03) and 48% (p = 0.03), respectively  following E. coli K12 treatment and 16% following infection with AIEC HM615 (p = 0.03); S. thermophilus NCIMB 41856 reduced death of epithelial cells following E. coli K12 and AIEC HM615 treatment by 10% (p = 0.01) and 18% (p = 0.007), respectively; E. coli Nissle 1917 reduced death of epithelial cells following E. coli K12 and treatment by 26% (p = 0.006) and 27% (p = 0.003), respectively (S. thermophilus NCIMB 41856 was able to reduce proliferation and cell death of untreated Caco-2 cells by 24% (p = 0.008) and 22% (p = 0.02) respectively  . A simil= 0.004) . Simulta= 0.001) . S. ther = 0.03) . In addi = 0.02) . S. therectively . Importaectively . In addiectively .E. coli strains in order to determine the effect of probiotic on either of these pro-inflammatory signalling events. NF-κB signalling in Caco-2 cells was upregulated by 491% (p = 0.002) compared to controls following infection with E. coli K12 and by 247% (p = 0.002) following AIEC HM427 treatment. However, this induction was reduced by the addition of all three probiotic strains: L. acidophilus ASF360 reduced the NF-κB response to E. coli K12 by 50% (p = 0.006) and AIEC HM427 by 28% (p = 0.007); S. thermophilus NCIMB 41856 reduced the NF-κB response to E. coli K12 by 58% (p = 0.002) and AIEC HM427 by 49% (p = 0.005); and E. coli Nissle 1917 reduced the NF-κB response to E. coli K12 by 79% (p = 0.0005) and AIEC HM427 by 68% (p = 0.004)  (E. coli K12 (p = 0.001), 1104% in response to AIEC HM427 (p = 0.004) and 363% in response to HM615 (p = 0.02), as well as a 1498% increase following addition of E. coli Nissle 1917 (p = 0.004). The IL-8 response to E. coli K12 was reduced by 22% following addition of S. thermophilus NCIMB 41856 (p = 0.02) and the response to AIEC HM427 was reduced by both L. acidophilus ASF360 and S. thermophilus NCIMB 41856 by 17% (p = 0.02) and 39% (p = 0.02) respectively  . S. ther= 0.003) . Followiectively .E. coli K12 or AIEC HM615 in combination with each of the potential probiotics; TEER and bacterial translocation were measured. Both Caco-2 and T84 cells formed stable monolayers after 8–10 days. Neither L. acidophilus ASF360 nor S. thermophilus NCIMB 41856 had any effect on TEER alone. However, S. thermophilus NCIMB 41856 blocked the passage of E. coli K12 through the monolayer, a phenomenon not seen with L. acidophilus ASF360 which enhanced the passage of E. coli K12 across the barrier , whereas L. acidophilus ASF360 and E. coli K12 together caused a decrease in TEER (peaking at 56% in Caco-2 cells at 8 hours and 11% in T84 cells at 6 hours compared to K12 stimulated cells)  . While Ac strain . The pot on TEER  but it w strains .S. thermophilus NCIMB 41856 was added to determine its effect on the tight cell junction protein occludin. AIEC HM615 caused the breakdown of tight cell junctions in Caco-2 and T84 monolayers, illustrated by decreased occludin (61% and 56% respectively)  . AIEC HMnolayers .E. coli K12 and AIEC HM615 to determine the effect of competing probiotic antigens from L. acidophilus ASF360, S. thermophilus NCIMB 41856 and E. coli Nissle 1917. Both E. coli K12 and AIEC HM615 induced a Th1 response, indicated by upregulation of mRNA encoding the Th1-specific transcription factor T-box21 in the population of cultured leukocytes (16% (p = 0.003) and 13% (p = 0.007) respectively); this was significantly reduced by the addition of L. acidophilus ASF360 or S. thermophilus NCIMB 41856. L. acidophilus ASF360 reduced the response to E. coli K12 and AIEC HM615 by 13% (p = 0.003) and 11% (p = 0.02), respectively. S. thermophilus NCIMB 41856 reduced the Th1 response to E. coli K12 and AIEC HM615 by 10% (p = 0.03) and 13% (p = 0.04), respectively. E. coli Nissle 1917 also downregulated the Th1 response to AIEC HM615 by 21% (p = 0.009). Furthermore, S. thermophilus NCIMB 41856 reduced the baseline level of transcription of T-box21 in untreated cells by 6% (p = 0.03). Neither E. coli strain induced a significant Th2 response but the Th17-specific transcription factor, RORC, was also upregulated following treatment with E. coli K12 or AIEC HM615 antigens (9% and 13% (p = 0.02), respectively). The Th17 response to E. coli K12 was reduced by 12% following the addition of L. acidophilus ASF360 antigens (p = 0.009) and the response to AIEC HM615 was reduced by the addition of any of the three potential probiotic strains: L. acidophilus ASF360 reduced the response by 18% (p = 0.0002), S. thermophilus NCIMB 41856 by 15% (p = 0.003) and E. coli Nissle 1917 by 26% (p = 0.003). In addition, both L. acidophilus ASF360 and S. thermophilus NCIMB 41856 were capable of reducing the baseline level of RORC transcription in untreated cells by 10% . AIEC HM615 also induced a strong Treg response, shown by the upregulation of Foxp3 by 30% (p = 0.0009) in cultured leukocytes. However, this was reduced by the addition of any of the three potential probiotic strains: L. acidophilus ASF360 caused an 18% reduction in Foxp3 expression (p = 0.02), S. thermophilus NCIMB 41856 a 21% reduction (p = 0.03) and E. coli Nissle 1917 a 27% reduction (p = 0.02)  .E. coli K12 and AIEC HM615 induced upregulation of TNFα mRNA in cultured leukocyte populations (9% and 12% respectively), indicating a Th1 response. Despite this, neither E. coli K12 or AIEC HM615 induced a significant IFNγ response, although IFNγ mRNA production was increased by 15% when E. coli K12 and Nissle 1917 were used in combination (p = 0.04) and decreased by 16% following treatment with L. acidophilus ASF360 (p = 0.01) or by 10% following treatment with S. thermophilus NCIMB 41856 (p = 0.02) alone. The TNFα response to E. coli K12 was reduced by 16% by the addition of L. acidophilus ASF360 (p = 0.03) whereas S. thermophilus NCIMB 41856 reduced the level of TNFα mRNA produced in response to AIEC HM615 by 20% (p = 0.03)  following treatment with antigens derived from AIEC HM615; this appeared to be reduced by the addition of any of the three probiotic strains, although this was not statistically significant. No significant changes were seen in the expression of IL-10 mRNA, although E. coli Nissle 1917 did reduce IL-10 expression by 29% when cultured in combination with AIEC HM615; however HM615 did not induce the expression of IL-10 (E. coli K12 (p = 0.01) and by 11% in response to AIEC HM615 (p = 0.009) treatment. L. acidophilus ASF360 and S. thermophilus NCIMB 41856 were able to reduce the induction of TGFβ mRNA by E. coli K12 by 12% (p = 0.01) and 9% (p = 0.006) to levels comparable to that of control cells. Similarly, L. acidophilus ASF360 and S. thermophilus NCIMB 41856 also reduced the TGFβ response to AIEC HM615 by 12% (p = 0.04) and 10% (p = 0.009) respectively. E. coli Nissle 1917 was also able to reduce the expression of TGFβ mRNA induced by AIEC HM615 by 23% (p = 0.01)  . As expeof IL-10 . The tra = 0.01) .Faecalibacterium prausnitzii A2–165 (DSM 17677) is capable of reducing the expression of the pro-inflammatory cytokines IL-12 and IFNγ by PBMCs L. acidophilus Bar 13 and B. longum Bar 33; L. plantarum Bar 10, S. thermophilus Bar 20 and B. animalis subsp. lactis Bar 30 L. acidophilus, L. casei, L. reuteri, B. bifidum and S. thermophilus downregulated Th1, Th2 and Th17 cytokine responses, induced generation of CD4+ Foxp3+ Tregs and promoted regulatory dendritic cells expressing high levels of the regulatory cytokines IL-10 and TGFβ The mechanisms through which probiotics have been hypothesised to act are numerous and include an influence on intestinal transit time, competition with pathogens and immunomodulation. It is not clear what effect on the immune system is most desirable. A pro-inflammatory response may be required in order to more effectively clear infection Lactobacillus acidophilus ASF360 and Strep. thermophilus NCIMB 41856. One of the principal mechanisms for the action of probiotics is thought to be competitive exclusion of pathogens. These results support the theory that LAB are inefficient during active inflammatory disease because of their inability to compete with pathogens in the presence of iron, due to bleeding or supplementation.LAB have uniquely very low requirements for iron S. thermophilus NCIMB 41856 shows probiotic potential. This S. thermophilus NCIMB 41856 performed at least as well, and in many cases, better than, the more widely researched strains of L. acidophilus and E. coli Nissle 1917. L. acidophilus ASF360 showed a growth increase in response to iron, but only after 48 h, with the increase not being sustained after 72 h. In order for an organism to be able to establish itself and compete against pathogens in an iron-rich environment, stimulation of growth should ideally occur within typical intestinal transit time and then be sustained. This organism did not meet this test. In contrast, E. coli is known to be able to respond to iron in this manner S. thermophilus NCIMB 41856 ; L. plantarum JB012 and L. helveticus JB012 (isolated from probiotic capsules); and the commensal isolates L. acidophilus ASF360 and L. salivarius ASF361 (components of the Schaedler flora). Two strains of S. thermophilus (JB004 and NCIMB 41856) were isolated from yoghurt. Two strains of AIEC were used: HM427 and HM615 , as was E. coli K12 and E. coli Nissle 1917 ). All E. coli strains were grown in 10 ml volumes of LB broth  at 37°C overnight. S. thermophilus was grown in M17 broth supplemented with lactose (Oxoid) and Lactobacillus spp were cultured in MRS broth (Oxoid) overnight in a microaerobic atmosphere. Lactic acid bacteria were cultured in serum-SAPI medium with and without 100 µM (-) noradrenaline  in order to determine if they were capable of responding to it. O.D. measurements were taken at 24, 48 and 72 hours in order to determine bacterial growth. Differences were analysed using a repeated-measures ANOVA.Species of lactic acid bacteria that have been employed as probiotics were used. These were: 4 cells per well. After 3 days incubation, the medium was changed for one that was antibiotic-free and cells were labelled with BrdU in order to quantify proliferation. Bacteria were added to the epithelial cell cultures at an MOI of 30 and incubated for 24 hours at 37°C with 5% CO2. After 24 hours supernatants were harvested to determine cytotoxic effects of bacteria using the Cytotox 96 non-radioactive cytotoxicity assay kit  as directed by the manufacturer's instructions. Quantification of BrdU incorporation into the cells was determined using the cell proliferation biotrack ELISA system  as per the manufacturer's instructions. Differences were analysed using paired t-tests .All cell culture reagents were purchased from PAA Laboratories (Austria) unless otherwise specified. T84 and Caco-2 human adenocarcinoma cells  were grown in DMEM/Ham's F-12 or DMEM respectively, supplemented with 10% FCS, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates at an initial density of 2.4×105 cells per well. After 2 days of culture, cells were transfected with a reporter plasmid having an NF-κB response element, pGL4.32 (Promega), and the internal control reporter pGL4.74 (Promega) using lipofectamine . 24 hours later, the medium was replaced with 1x HBSS (Invitrogen) and bacteria were added at an MOI of 30. After 40 hours, cells were lysed and luciferase activity was quantified using a Dual Luciferase Reporter Assay (Promega) as per the manufacturer's instructions. Differences were analysed using paired t-tests (GraphPad Prism 5). T84 cells could not be efficiently transfected using these reporter plasmids and therefore results are not shown.Caco-2 cells were seeded into 12-well tissue culture plates at an initial density of 6×105 cells per well. After 3 days of culture, the medium was replaced with antibiotic-free medium and bacteria added at an MOI of 30. After 6 hours supernatants were harvested. Cytotoxicity was determined as above and production of IL-8 was quantified by ELISA using the human IL-8/CXCL8 DuoSet  as per the manufacturer's instructions. IL-8 production was corrected for cell death and differences were analysed using paired t-tests (GraphPad Prism 5). Caco-2 cells did not produce sufficient levels of IL-8 and therefore results are not shown.In order to determine the effect of potential probiotic strains on IL-8 production T84 cells were seeded into 12-well tissue culture plates at an initial density of 6×105 cells per insert. Plates were incubated at 37°C in 5% CO2 for 8–10 days until the cells formed confluent monolayers and the transepithelial resistance (TEER) was greater than 300 Ω/cm2 as measured with an epithelial voltmeter as an indicator of membrane permeability. The medium was then changed to one that was antibiotic free and bacteria were added to the apical well of the Transwell insert at an MOI of 30. TEER of all monolayers was measured at 2 hour intervals up to 12 hours and bacterial in the basal well were enumerated every 2 hours up to 10 hours. Differences were evaluated at each time point using paired t-tests (GraphPad Prism 5). In a separate experiment, medium was removed from the Transwells after 10 hours and the monolayers were examined as detailed below.Caco-2 and T84 cells were seeded onto 12-mm Transwell membranes  in 12-well tissue culture plates at an initial density of 3×10http://rsb.info.nih.gov/ij) and positive pixels automatically counted as previously described Transwell inserts were fixed in ice-cold methanol at 4°C overnight. Inserts were washed in PBS and the cells permeabilised with 0.1% Triton X-100 for 10 minutes before being washed again. Mouse anti-occludin monoclonal antibody  was diluted 1/200 and added to the apical chamber of the insert and incubated at room temperature for 45 minutes. Inserts were then washed in PBS and incubated for a further 45 minutes with TRITC-conjugated isotype-specific goat anti-mouse antibody  diluted 1/100 in the apical chamber. The inserts were then washed with PBS and the membranes were cut out of the inserts with a scalpel and mounted on slides with Vectashield containing DAPI . Fluorescent staining was examined on a Leica DMRA microscope equipped with a Hamamatsu Orca-ER monochrome camera. Ten fields of view per slide at 40x magnification were digitised using Leica Q-Fluoro software. Images were viewed using ImageJ software . It has previously been shown that co-culture of lamina propria T cells with ECM components prevents activation-induced apoptosis 1 integrins 2. After 5 days leukocytes were liberated from the collagen gel by the addition of collagenase (1000 U/ml). Cells were washed and counted before RNA extraction.Resected intestinal tissue was collected from patients undergoing surgery for complications associated with Crohn's disease or ulcerative colitis and from patients with colorectal cancer after informed written consent and with appropriate ethical approval (Somerset Research Ethics Committee). The mucosa was separated from the muscle, cut into small fragments and incubated in collagenase  for 2 hours at 37°C. Cells were washed in PBS and leukocytes purified over discontinuous Percoll gradients . The cell count and viability was determined by trypan blue exclusion. Cells were resuspended to a final concentration of 5×10http://frodo.wi.mit.edu/primer3) and M-Fold using the human specific GenBank sequences for T-box21 (accession number NM_013351), GATA-3 (accession number NM_001002295), RORC (accession number NM_005060), Foxp3 (accession number NM_017009), IFNγ (accession number NM_000619), TNFα (accession number NM_000594), IL-4 (accession number NM_000589), IL-4δ2 (accession number NM_172348), IL-17A (accession number NM_002190), IL-10 (accession number NM_000572) and TGFβ (accession number NM_000660). The housekeeper gene hydroxymethylbilane synthase (HMBS) was used as an internal control. Quantitative PCR (qPCR) was performed using HotStarTaq Master Mix (Qiagen Ltd.). Gene specific amplification was performed using 0.2µM of each primer, 0.1µM of probe or SYBR Green 1  and 5μl of diluted cDNA in a final volume of 25μl. Magnesium chloride concentrations were adjusted to 4.5 mM in the final reaction by addition of 50 mM MgCl2. Sample incubations were performed in an MxPro3005P  at 95°C for 15 minutes and then 45 cycles of 95°C for 15 seconds, and 60°C for 30 seconds during which the fluorescence data were collected. Data is expressed as the relative change in mRNA transcription following treatment and is normalised for cell number. No significant differences were seen between cells isolated from the three disease states and therefore results were pooled for analysis. Differences were analysed using paired t-tests (GraphPad Prism 5).RNA was extracted from the cultured leukocytes using a Macherey-Nagel NucleoSpin RNA II Isolation Kit . Synthesis of cDNA was carried out using 500 ng of random hexamers using the ImProm-II Reverse Transcription System (Promega) in a final volume of 20µl. All reactions were prepared according to the manufacturer's instructions giving a final magnesium chloride concentration of 3 mM. All cDNAs were diluted to a final volume of 100μl (1/5 dilution) using EB buffer . Primers and probes were designed using Primer 3  and Caco-2 cells (B). * p≤0.05, ** p≤0.01 and *** p≤0.001.(TIF)Click here for additional data file.Figure S2Percent change in cytokine mRNA levels in response to E. coli with and without probiotic. Results are expressed as mean±S.E.M. * p≤0.05.(TIF)Click here for additional data file.


Cardamine hupingshanesis (Brassicaceae), Ligulariafischeri (Ledeb.) turcz (Steraceae) and their underlying top sediments were collected from selenium (Se) mine drainage areas in Enshi, China. Concentrations of total Se were measured using Hydride Generation-Atomic Fluorescence Spectrometry (HG-AFS) and Se speciation were determined using liquid chromatography/UV irradiation-hydride generation-atomic fluorescence spectrometry (LC-UV-HG-AFS). The results showed that C. hupingshanesis could accumulate Se to 239±201 mg/kg DW in roots, 316±184 mg/kg DW in stems, and 380±323 mg/kg DW in leaves, which identifies it as Se secondary accumulator. Particularly, it could accumulate Se up to 1965±271 mg/kg DW in leaves, 1787±167 mg/kg DW in stem and 4414±3446 mg/kg DW in roots, living near Se mine tailing. Moreover, over 70% of the total Se accumulated in C. hupingshanesis were in the form of selenocystine (SeCys2), increasing with increased total Se concentration in plant, in contrast to selenomethionine (SeMet) in non-accumulators (eg. Arabidopsis) and secondary accumulators (eg. Brassica juncea), and selenomethylcysteine (SeMeCys) in hyperaccumulators (eg. Stanleya pinnata). There is no convincing explanation on SeCys2 accumulation in C. hupingshanesis based on current Se metabolism theory in higher plants, and further study will be needed.Plant samples of  Enshi is located in western Hubei province, China Fig. 1a.Stanleya pinnata) and twogrooved milkvetch  were found to contain Se up to 0.1–1.5% (dry weight) and 0.6% (dry weight), respectively S. pinnata were found to be related to the levels of ascorbic acid, glutathione, total sulfur, and nonprotein thiols, and may in part be due to increased antioxidants and up-regulated sulfur assimilation Pilon-Smits and Quinn (2010)Earlier studies indicated that the soil Se concentrations varied significantly in Enshi with the highest Se concentration found in the Se mine drainage areas The specific objectives of this study were to identify local Se-accumulating plant species, and to determine the dominant chemical forms of Se accumulated in plant tissues of different species in Enshi, China. The research findings of this study could be helpful in the development and application of Se phytoremediation and biofortification technologies.Permissions for field work were obtained from local government. As this was a purely scientific study, no specific permit was needed. This field study did not involve endangering to protected species.The study site was located in Yutangba , northwestern Enshi, Hubei Province, including three Se mine drainage creeks Fig. 1b.Cardamine hupingshanesis (Brassicaceae) and Ligulariafischeri (Ledeb.) turcz (Steraceae). These two plant species were collected along with their underlying top sediment samples (0–3 cm depth). The collected samples and their locations are shown in andThere are two Se mine outcrops in Yutangba, Enshi Fig. 1b.Plant samples were washed in tap water to remove soils from the root surface, and then rinsed in deionized water. Plants were separated into roots, stem, and leaves, oven-dried at approximately 50°C for 24 hours, and ground to pass through a 0.2 mm sieve . Sediment samples, free of plant roots and detritus, were oven-dried at approximately 50°C for 24 hours, and then ground in an agate-mortar to pass through a 0.15 mm sieve.3 and HClO4  were added to each flask and covered with a glass funnel. The flasks were kept overnight at the room temperature, heated at 100°C for one hour, 120°C for two hours, and 180°C for one hour on an electrical hot plate. The samples were then heated at 210°C until the white fume formed and the volume of solution was approximately 2 ml. After acid digestion, the digest was cooled to room temperature and 5 ml HCl (12 M) was added to reduce Se(VI) to Se(IV) for 3–4 h as following pathway: H2SeO4+2HCl (Concentrated) = H2SeO3+Cl2+H2O Gao et al. (2011) 0.5–3.0 g samples were weighed into a 50 ml conical flask. Ten ml of concentrated HNO4H2PO4 (pH 6.0) with a flow rate of 1 mL/min. The eluent from the column was mixed with concentrated HCl (flow rate: 3 mL/min) and then passed through the UV unit. 1.2% NaBH4 in 0.1 mol/L NaOH (flow rate: 3 mL/min) was added after the UV unit. Argon was used as carrier gas (260 mL/min) to transfer H2Se from the gas liquid separator through the dryer into the AFS detector . The dryer gas was nitrogen with a flow rate of 3 L/min. The organic Se standards  were purchased from Tokyo Chemical Industry, Co., Japan, and the inorganic Se standards (Selenite and Selenate) were purchased from National Reference Material Centre, China. Since Se(VI) could be reduced to Se(IV) by concentrated HCl during elution, the inorganic Se of sample was represented by measured Se(IV). Thus, four Se species were detected in plant tissues, including selenocystine (SeCys2), selenomethylselenocysteine (SeMeCys), selenomethionine (SeMet) and Se(IV) species were determined with precisions of 5%, 6%, 10% and 5%, respectively. The instrument detection limits were 2 µg/L, 5 µg/L, 10 µg/L and 2 µg/L , respectively. The detailed description of the procedure was given by Liang et al. (2006)and Mazej et al. (2006)2), was used since the SeCys could be co-eluted with SeCys2 in LC-UV-HG-AFS 2 here will be the total concentrations of SeCys and SeCys2 in plant tissues.The leaf, stem and root samples were extracted with 100 mM Tris-HCl buffer (pH 7.5) in an ultrasonic tank for 10 min. The enzyme Protease XIV was added and the mixture was shaken for 24 h at 37°C. After extraction, the mixture was centrifuged at 10000 rpm/min for 30 min at 4°C. The supernatant was collected and filtered through a 0.22 µm filter for Se speciation analysis. The separation of different Se compounds was performed using a Hamilton PRP X-100 anion exchange column (4.1 mm×250 mm×10 µm). The mobile phase was 40 mM NHNormality test, two sample t test on normally distributed data, nonparametric test (Two sample Kolmogorov-Smirnov Test/K-S Test) on non-normally distributed data, and correlation analyses were performed by OriginPro 8.C. hupingshanesis are shown in C. hupingshanesis were found in Creek 2 (YTB 7–11 sampling sites), with 56.85±19.20 mg/kg DW (n = 5) in roots, 132.67±21.48 mg/kg DW (n = 5) in stems, and 117.70±52.84 mg/kg DW (n = 5) in leaves. Concentrations of total Se in L. (Ledeb.) turcz (Steraceae) at YTB 4–6 and 8–10 sampling sites were compiled in L. (Ledeb.) turcz grown in Creeks 1 and 2 (n = 6) were 18.98±8.13 mg/kg in root, 14.01±7.78 mg/kg in stem, and 25.62±18.05 mg/kg in leaves.Concentrations of total Se accumulated in plant tissues of C. hupingshanesis has significant higher Se concentrations in leaves (432±471) (n = 27) (mg/kg DW), stems (425±447) (n = 27) (mg/kg DW), and roots (462±709) (n = 19) (mg/kg DW) than those of L. (Ledeb.) turcz in leaves (25.62±18.05) (n = 6) (mg/kg DW) , stems (14.01±7.78) (n = 6) (mg/kg DW) , and roots (18.98±8.13) (n = 6) (mg/kg DW) , respectively. In contrast, there were no significant differences  in Se distributions in plant tissues of C. hupingshanesis.The statistical analysis revealed that C. hupingshanesis and L. (Ledeb.) turcz are shown in hupingshanesis and 28.78±14.76 (n = 6) mg/kg DW for L. (Ledeb.) turcz, and there was no significant  difference between them though the total Se concentrations in C. hupingshanesis tissues were significant higher than those of L. (Ledeb.) turcz tissues.Total Se concentrations in underlying top sediments (0–3 cm depth) of C. hupingshanesis are shown in L. (Ledeb.) turcz is presented in C. hupingshanesis included SeCys2 with 78±16% in leaves, 74±17% in stems, and 78±23% in roots, followed by SeMeCys with 12.91±6.74% in leaves, 16.65±9.21% in stems, and 12.43±8.88% in roots, and Se(IV) with 5.12±6.77% in leaves, 4.07±4.31% in stems, and 8.71±10.23% in roots. Moreover, the accumulation of SeCys2 was greater in the plant having higher contents of total Se. The Se speciation in C. hupingshanesis also varied among different sampling sites. At Site 3 (or YTB 3) where the highest plant Se concentrations were observed, SeCys2 accounted for almost all the Se accumulated in C. hupingshanesis. In contrast, the lowest proportion of SeCys2 in C. hupingshanesis was recorded at YTB 8 sampling site, with 30.37% in stem and 38.37% in leaves, which were corresponding to the lowest total Se contents with 128 mg/kg DW in stem and 66.67 mg/kg DW in leaves. Furthermore, there were no significant differences in Se speciation of plant tissues between roots and leaves , and between stem and leaves .The chemical compositions of Se in L. turcz were compiled in L. turcz, followed by SeMeCys Corn seeds, 1.48±1.41 mg/kg DW (n = 20); Agry wormwood, 1.68±1.27 mg/kg DW (n = 30); Bracken fern, 0.63±1.61 mg/kg DW (n = 57); Central China dryoathyrium, 0.48±0.72 mg/kg DW (n = 39) and Hupeh beautyberry, 0.19±0.03 mg/kg DW (n = 5). But in the present study, C. hupingshanesis collected from the same study area in Enshi accumulated Se as high as 432±471 mg/kg DW (n = 27) in leaves, 425±447 mg/kg DW (n = 27) in stems and 462±709 mg/kg DW (n = 19) in roots. In fact, C. hupingshanesis could be identified as a new Se accumulator, or Se secondary accumulator, which could grow on soils contaminated with moderate levels of Se (10–70 mg/kg DW) in the Se mine drainage area, and accumulate Se up to 100–1000 mg/kg DW C. hupingshanesis could accumulate Se up to 1965±271 mg/kg DW in leaves, 1787±167 mg/kg DW in stem and 4414±3446 mg/kg DW in roots near Se mine tailing (Sampling site YTB-3), which were comparable to those of typical Se hyperaccumulating plants, Stanleya pinnata (prince's plume) (1000–15000 mg Se/kg DW) and Astragalus bisulcatus (twogrooved milkvetch) (about 6000 mg Se/kg DW) C. hupingshanesis displayed exceptionally high BCF values of 85 in roots on site of YTB-3, 39 in stem and 38 in leaves on site of YTB-4, indicating soil Se could be efficiently taken up by it. Overall, C. hupingshanesis had BCFs with ranges of 9.31±9.07 (n = 28) in leaves, 8.70±7.69 (n = 28) in stems and 10.71±16.90 (n = 24) in roots. In contrast, L. turcz had relatively low BCFs values of <2, significantly lower  than those of C. hupingshanesis (Brassica juncea (Indian mustard) and Brassica napus (Canola) shoots, the BCFs were less than 10 Stanleya pinnata (prince's plume) and Astragalus bisulcatus (twogrooved milkvetch) had BCFs typically >100 To characterize the Se-translocation from soil to plant, the bio-concentration factor (BCF) of Se was calculated Table 3.con/rootcon values and the leafcon/rootcon values were very close in C. hupingshanesis with 1.72±0.95 (n = 24) and 1.76±0.98 (n = 24), respectively, which likely indicated that stems of C. hupingshanesis were not effective Se channels. Moreover, the stemcon/rootcon values in C. hupingshanesis were significant higher  than those in L. turcz, although there were no significant  differences on the leafcon/rootcon values, which revealed that C. hupingshanesis had much better performance to transport Se from root to stem. It is known that translocation of Se from root to shoot depends on the forms of Se supplied from the soil, in which selenate is much more easier to transport, then selenite and SeMet Zayed et al. (1998)The translocation of Se from root to shoot by plants could be indicated by Se concentrations from stem/root and leaf/root ratios; thus calculated values were summarized in 0), or converted to selenomethionine (SeMet) or selenomethylcysteine (SeMeCys), then methylated to volatile Se compounds as dimethylselenide (DMSe) or dimethyldiselenide (DMDSe) Arabidopsis and Secondary accumulating plants such as Indian mustard (Brassica juncea) store Se mainly as selenate when the plants are fed with selenate. In contrast, selenate-supplied biofortified vegetables, such as garlic (Allium sativum), onion (Allium cepa), leek (Allium ampeloprasum) and broccoli (Brassica oleracea), store Se predominantly as SeMeCys Stanleya pinnata) (Fabaceae) and twogrooved milkvetch  (Brassicaceae), around 90% of the accumulated Se is present as SeMeCys in specialized cells in the leaf epidermis or in leaf hairs Freeman et al. (2010)Stanleya albescens (Brassicaceae), as a secondary Se accumulator, accumulated Se mainly as free amino acid selenocystathionine (SeCyst). Therefore, this is the first study reporting up to 99% of total Se as SeCys2 or SeCys in a higher plant.Selenocysteine (SeCys) is a key component in Se metabolism for plant, which will be incorporated into proteins, transformed to elemental Se  and Stanleya pinnata (Brassicaceae); SMT methylates SeCys and diverts Se away from proteins, thereby reducing Se toxicity in plant, which could provide Se tolerance and hyperaccumulation A. bisulcatus has been successfully overexpressed in two different non-hyperaccumulating plants, Arabidopsis thaliana and Brassica juncea (Indian mustard), via transgenic approaches, confirming that SMT is one of key enzymes for Se hyperaccumulation S. pinnata showed that it related with a constitutively higher expression of genes involved in sulfur assimilation, antioxidant activities, defense, and response to (methyl)jasmonic acid, salicylic acid, or ethylene In fact, although Se is an essential human and animal nutrient that is needed for several proteins, such as glutathione peroxidase, thioredoxin reductase and at least 23 other essential selenoproteins, no such requirement for Se has been shown for higher plants 2, which indicated SeCys methyltransferase, cysthathionine-γ-synthase, cysthathionine-β-lyase, and methionine synthase did not play a significant role in Se accumulation or tolerance for C. hupingshanesis. So what is the possible mechanism for C. hupingshanesis to survive in the Se-mine drainage area of Enshi? The accumulation Se in peripheral tissues of young leaves and reproductive organs may be a strategy to accumulate high concentrations of Se as SeCys22 in tissues 2 synthase or enhancement by rhizospheric microorganisms to explain high SeCys2 accumulation in C. hupingshanesis.In the present study, SeMet and SeMeCys were detected in much lower proportions than SeCysCardamine hupingshanesis (Brassicaceae) was identified as a new Se secondary accumulator species. It could accumulate Se to concentrations ranging as 239±201 mg/kg DW in roots, 316±184 mg/kg DW in stems, and 380±323 mg/kg DW in leaves, with the underlying top sediment Se contents of 35.79±17.95 mg/kg DW. Particularly, this plant species could accumulate Se up to 1965±271 mg/kg DW in leaves, 1787±167 mg/kg DW in stem and 4414±3446 mg/kg DW in roots, which were comparable to typical Se hyperaccumulating plants, Stanleya pinnata and Astragalus bisulcatus. Furthermore, C. hupingshanesis had a high ability to accumulate Se from sediment with bio-concentration factors of about 10, and it could accumulate up to 99% of total Se as SeCys2 in plant tissues without showing phytotoxic symptoms. The current Se metabolisms knowledge could not give a reasonable explanation on it. More research should be needed to elucidate the possible mechanism on it.In the Se-mine drainage area from Enshi, China,


To assess the dose-response relationships between cause-specific mortality and exercise energy expenditure in a prospective epidemiological cohort of walkers.2/kg/min), which were used to divide the cohort into four exercise categories: category 1 (≤1.07 MET-hours/d), category 2 (1.07 to 1.8 MET-hours/d), category 3 (1.8 to 3.6 MET-hours/d), and category 4 (≥3.6 MET-hours/d). Competing risk regression analyses were use to calculate the risk of mortality for categories 2, 3 and 4 relative to category 1.The sample consisted of the 8,436 male and 33,586 female participants of the National Walkers' Health Study. Walking energy expenditure was calculated in metabolic equivalents  and 32.9% lower in category 4 (P = 10−11) than in category 1. For underlying causes of death, the respective risk reductions for categories 2, 3 and 4 were 23.6% (P = 0.008), 35.2% (P<10−5), and 34.9% (P = 0.0001) for cardiovascular disease mortality; 27.8% (P = 0.18), 20.6% (P = 0.07), and 31.4% (P = 0.009) for ischemic heart disease mortality; and 39.4% (P = 0.18), 63.8% (P = 0.005), and 90.6% (P = 0.002) for diabetes mortality when compared to category 1. For all related mortality , the respective risk reductions for categories 2, 3 and 4 were 18.7% (P = 0.22), 42.5% (P = 0.001), and 57.5% (P = 0.0001) for heart failure; 9.4% (P = 0.56), 44.3% (P = 0.0004), and 33.5% (P = 0.02) for hypertensive diseases; 11.5% (P = 0.38), 41.0% (P<10−4), and 35.5% (P = 0.001) for dysrhythmias: and 23.2% (P = 0.13), 45.8% (P = 0.0002), and 41.1% (P = 0.005) for cerebrovascular diseases when compared to category 1.22.9% of the subjects were in category 1, 16.1% in category 2, 33.3% in category 3, and 27.7% in category 4. There were 2,448 deaths during the 9.6 average years of follow-up. Total mortality was 11.2% lower in category 2 (P = 0.04), 32.4% lower in category 3 (P<10There are substantial health benefits to exceeding the current exercise guidelines. The health benefits produced by physical activity are well-documented The current report examines the dose-response relationships between exercise energy expenditure and mortality in the National Walkers' Health Study The National Walkers' Health Study is a prospective epidemiological cohort that has been described in detail in several publications Intakes of meat, fish and fruit were based on the questions “During an average week, how many servings of beef, lamb, or pork do you eat”, and “…pieces of fruit do you eat”. Alcohol intake was estimated from the corresponding questions for 4-oz. (112 ml) glasses of wine, 12-oz. (336 ml) bottles of beer, and mixed drinks and liqueurs. Alcohol was computed as 10.8 g per 4-oz glass of wine, 13.2 g per 12 oz. bottle of beer and 15.1 g per mixed drink Mortality surveillance was completed through December 31, 2008 using the National Death Index , race, sex, years of education, baseline smoking status (yes/no), ever smoked (yes/no), prior history of a heart attack, prior history of a cancer, aspirin use, and intakes of meat, fruit, and alcohol. Semi-hazard ratios (SHR) from competing risks regression were used to test whether cause-specific deaths were significantly related to exercise energy expenditure when adjusted for these covariates Hazard ratios (HR) from Cox proportional hazard analyses were used to compare all-cause mortality to exercise energy expenditure when adjusted for baseline age (age and age2) was included to test whether the dose-response relationship between mortality and MET-hours/d of exercise was significantly nonlinear. The two- degree of freedom Chi square test was used to test whether men and women showed the same dose-response relationship between mortality and exercise. Specifically, the Chi square test was applied to the difference in the log likelihoods between a model that included separate linear and quadratic regression coefficients for men and women with one that had the same linear and quadratic regression coefficients for both sexes. Results are presented as relative risk  and their percent reductions in risk  or 100*(SHR-1)) per MET-hours/d, and for four categories of exercise energy expenditure: category 1 is falling short of the 450 MET minutes per week recommended for health (≤1.07 MET-hours/d), category 2 is meeting the 450 to 750 MET min/wk guideline exercise recommendations  was available for 96.8% of the sample. Greater exercise was associated with younger age, less smoking, fewer prior heart attacks or cancers , less meat and greater fruit and alcohol consumption, and lower BMIbaseline. BMI when first began walking 12 or more miles per week  was available for 72.4% of the sample. Most of those with missing BMIstarting walking were subjects who simply never achieved that level of activity, i.e. BMIstarting walking was unreported for 54.7% of the sample who exercised <1.07 MET-hours/d at baseline, 45.0% of the sample who exercised 1.07 to 1.8 MET-hours/d, 13.2% of the sample who exercised 1.8 to 3.6 MET-hours/d, and 12.6% of the sample who exercised ≥3.6 MET-hours/d.baseline somewhat attenuated the risk reductions. There was no significant difference in the dose-response relationship between males and females , so their data were combined and analyzed adjusted for sex.One-thousand two hundred thirteen of the 8,436 men (14.4%) and 1235 of the 33,586 women surveyed at baseline (3.7%) died during the average 9.6-year follow-up. baseline .When the sexes were combined, baseline  or BMIst walking . ExceediSex also did not significantly affect the dose-response relationships between MET-hours/d of exercise and the risk for CVD (P = 0.88 for sex difference), ischemic heart disease , dysrhythmias (P = 0.99), heart failure (P = 0.81), hypertensive disease (P = 0.86), cerebrovascular disease (P = 0.89), or diabetes mortality (P = 0.66). Therefore, the male and female data for these deaths were also combined and adjusted for sex when analyzed. starting walking .IHD represented about one-half of all CVD deaths. The reduction in risk for all IHD-related mortality was similar to that of all CVD-related mortality . Adjustm in risk , whereasline did . Neverthbaseline . The total number of events was increased substantially by including deaths with cerebrovascular disease listed as a contributing cause. baseline or BMIstarting walking). Two-thirds of the diagnoses were “stroke not specified as hemorrhage or infarction” or “unspecified cerebrovascular disease” that prevented a more refined endpoint analyses.The risk for death due to cerebrovascular disease as an underlying cause was significantly less in those who exercised ≥1.8 MET-hours/d vs. <1.07 MET-hours/d , which persisted when adjusted for medication use and BMIbaseline , and when adjusted for BMIbaseline . There were ten times as many dysrhythmia-related deaths (underlying and contributing) than dysrhythmias as an underlying cause. The risk for dysrhythmia-related deaths was significantly lower for 1.8 to 3.6 MET-hours/d and ≥3.6 MET-hours/d vis-à-vis <1.07 MET-hours/d, with or without BMI adjustment.Despite only 48 deaths ascribed to dysrhythmias as the underlying cause, their risk was significantly less in those who exercised ≥1.8 MET-hours/d vs. <1.07 MET-hours/d , which gbaseline , or BMIs walking . There wThere were only 53 deaths due to heart failure as an underlying cause, but five times as many heart failure-related deaths, and their risk was significantly reduced by exercising 1.8 to 3.6 MET-hours/d and ≥3.6 MET-hours/d vis-à-vis <1.07 MET-hours/d, with or without BMI adjustment –8. ExceeDeaths due to other circulatory diseases  were unrelated to physical activity.The risk for diabetes as an underlying cause of death was about two-thirds lower for 1.8 to 3.6 MET/d, and 90% lower for ≥3.6 MET/h vis-à-vis inadequate exercise, with the reduction in risk for ≥3.6 MET-hours/d remaining significant when adjusted for BMI. The larger number of all diabetes-related deaths strengthen the significance of the risk reduction for both 1.8 to 3.6 MET/d and ≥3.6 MET/h vs. inadequate exercise, with or with BMI adjustment –8.There were 467 deaths before age 65, including 165 CVD-related, 66 dysrhythmia-related, 63 IHD-related, 35 hypertensive disease-related, 14 heart failure-related- and 35 diabetes-related deaths. No one died from a cerebrovascular disease-related death before the age of 65. Relative to exercising inadequately, expending between 1.07 to 1.8 MET-hours/d, 1.8 to 3.6 MET-hours/d, and ≥3.6 MET-hours/d was associated with: 1) 25.4% (P = 0.04), 27.3% (P = 0.005), and 29.7% (P = 0.01) lower risk for all-cause premature mortality, respectively; 2) 11.7% (P = 0.58), 34.0% (P = 0.04), and 38.6% (P = 0.03) lower risk for all premature CVD-related mortality, respectively; 3) 47.7% (P = 0.58), 44.2% (P = 0.04), and 49.8% (P = 0.03) lower risk for all premature IHD-related mortality, respectively; 4) 22.9% (P = 0.45), 59.8% (P = 0.007), and 52.4% (P = 0.03) lower risk for all premature dysrhythmia-related mortality, respectively; 5) 33.1% (P = 0.38), 53.9% (P = 0.08), and 36.6% (P = 0.03) lower risk for all premature hypertensive disease-related mortality, respectively; 6) 65.1% (P = 0.20), 83.2% (P = 0.03), and 89.8% (P = 0.02) lower risk for all premature heart failure-related mortality, respectively; and 7) 52.8% (P = 0.14), 60.3% (P = 0.04), and 76.1% (P = 0.005) lower risk for all premature diabetes-related mortality, respectively.−7), and 16.5% (P = 0.01) lower risk for total mortality, respectively; 2) 12.8% (P = 0.12), 37.3% (P = 10−8), and 31.8% (P = 10−5) lower risk for all CVD-related mortality, respectively; 3) 16.4% (P = 0.17), 30.7% (P = 0.002), and 29.5% (P = 0.009) lower risk for all IHD-related mortality, respectively; 4) 7.0% (P = 0.60), 40.2% (P = 0.0001), and 31.6% (P = 0.01) lower risk for all dysrhythmia-related mortality, respectively; 5) 1.7% , 43.3% (P = 0.002), and 25.2% (P = 0.13) lower risk for all hypertensive disease-related mortality, respectively; 6) 21.0% (P = 0.22), 45.7% (P = 0.0006), and 31.3% (P = 0.07) lower risk for all cerebrovascular disease-related mortality, respectively; 7) 14.8% (P = 0.39), 39.6% (P = 0.007), 55.0% (P = 0.0004) lower risk for all heart failure-related mortality, respectively; and 8) 11.2% (P = 0.55), 59.7% (P = 10−6), and 76.9% (P = 10−6) lower risk for all diabetes-related mortality, respectively.All of the preceding analyses excluded mortality within the first year of follow-up. Excluding deaths during the first three years of follow-up left 2029 total deaths, including 1084 CVD-related, 494 IHD-related, 414 dysrhythmia-related, 231 hypertensive disease-related, 216 heart failure-related, 207 cerebrovascular disease-related, and 160 diabetes-related deaths. Relative to exercising inadequately, expending between 1.07 to 1.8 MET-hours/d, 1.8 to 3.6, MET-hours/d and ≥3.6 MET-hours/d were associated with: 1) 6.0% (P = 0.32), 27.2%  per day of brisk walking. This exercise category is substantially greater than those included in other studies. It is greater than all but one of the studies included in Hamer and Chida's meta-analyses of CVD Consistent with prior studies Unlike other studies, the current analyses were not restricted to the underlying cause of death. There is no evidence from th percentile of their walking distribution. Similarly, the Health Professionals' Follow-up Study reported that there was a 40% reduction in total mortality by the 60th percentile of their walking distribution Our cohort did not include a totally sedentary group. Therefore, the risk reductions reported here probably underestimate the potential for risk reduction relative to completely sedentary populations. Specifically, others report substantial risk differences that fall within our lowest exercise category (0 to 1.07 MET-hours/d). For example, the Women's Health Study reported that there was a 63% difference in coronary heart disease risk and 44% difference in diabetes risk between non-walkers and those walking between 1 and 1.5 h/wk The current findings for walkers differ somewhat from our prior analyses of runners, in which we showed prospectively that the risk for nonfatal coronary heart disease decreased 30% (P = 0.006) by running ≥6 MET-hours/d when compared to running 3–6 MET-hours/d baseline and medication use. The reductions in risk were somewhat diminished by the adjustment, but nevertheless remained clinically and statistically very significant. These analyses likely represent over-adjustment because running and walking are known to reduce hypertension, high cholesterol and type 2 diabetes risk starting walking in baseline, given that weight control may mediate some of the health benefits of walking.There are important limitations to these analyses. Vital status in known only from the National Death Index and therefore some subjects who have died are likely to be misclassified as alive. The set of covariates used in the analyses are somewhat restricted and all are measured with error, which will cause their effect to be underestimated by the adjustment. The ability to exercise harder may be an innate quality of health and not directly related to habitual physical activity. The greater risk for heart failure mortality with less exercise could be a consequence of cardiac insufficiency at an early, preclinical, stage of the disease.These analyses suggest there are important health benefits to exceeding the current exercise recommendations for health (≥750 MET minutes per week or ≥1.8 MET-hours/d


In the brain, astrocytes signal to the neighboring cells by the release of chemical messengers (gliotransmitters) via regulated exocytosis. Recent studies uncovered a potential role of signaling lipids in modulation of exocytosis. Hence, we investigated whether sphingosine and the structural analog fingolimod/FTY720, a recently introduced therapeutic for multiple sclerosis, affect (i) intracellular vesicle mobility and (ii) vesicle cargo discharge from cultured rat astrocytes. Distinct types of vesicles, peptidergic, glutamatergic, and endosomes/lysosomes, were fluorescently prelabeled by cell transfection with plasmids encoding atrial natriuretic peptide tagged with mutant green fluorescent protein and vesicular glutamate transporter tagged with enhanced green fluorescent protein or by LysoTracker staining, respectively. The confocal and total internal reflection fluorescence microscopies were used to monitor vesicle mobility in the cytoplasm and near the basal plasma membrane, respectively. Sphingosine and FTY720, but not the membrane impermeable lipid analogs, dose-dependently attenuated vesicle mobility in the subcellular regions studied, and significantly inhibited stimulated exocytotic peptide and glutamate release. We conclude that in astrocytes, cell permeable sphingosine-like lipids affect regulated exocytosis by attenuating vesicle mobility, thereby preventing effective vesicle access/interaction with the plasma membrane docking/release sites. © 2012 Wiley Periodicals, Inc. Acidic endosomes/lysosomes were stained by incubating astrocytes in the culture medium containing 200 nM LysoTracker Red DND-99 (Invitrogen) for 5 min at 37°C.Cells were transfected with the plasmid encoding atrial natriuretic peptide tagged with emerald green fluorescent protein , using Nucleofector II (Amaxa Biosystems) and Rat Astrocyte Nucleofector Kit (Lonza) according to the manufacturer's instructions. The transfected cells were subcultured onto round 22 mm diameter poly-2, 1 mM MgCl2, 10 mM d-glucose, and 10 mM HEPES, pH 7.2, mounted onto the recording chamber and transferred to a confocal microscope . The cells were observed by a plan-apochromatic oil-immersion objective 63×/NA 1.4. ANP.emd and VGLUT1-EGFP were excited by 488 nm argon laser line and emission fluorescence was filtered with 505–530 nm band-pass filter. LysoTracker Red DND-99 was excited with 543 nm He-Ne laser line and emission fluorescence filtered with long pass 560 nm filter. In initial experiments, the astrocyte plasma membrane was stained with FM 4-64  dissolved in the extracellular solution, to visualize the cell perimeter. FM 4-64 was excited by 488 nm argon laser line and emission fluorescence filtered with 585 nm long-pass emission filter.To observe the mobility of ANP.emd, VGLUT1-EGFP and LysoTracker labeled vesicles, astrocyte-loaded coverslips were washed two times with extracellular solution containing 130 mM NaCl, 5 mM KCl, 2 mM CaClL-glutamic acid (L-Glu) and ATP  was applied as a bolus into the extracellular solution to all examined cells and the vesicle mobility recorded before and after stimulation.To study the mobility of fluorescently labeled vesicles, time-lapse images were acquired every 493 ms for 1–3 min in nontreated cells and cells treated with 1–20 μM FTY720 (Enzo Life Sciences), 10 μM sphingosine (Biomol), 10 μM FTY720-P (Echelon) or 10 μM thonzonium dissolved in the cell culture medium for 10 min at 37°C. In addition, exocytotic stimulus composed of In a separate set of experiments coverslips with ANP.emd transfected astrocytes were observed by the inverted microscope  equipped with beam-splitter/emission-filter cubes for epifluorescence imaging, and diode-pumped solid-state (DPSS) 488 nm laser (Toptica) for TIRF imaging. The emission fluorescence was collected with α-Plan-Apochromat oil-immersion objective 100×/NA 1.46 and alternate, dual mode (TIRF and epifluorescence) images were acquired every 500 ms for 1 min prior to and 10 min after treatment with 10 μM FTY720. In epifluorescence mode ANP.emd was excited with 488 nm laser line and emission was band-pass filtered (525/31 nm). In TIRF mode ANP.emd was excited with the evanescent field formed above the coverslip glass due to the total internal reflection of 488 nm laser beam. Depending on the angle of the incident light used for TIRF imaging, the calculated Axelrod, penetratz-axis translocation of individual vesicles imaged in the TIRF plane, we considered the exponential dependence of the TIRF excitation intensity of the fluorescent objects positioned along the z-axis  software t-TIME.To determine the time-course and the relative magnitude of the  Axelrod,. Any chax, y coordinates (peak of the curve), which were then connected to obtain the pathways that vesicles traveled within the total recording time. Typically, ∼50 randomly selected vesicles were tracked per cell. The mobility parameters were estimated for 15 s epochs. For each vesicle the track length  and maximal displacement  were determined. The analysis of the vesicle mobility was performed in nontreated cells and in cells either acutely treated with 10 μM FTY720, or pretreated with 1–20 μM FTY720, 10 μM sphingosine, 10 μM FTY720-P, or 10 μM thonzonium for 10 min. The mean (±s.e.) vesicle TL and MD were determined in 5–15 nontreated and treated cells.The mobility of peptidergic (ANP.emd), glutamatergic vesicles (VGLUT1-EGFP), and endosomes/lysomes (LysoTracker labeled) was analyzed by ParticleTR software  in exported tiff files . Any glutamate release from nontreated and FTY720 treated cells stimulated by 1 mM ATP was detected as an increase in NADH fluorescence. Time-lapse images were taken at 2 s intervals (250 ms exposure time) by Andor Clara camera. The circular regions (2r = 5 μm) were positioned over the imaged cells by Till Offline Analysis software to assess NADH fluorescence increase and the fluorescence peak (in A.U.) measured.The release of glutamate from confluent cultures was measured fluorimetrically  derived from 9 to 15 cells exposed to control conditions and to different FTY720 concentrations  was analyzed. The mean vesicle TL in nontreated cells was 4.56 ± 0.36 μm (n = 11), while in FTY720 treated cells the TL decreased in a dose-dependent manner from 4.47 ± 0.40 μm (n = 9) to 3.28 ± 0.14 μm (n = 9), 2.56 ± 0.21 μm (n = 9), and 2.19 ± 0.14 μm (n = 15) for 1, 5, 10, and 20 μM FTY720, respectively (P = 0.877), the TL in FTY720 treated cells diminished significantly when compared with nontreated cells . The MD significantly (P < 0.01 for 1 μM FTY720 and P < 0.001 for higher concentrations) diminished in a dose-dependent fashion from 2.15 ± 0.18 μm in nontreated cells to 1.30 ± 0.22 μm, 0.77 ± 0.06 μm, 0.44 ± 0.06 μm, and 0.39 ± 0.05 μm in cells treated with 1, 5, 10, and 20 μM FTY720, respectively  and the maximal displacement (MD) were determined for each tracked vesicle within 15 s. In total, the mobility of vesicles /b)2), whereby for the TL plot the parameters were a = 41.63 ± 2.28, b = 0.40 ± 0.02 μm, x0 = 1.87 ± 0.03 μm and for the MD plot a = 55.22 ± 0.58, b = 0.19 ± 0.00 μm, x0 = 0.22 ± 0.00 μm , 2.80 ± 0.30 μm (n = 5), and 1.69 ± 0.23 μm (n = 5), respectively , ∼36% , and ∼39%  (P < 0.001), ∼69% , and ∼54%  for ANP, VGLUT-1, and LysoTracker-labeled vesicles, respectively.Next, we examined whether FTY720 also affects the mobility of glutamatergic vesicles (VGLUT1-EGFP) and acidic vesicles (endosomes/lysosomes) labeled by LysoTracker in addition to peptidergic vesicles. As observed before, the mobility of different vesicle types in nontreated astrocytes varied substantially. The ANP vesicles were the fastest vesicles observed (0.31 ± 0.02 μm/s), followed by VGLUT1 (0.19 ± 0.02 μm/s) and then by LysoTracker-labeled vesicles (0.11 ± 0.02 μm/s) that were nearly three times slower than the ANP vesicles. These results are in agreement with the previous reports  and ∼53%  (P < 0.05) and ∼64%  in 3T3-L1 and lactotrophs, respectively. Our results thus suggest that FTY720 acts as a general mobility attenuating agent affecting various vesicles in three different cell types.Furthermore, we examined whether FTY720 affects the mobility of ANP.emd-loaded vesicles in additional two cell types—rat lactotrophs and mouse embryonic fibroblast-adipose-like 3T3-L1 cells. Although the same vesicle type was observed, the spontaneous mobility substantially differed in resting 3T3-L1 cells and cultured lactotrophs  was reduced by ∼52% (P < 0.001) when compared with nontreated cells . A similar outcome was also observed in vesicle MD, where 10 μM sphingosine (0.34 ± 0.02 μm) and 10 μM FTY720 (0.44 ± 0.06 μm) reduced the mean vesicle MD by ∼84% and 80%, respectively, when compared with nontreated cells , while 10 μM FYT720-P and 10 μM thonzonium did not significantly alter vesicle MD  and thonzonium—a membrane impermeable positively charged lipid C. In celTo investigate whether FTY720 differently affects vesicle mobility in the subplasmalemal region and deeper in the cytoplasm, we used total internal reflection fluorescence (TIRF) microscopy. Alternate time-lapse acquisition of TIRF A and epix–y mobility of vesicles, we assessed vesicle movements along the z-axis by analyzing time-resolved average fluorescence intensity of the selected vesicles. In nontreated astrocytes, the vesicles . Treatment of cells with 10 μM FTY720 caused significant diminution of vesicle mobility, by reducing the mean TL in EPI and TIRF plane by 48%  and 37% , respectively, while the corresponding MD diminished by 70%  and 63%   and TIRF imaging G,H. The L-Glu and ATP, which was shown to trigger a strong and Ca2+-dependent exocytotic release of neuropeptide Y from cultured astrocytes  that were not treated or pretreated with sphingosine, FTY720, or thonzonium (n = 10) vesicles apparently discharged cargo within 1 min. This was likely due to spontaneous vesicle fusion . Stimulation of nontreated cells with L-Glu and ATP triggered a substantial (P < 0.001) increase in vesicle cargo discharge . In contrast, the treatment of cells with sphingosine or FTY720 apparently prevented stimulated cargo discharge triggered by L-Glu and ATP; treatment with sphingosine or FTY720 resulted in 7.83% ± 1.58% (n = 4) and 6.97% ± 0.99% (n = 7) responses upon stimulation, respectively. Interestingly, the treatment of cells with thonzonium did not affect stimulated exocytotic release , as the vesicle discharge triggered by L-Glu and ATP was similar to the one observed in nontreated cells.Further, we statistically evaluated the extent of vesicle cargo discharge by counting vesicles before and 1 minute following the stimulation of astrocytes (onzonium A. In addn = 20) and FTY720 treated cells (n = 30) indicated a ∼42% reduction of glutamate release monitored as the regional reduction in the NADH peak fluorescence, which was 4.05 ± 0.32 A.U. (n = 60) vs. 2.35±0.19 A.U. (n = 90) in nontreated versus FTY720-treated cells (P < 0.001), respectively.Finally, we examined whether short-term 10 μM FTY720 treatment affects astrocytic glutamate release measured fluorimetrically , which is inhibited by both, sphingosine (Bazzi and Nelsestuen,2+-regulated exocytosis in astrocytes is ∼2 orders of magnitude slower (Kreft et al.,An interesting finding is that FTY720 and sphingosine inhibit stimulation-dependent vesicle peptide discharge from astrocytes. This is in contrast to neurons and neuroendocrine cells where sphingosine upregulated stimulated exocytotic cargo releases by facilitating the SNARE complex assembly (Darios et al.,An important question is whether the FTY720 effects described here can occur in patients taking this drug. While the therapeutic dose of the drug is expected to yield submicromolar range in the blood plasma (Meno-Tetang et al.,In conclusion, the results of this study have shown that the sphingosine-like molecule FTY720 plays a regulatory role in astrocytic vesicle dynamics, affecting regulated exocytosis of peptides, which was also observed in other cell types studied. We propose that the attenuation of vesicle mobility hinders the delivery of vesicles to the fusion competent sites at the plasma membrane, which may play a role in the therapy of multiple sclerosis and other immunoneurological disorders.


Bixa orellana, against Leishmania amazonensis. Geranylgeraniol was identified through 1H and 13C nuclear magnetic resonance imaging and DEPT. The compound inhibited the promastigote and intracellular amastigote forms, with IC50 of 11 ± 1.0 and 17.5 ± 0.7 μg/mL, respectively. This compound was also more toxic to parasites than to macrophages and did not cause lysis in human blood cells. Morphological and ultrastructural changes induced by geranylgeraniol were observed in the protozoan by electronic microscopy and included mainly mitochondria alterations and an abnormal chromatin condensation in the nucleus. These alterations were confirmed by Rh 123 and TUNEL assays. Additionally, geranylgeraniol induces an increase in superoxide anion production. Collectively, our in vitro studies indicate geranylgeraniol as a selective antileishmanial that appears to be mediated by apoptosis-like cell death.Here we demonstrate the activity of geranylgeraniol, the major bioactive constituent from seeds of  Leishmania genus, endemic in tropical and subtropical regions. Two million new cases of cutaneous and 0.5 million of visceral leishmaniasis are reported each year  . The parasites were incubated for 10 min at room temperature (22°C) and protected from light. After incubation with MitoSOX reagent, the parasites were washed two times with KH buffer and untreated or treated with 11 μg/mL and 30 μg/mL. Antimycin A at 10 μM, a stimulus known to induce superoxide anion (O2∙−) production by mitochondria, was used as positive control. MitoSOX detection was performed using black 96-well plates for 2 h. Fluorescence was measured in a fluorescence microplate reader (Victor X3 - PerkinElmer) at λex = 510 nm and λem = 580 nm [Promastigote forms . For this, promastigote forms (1 × 106 parasites/mL) were incubated in the absence or in the presence of geranylgeraniol at 11 μg/mL for 48 h. The parasites were fixed with paraformaldehyde 1% and subjected to the TUNEL assay as recommended by the manufacturer . Parasites that have undergone DNA double-strand ruptures should fluoresce brightly, unlike the untreated parasites. Fluorescence was observed in a fluorescence microscope Olympus BX51 (Olympus) and pictures were captured with a UC30 camera (Olympus). DNA double-strand ruptures were analyzedP ≤ 0.05 was adopted as the minimum criterion of significance. Data were analyzed with one-way analysis of variance (ANOVA) and significant differences among means were identified with post hoc Tukey testing. Statistical analyses were performed using the program GraphPad Prism 4 . The data shown in the graph was expressed as means ± standard deviation of the mean of independent experiments.  Bixa orellana was identified as geranylgeraniol by 1H-NMR, 13C-NMR and DEPT analyses and the data were compared to data from literature [+ (100); 1H nuclear magnetic resonance (NMR)  internal standard) δ (ppm), 3.72 , 5.09-5.11 , 1.99-2.07 , 1.60 , 1.68 , 1.24 , and 0.94 ; 13C NMR  δ (ppm), 16.19 (C-8′), 16.47 (C-12′), 18.53 (C-16′), 17.87 (C-4′), 26.50 (C-9), 25.89 (C-16), 26.80 (C-13), 26.94 (C-5), 39.75 (C-4), 39.87 (C-8), 39.90 (C-12), 59.52 (C-1), 123.39 (C-2), 123.90 (C-6), 124.35 (C-10), 124.57 (C-14), 131.50 (C-15), 135.18 (C-7), 135.32 (C-11), and 140.09 (C-3). The compound obtained from the seeds ofterature . ElectroB. orellana was tested initially against the promastigote form of L. amazonensis. The results indicated a progressive inhibition in a dependent concentration growth  (Geranylgeraniol obtained from the seeds of n growth . The IC5 (38 μM) .μg/mL of the drug, respectively. Survival indexes of treated amastigotes were significantly (P < 0.05) different from that of non-treated macrophages. The compound showed an IC50 value of 17.5 ± 0.7 μg/mL (60 μM).The effect of geranylgeraniol on intracellular amastigotes was observed after 24 h of incubation . The sur50 for the positive control, amphotericin B, was 0.058 μg/mL (0.06 μM) and 0.26 μg/mL (0.3 μM) against the promastigotes and amastigotes, respectively (data not shown).The ICμg/mL (143 μM) (data not shown). The toxicity for J774G8 macrophages was compared with the activity against the promastigote form, obtaining the selectivity index (SI) (CC50 for J774G8 cells/IC50 for protozoa). Geranylgeraniol was more selective against the promastigotes than the mammalian cells, with an SI ratio of 3.8.This assay evaluated the potential toxic effects of geranylgeraniol on the J774G8 murine macrophages, after 48 h of treatment. When macrophages were treated with geranylgeraniol, the 50% cytotoxic concentration was 41.5 ± 3.5 P < 0.05) of 26.4% ± 6.5 for geranylgeraniol at 500 μg/mL (data not shown). In contrast, the reference drug showed a hemolytic effect of 81.9% ± 7.7 at the same concentration. The hemolytic potentials of geranylgeraniol and the reference drug Amphotericin B were tested and compared with positive (Triton X-100) and negative control (erythrocyte suspension). After 2 h of incubation at 37°C, the results indicated a significantly low hemolysis percentage  with 2 h of incubation, when compared to the control group. The positive control with AA also induced an increase of mitochondria O2∙− production.As shown in μg/mL of geranylgeraniol for 48 h (d) when compared to the control group (untreated parasites) (b).The DNA fragmentation experiment, documented through the DNA labelling, confirmed the electron microscopy results in geranylgeraniol-treated promastigotes. As shown in B. orellana showed antileishmanial activity against L. amazonensis and L. chagasi with an IC50 value of 22 and 250 μg/mL, respectively [B. orellana seeds, against L. amazonensis, in vitro. Geranylgeraniol was identified by NMR and DEPT analyses and the data agree with those reported by Coates et al. [Pterodon pubescens seeds and fruit oil and Croton lobatus leaves [B. orellana contains 57% of geranylgeraniol. Furthermore, the farnesyalcetate was the second compound identified in the hexane extract and it seems that it is a product from geranylgeraniol degradation [Pterodon pubescens [In a previous study, a methanol extract from the seeds of ectively . In the s et al. . This cos leaves . Previouradation . Additioubescens . L. amazonensis and these data are very similar to the activity of geranylgeraniol on T. cruzi [Geranylgeraniol showed alterations in mitochondrial membrane potential (ΔΨm) and fragmentation of DNA of T. cruzi . AdditioLeishmania mitochondria [2∙− production in mitochondria, by MitoSOX assay, similar to the effect of a compound studied by Britta et al. [Based on the results described above, we further investigated the leishmanicidal mechanism of geranylgeraniol. We observed, by electron microscopy, drastic ultrastructural alterations especially in the mitochondria and in the nuclei of the treated parasites. In fact, the mitochondria of Trypanosomatids exhibit unique characteristics that are distinct from mammalian mitochondria, making this organelle a major target of chemotherapeutic agents . Additiochondria –28. Thesa et al. . 2∙− production as soon as treatment began. In contrast, geranylgeraniol induced mitochondrial membrane depolarization after 3 h treatment and only with higher concentration, which was observed in the Rh 123 assay. Our overall hypothesis is that the mechanism of action of geranylgeraniol involves an increase in reactive oxygen species (ROS) that acts in any membrane of the parasite, including the mitochondrial membrane leading to membrane permeabilization followed by mitochondrial depolarization and increase in mitochondrial ROS production through the electron transport chain. Up to here, we observed that geranylgeraniol is able to induce oxidative imbalance, a disorder induced also by promising leishmanicidal compounds [The experiments presented herein showed that geranylgeraniol increased Oompounds , 29. In B. orellana, in L. amazonensis promastigotes is typical of cells undergoing apoptosis-like death [Our results suggest that the type of alterations induced by geranylgeraniol, isolated from the seeds of ke death . Apoptoske death .Leishmania can undergo programmed cell death (PCD) in response to stress and drug application [L. donovani, after treatment with diospyrin and ethanolamide, exhibited depolarization of the ΔΨm and fragmentation of nuclear DNA, characteristics typical of PCD, indicating the initiation of “apoptosis-like death” in the promastigotes [The literature provides a number of examples that protozoa parasites such as lication –34. Theslication . Geranyllication , 36. Prostigotes . This efstigotes , 32, 34.


Cardiac magnetic resonance (CMR) T1 mapping has been used to characterize myocardial diffuse fibrosis. The aim of this study is to determine the reproducibility and sample size of CMR fibrosis measurements for use in clinical trials.A modified Look-Locker with inversion recovery (MOLLI) sequence was used to determine myocardial T1 values pre-, and 12 and 25 min post-administration of a gadolinium-based contrast agent at 3 Tesla. For 24 healthy subjects , two separate scans were obtained a) with a bolus of 0.15 mmol/kg of gadopentate dimeglumine and b) 0.1 mmol/kg of gadobenate dimeglumine, respectively, with averaged of 51±34 days between two scans. Separately, 25 heart failure subjects , were evaluated after a bolus of 0.15mmol/kg of gadopentate dimeglumine. Myocardial partition coefficient (λ) was calculated according to (ΔR1myocardium/ΔR1blood), and ECV was derived from λ by adjusting (1-hematocrit).Mean ECV and λ were both significantly higher in HF subjects than healthy . For healthy subjects, the mean intra-study changes in ECV and λ between 12 and 25 minutes were 0.007±0.006 and 0.012±0.009, respectively. The mean inter-study changes in ECV and λ were 0.006±0.017 and 0.016±0.025, respectively. Thus, the inter-study ECV and λ variation was about 2.8 times greater than the intra-study ECV and λ variation in healthy subjects . In heart failure subjects, the intra-study differences between 12 and 25min ECV and λ were 0.007±0.017 and 0.012±0.028, respectively. The estimated sample size to detect a one standard deviation (SD) change of ECV (0.035) or λ (0.05) with a power of 80% and an alpha error of 0.05 for heart failure subjects using a two group design was 31 and 40 in each group, respectively.ECV and λ quantification have a low variability across scans, and could be a viable tool for evaluating clinical trial outcome. ECV requires a smaller sample size than λ to detect group differences from treatment.National Institutes of Health Intramural program


Marivirga tractuosa (Lewin 1969) Nedashkovskaya et al. 2010 is the type species of the genus Marivirga, which belongs to the family Flammeovirgaceae. Members of this genus are of interest because of their gliding motility. The species is of interest because representative strains show resistance to several antibiotics, including gentamicin, kanamycin, neomycin, polymixin and streptomycin. This is the first complete genome sequence of a member of the family Flammeovirgaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,511,574 bp long chromosome and the 4,916 bp plasmid with their 3,808 protein-coding and 49 RNA genes are a part of the  Genomic  Encyclopedia of  Bacteria and  Archaea  project. T (= DSM 4126 = ATCC 23168 = NBRC 15989) is the type strain of the species Marivirga tractuosa. The genus Marivirga, whose type species is M. tractuosa, contains only one additional species: M. sericea. The generic name ‘Marivirga’ derives from Latin words ‘mare’, the sea and ‘virga’, rod, meaning ‘a rod that inhabits marine environments’ , an, anT shaad zones . A repredatabase  and the database ) were deM. tractuosa H-43T in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome do not differ from the previously published 16S rRNA sequence (AB078072).T are long, slender and flexible rods 0.4-0.5 µm in diameter and 10-50 µm in length or longer  are not produced [et al. reported that the strain does not hydrolyze Tween 20, Tween 40 or Tween 80 [T, while sodium acetate and sodium lactate do not [3- [T is sensitive to ampicillin (10 µg), benzylpenicillin (10 U), carbenicillin (100 µg), chloramphenicol (30 µg), doxycycline (10 µg), erythromycin (15 µg), lincomycin (15 µg), oleandomycin (15 µg) and tetracycline (30 µg), but resistant to gentamicin (10 µg), kanamycin (30 µg), neomycin (30 µg), polymixin (300 U) and streptomycin (30 µg) [et al. [The cells of strain H-43r longer . Strain acterium  that exhoxanthin . In n-he [et al.  found thTween 80 . Acid is [et al.  found onT were iso-C15:0 (36.8%), iso-C15:1 (23.0%) and iso-C17:03-OH (12.2%), with a detailed listing given in Nedashkovskaya et al. [et al. reported fundamentally different observations for strain H-43T, with the C16:0  to be the most important fatty acids in the strain H-43T, whereas iso-C15:0 was not detectable [The predominant cellular fatty acid of the strain H-43tectable . The mai Genomic  Encyclopedia of  Bacteria and  Archaea  project [This organism was selected for sequencing on the basis of its phylogenetic position , and is  project . The gen project  and the M. tractuosa H-43T, DSM 4126, was grown in DSMZ medium 172 (Cytophaga (marine) medium) [et al. [ medium)  at 25°C. [et al. . DNA is  [et al. .The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website . PyroseqGenes were identified using Prodigal  as part The genome consists of a 4,511,574 bp long chromosome with a 35.5% G+C content and a 4,916 bp plasmid with 40% G+C content . Of the


Ten-week-old rats received implantation of HA-, 2% Ag-HA-, or 50% Ag-HA-coated titanium rods. Concentrations of silver in serum, brain, liver, kidneys, and spleen were measured in the acute phase (2–4 days after treatment) and subacute phase (4–12 weeks after treatment). Biochemical and histological examinations of those organs were also performed. Mean serum silver concentration peaked in the acute phase and then gradually decreased. Mean silver concentrations in all examined organs from the 2% Ag-HA coating groups showed no significant differences compared with the HA coating group. No significant differences in mean levels of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, creatinine, or blood urea nitrogen were seen between the three groups and controls. Histological examinations of all organs revealed no abnormal pathologic findings. No acute or subacute toxicity was seen in vivo for 2% Ag-HA coating or HA coating. Ag-HA coatings on implants may represent biologically safe antibacterial biomaterials and may be of value for reducing surgical-site infections related to implantation.To reduce the incidence of implant-associated infection, we previously developed a novel coating technology using hydroxyapatite (HA) containing silver (Ag). This study examined  Orthopedic implants are widely used in many countries to provide pain relief and improve range of motion and quality of life for patients with arthritic disorder. However, one of the serious postoperative complications associated with these procedures is bacterial infection, including surgical-site infection (SSI). The rate of primary infection for joint replacement is between 0.86% and 2.52% according to the National Nosocomial Infections Surveillance System . Infecti in vitro [ Staphylococcus aureus in vivo. Furthermore, the 3% Ag-HA coating has been shown to provide good osteoconductivity and has been comparable with an HA coating and a 50% Ag-HA coating in the inhibition of bone formation because silver toxicity depended on the dose [ in vivo is required.One way to reduce the incidence of implant-related infection is to use implants possessing innate antibacterial properties. Various antibacterial coatings, such as vancomycin , gentamiin vitro . In addiin vitro  and tibiin vitro , we demothe dose . TherefoThe aim of this study was to clarify local and systemic adverse effects of Ag-HA-coated implants in rat tibiae.2O powder  was mixed with HA powder  in concentrations of 2% and 50% and then shaken for 5 min in a plastic bag. The mix was then sprayed onto the surface of the Ti wire using the Frame Sprayed System , which uses an acetylene torch. Temperature of the flame was about 2700°C. This procedure was performed under normal atmosphere. The properties of this coating have been reported by Akiyama et al. [μm thick layer of Ag-HA was uniformly coated on the substrate. Amorphous structure of the Ag-HA coating was confirmed for concentrations ≤50% Ag. Qualitative analysis revealed that HA and Ag-HA coatings contained similar levels of Ca and P, although Ag was not identified. According to quantitative elementary analysis, the Ca/P ratio of the Ag-HA coating (1.74) was almost the same as that of the HA coating (1.67).The implants used were Ti wire 20 mm in length and 1 mm in diameter . Aga et al. . In brieWe prepared three types of implant as follows: (i) HA coating on Ti; (ii) 2% Ag-HA coating on Ti; and (iii) 50% Ag-HA coating on Ti. Implants were packaged singly and sterilized using a JS-8500 gamma sterilizer . The implants were obtained from KYOCERA Medical Materials Corporation .We used 10-week-old male Sprague-Dawley rats  with a mean weight of 382.5 ± 13.7 g. Rats were acclimatized for 5 days prior to use, in a room in which a suitable environment was maintained. All animal procedures were conducted with the approval of the Animal Research Ethics Committee at Saga University . According to their recommendation that the number of experimental animals be kept as low as possible, the sample was limited to three or six rats per group, as the minimum number needed to calculate the mean and standard deviation. ad libitum access to food and water. Clinical parameters and general condition were documented weekly. Body weight was measured after 2, 4, 6, 8, 10, and 12 weeks. Three rats from each group were euthanized at 2, 3, and 4 days after treatment. Six rats from each group were euthanized at 4, 8, and 12 weeks after treatment. The acute phase was defined as 2–4 days after treatment, and the subacute phase was defined as 4–12 weeks after treatment.Rats were anesthetized using a 50 mg/kg intraperitoneal injection of pentobarbital sodium solution prior to the surgical procedure. Both hind legs were shaved, cleaned with povidone-iodine, and dried. One transverse 10 mm incision was made on both knees. To access the medullary cavity, a hole was drilled with an 18-gauge needle through tibial tuberosity, and the same implants were inserted into the medullary cavity of both tibiae in each rat. Rats were divided into three groups according to the implant inserted. They were housed individually withThe abdomen was then opened under general anesthesia and 10 mL of blood was collected from the right common iliac vein. The liver, kidneys, spleen, and brain were obtained after euthanasia. Postmortem examinations included macroscopic inspection of the skin after shaving. The inspection focused on the presence of ash-colored skin, which can occur in argyria (systemic silver intoxication).μm were stained with hematoxylin and eosin. Stained sections from each test sample were then examined under light microscopy for analysis of tissue inflammatory reaction, fibrosis, and the presence of silver sedimentation.Histological examinations used nine rats from each group, with three rats each euthanized at 4, 8, and 12 weeks postoperatively. Histological samples were obtained from the brain, liver, kidneys, and spleen. These organs were fixed in 10% phosphate-buffered formaldehyde and embedded in paraffin wax. Sections of ~5 The concentration of silver in serum was determined by inductively coupled plasma-mass spectrometric analysis (ICP-MS) . In addition, the following parameters were determined: glutamic-oxaloacetic transaminase (GOT); glutamic-pyruvic transaminase (GPT); lactate dehydrogenase (LDH); blood urea nitrogen (BUN); and creatinine. Since no standard values were available for GOT, GPT, LDH, BUN, and creatinine for 10-, 14-, 18-, and 22-week-old Sprague-Dawley rats, these values were determined preoperatively in 20 healthy rats. Standard values were assumed if they ranged between 2.5% and 97.5% percentiles.Organ analysis involved the measurement of concentrations of silver in the brain, liver, kidneys, and spleen. Three rats from each group were euthanized for organ analysis at 2, 3, and 4 days and 4, 8, and 12 weeks postoperatively. These organs were obtained from rats and washed using ultrapure water provided by a Milli-Q water purification system . Concentrations of silver in the brain, liver, kidneys, and spleen were measured using an Agilent 7500c inductively coupled plasma-mass spectrometer (ICP-MS) . post hoc test. Values of P ≤ 0.05 were considered significant.This was performed using Excel and SPSS version 19 software . One-way analysis of variance was used to evaluate body weight, blood serum analysis, and organ analysis. Where this was significant, Tukey's honestly significant difference (HSD) was applied as aMean body weights preoperatively in the HA, 2% Ag-HA coating, and 50% Ag-HA coating groups were 383.1 ± 10.6 g, 382.9 ± 16.4 g, and 381.7 ± 13.0 g, respectively . Mean boNo animals in any of the three groups died during the measurement period. No animals showed any signs of SSI. No animals displayed any signs of local or systemic argyrosis (data not shown).P < 0.01 for all comparisons at all periods, Tukey's HSD test). Mean concentration of silver in the 2% Ag-HA coating group showed no significant difference compared with the HA coating group in any experimental periods.The HA, 2% Ag-HA, and 50% Ag-HA coating groups showed mean serum silver concentrations of 0.60 ± 0.17 ppb, 1.75 ± 1.08 ppb, and 13.8 ± 3.95 ppb at 2 days, respectively, and 0.37 ± 0.06 ppb, 0.95 ± 0.63 ppb, and 16.2 ± 5.85 ppb at 3 days, respectively . The silLiver function was assessed by determination of GOT, GPT, and LDH levels. Kidney function was assessed by measurement of creatinine and BUN levels.Mean GOT, GPT, LDH, creatinine, and BUN of the HA, 2% Ag-HA, and 50% Ag-HA coating groups were not significantly higher than in controls at 2, 3, and 4 days .At 4 weeks, mean GOT differed significantly between the control and HA coating groups, but mean GPT and LDH in the HA coating group were not significantly different from that in the control group . No signμg/g, 0.01 ± 0.01 μg/g, and 0.06 ± 0.02 μg/g, respectively . No significant difference was apparent between HA and 2% Ag-HA coating groups at 2, 3, and 4 days.Mean silver concentrations in the brain for the HA, 2% Ag-HA, and 50% Ag-HA coating groups at 2 days were 0.02 ± 0.00 ectively . At 3 daμg/g, 0.02 ± 0.00 μg/g, and 0.05 ± 0.01 μg/g, respectively. At 8 weeks, mean silver concentrations in the brain for HA, 2% Ag-HA, and 50% Ag-HA coating groups were 0.01 ± 0.01 μg/g, 0.01 ± 0.00 μg/g, and 0.05 ± 0.02 μg/g, respectively. The values in the 50% Ag-HA coating group differed significantly from those in the HA and 2% Ag-HA coating groups at 4 and 8 weeks . No significant differences were found between the HA and 2% Ag-HA coating groups at 4 and 8 weeks. At 12 weeks, mean silver concentrations in the brain for HA, 2% Ag-HA, and 50% Ag-HA coating groups were 0.01 ± 0.01 μg/g, 0.01 ± 0.01 μg/g, and 0.05 ± 0.03 μg/g, respectively. No significant differences were found between the three groups.Furthermore, mean silver concentrations in the brain for HA, 2% Ag-HA, and 50% Ag-HA coating groups at 4 weeks were 0.02 ± 0.01 μg/g, 0.01 ± 0.00 μg/g, and 0.04 ± 0.02 μg/g, respectively . No significant differences between the HA and 2% Ag-HA coating groups were seen at 4 and 8 weeks. At 12 weeks, mean silver concentrations in the HA, 2% Ag-HA, and 50% Ag-HA coating groups were 0.01 ± 0.01 μg/g, 0.02 ± 0.01 μg/g, and 0.02 ± 0.02 μg/g, respectively. No significant differences were found between the three groups.Mean silver concentrations in the liver for the HA and 2% Ag-HA coating groups were 0.01 ± 0.00 μg/g, 0.01 ± 0.00 μg/g, and 0.01 ± 0.00 μg/g, respectively . No significant differences were found between the three groups at 12 weeks.The highest silver concentrations were found in the kidneys of the 50% Ag-HA coating group at 4 weeks, with a mean concentration of 0.04 ± 0.01 μg/g, 0.03 ± 0.01 μg/g, and 0.10 ± 0.04 μg/g, respectively . No significant differences were found between the HA and 2% Ag-HA coating groups at 2, 3, or 4 days.Mean silver concentrations in the spleen for the HA, 2% Ag-HA, and 50% Ag-HA coating groups at 2 days were 0.01 ± 0.00 ectively . At 3 daμg/g. The concentration of Ag decreased gradually over the experimental period. Mean silver concentration for the 50% Ag-HA coating group at 8 weeks was 0.20 ± 0.13 μg/g. Silver concentration for the HA and 2% Ag-HA coating groups was 0.01 ± 0.00 μg/g at 4 and 8 weeks. At 12 weeks, mean silver concentrations for the HA, 2% Ag-HA, and 50% Ag-HA coating groups were 0.03 ± 0.03 μg/g, 0.01 ± 0.01 μg/g, and 0.08 ± 0.05 μg/g, respectively. The value in the 50% Ag-HA coating group differed significantly from those in the HA and 2% Ag-HA coating groups at 4 and 8 weeks . No significant differences were found between the three groups at 12 weeks.The highest silver concentrations were found in the spleen for the 50% Ag-HA coating group at 4 weeks, with a mean concentration of 0.21 ± 0.05 Although data are not shown, growth of fibrous tissue, chronic inflammatory infiltrates, foreign body granulomas, and fatty degeneration was not detected in the brain, liver, kidneys, or spleen of the HA, 2% Ag-HA, and 50% Ag-HA coating groups at any time during the experimental period. Histological examination of organs revealed no abnormal pathological findings. in vitro [ in vivo toxicity of Ag-HA. Our results shown here suggest that no acute or subacute toxicity occurs in vivo with 2% Ag-HA coating as well as HA coating. This is the first report about the in vivo toxicity of Ag-HA.Silver has a broad antibacterial spectrum, strong antibacterial activity, and low toxicity. Devices such as heart valves , centralin vitro . Howeverμg/cm2. The total amount of silver necessary for the 2% Ag-HA coating on femoral replacements in humans  is, therefore, about 1.14 mg. The small amount of silver used here would not cause argyria.Wan et al. reported that silver levels below 200 ppb in blood could be considered normal, because humans incorporate orally each day small amounts of silver . Only smA concentration of silver in the blood of more than 300 ppb has been reported to cause argyria, argyrosis, and liver and kidney damage . Drake aμg/g, 0.05 μg/g, 0.04 μg/g, and 0.21 μg/g, respectively. The concentration of silver decreased gradually over the experimental period, and no significant differences were found between the three groups at 12 weeks, with neither pathological changes in laboratory parameters nor histological changes in tissues. We reported in a previous study that silver toxicity depends on the dose [In this study, mean silver concentrations in all analyzed organs of the 2% Ag-HA coating group showed no significant differences compared with those in the HA coating group at all experimental periods. The silver concentration of 50% Ag-HA was significantly elevated in all analyzed organs. The highest silver concentrations were found in the brain, liver, kidneys, and spleen of the 50% Ag-HA coating group at 4 weeks, with mean concentrations of 0.05 2O powder mixed with HA powder of around 2-3% appear appropriate. Ag-HA coating on an implant may offer a biologically safe antibacterial biomaterial and may be of value in reducing SSI related to implantation.We have shown that the use of 2% and 50% Ag-HA-coated implants is associated with neither acute nor subacute toxicity in the rat tibia. However, data about chronic toxicity are still missing. Since the 50% Ag-HA coating inhibits bone formation , silver


In animal models of systemic inflammation, the endogenous nucleoside adenosine controls inflammation and prevents organ injury. Dipyridamole blocks the cellular uptake of endogenous adenosine and increases the extracellular adenosine concentration. We studied the effects of oral dipyridamole treatment on innate immunity and organ injury during human experimental endotoxemia.Escherichia coli endotoxin  intravenously after 7-day pretreatment with dipyridamole, 200 mg slow release twice daily, or placebo.In a randomized double-blind placebo-controlled study, 20 healthy male subjects received 2 ng/kg P < 0.0001), and the circulating endogenous adenosine concentration was increased. Treatment with dipyridamole augmented the LPS-induced increase in the antiinflammatory cytokine interleukin (IL)-10 with 274%, and resulted in a more rapid decrease in proinflammatory cytokines tumor necrosis factor-α (TNF-α) and IL-6 levels directly after their peak level . A strong correlation was found between the plasma dipyridamole concentration and the adenosine concentration , and between the adenosine concentration and the IL-10 concentration , and the subsequent decrease in TNF-α . Dipyridamole treatment did not affect the LPS-induced endothelial dysfunction or renal injury during experimental endotoxemia.Nucleoside transporter activity on circulating erythrocytes was reduced by dipyridamole with 89% ± 2% :  During sepsis, unopposed and prolonged activation of the innate immune system can induce significant collateral damage to host tissues, resulting in a high mortality rate. During inflammation, the extracellular concentration of the purine nucleoside adenosine rapidly increases -3. SubseDipyridamole blocks the equilibrative nucleoside transporter (ENT), which facilitates the transmembranous diffusion of adenosine Figure . DipyridWe hypothesized that dipyridamole may ameliorate the excessive and prolonged activation of the immune response that can occur during systemic inflammation. Therefore, in a proof-of-concept study in healthy volunteers, we examined whether pretreatment with dipyridamole curtails the activation of the innate immune system during experimental endotoxemia and prevents  organ damage.http://www.clinicaltrials.gov, NCT01091571). After signing for informed consent, 20 healthy male volunteers participated. Because of significant differences in the innate immune response between male and female subjects during experimental endotoxemia, we included only male subjects [n = 10) or placebo , based on the fact that a steady state occurs after 3 days, and previous studies that examined the effects of dipyridamole during ischemia were also performed after 7 days of treatment [This study was approved by the local ethics committee and registered ) was administered as a bolus infusion in 1 minute (2 ng/kg) at t = 0 hours, after vortex mixing for 30 minutes. The protocol is illustrated in Figure After local anesthesia, the brachial artery of the nondominant arm was cannulated for blood pressure monitoring, blood sampling, and administration of vasoactive drugs . A secon1 minute  ng/kg atPlasma caffeine and dipyridamole concentrations were determined by reversed-phase high-power liquid chromatography (HPLC) . CirculaForearm blood flow  was measured 2 hours before and 4 hours after LPS administration with venous occlusion plethysmography  . The vasAcetylcholine  and norepinephrine  were dissolved in normal saline, and nitroprusside  was dissolved in a 5% glucose solution. All solutions were freshly prepared at the day of the experiment.S-transferase (GST) alpha (A1-1) and pi (P1-1), as markers of proximal and distal tubule injury, respectively, were measured [Subjects collected their morning urine before treatment with dipyridamole or placebo and on the day of the LPS experiment. After start of the LPS infusion, urine was collected in four 3-hour periods and during a period of 12 to 24 hours . Further to substantiate the possible mechanism of action of dipyridamole, Pearson correlations were conducted to explore the correlation between plasma levels of dipyridamole, adenosine, and cytokines. The lines were calculated from linear regression analyses.The effect of dipyridamole was analyzed by using a repeated measures analysis of variance (ANOVA), with t test. The effect of endotoxemia on FRAP was tested by using a repeated measures ANOVA. Because data had a gaussian distribution, data are expressed as mean ± SEM, unless specified otherwise. Nonparametric data are illustrated as box-and-whiskers. A P value < 0.05 was considered statistically significant.The area under the curve (AUC) of the increase in FBF was calculated (before and after LPS administration). The LPS-mediated difference was compared between groups by using an unpaired Student The demographic characteristics were comparable between groups Table . Plasma t = 0) averaged 1.8 ± 0.3 and 0.0 ± 0.0 mg/L for the dipyridamole and placebo groups, respectively.The plasma dipyridamole concentration at the moment of LPS administration (9 erythrocytes/min at baseline to 11 ± 2 nmol/109 erythrocytes/min immediately before the LPS experiment (P < 0.0001). In placebo-treated subjects, uridine transport was 112 ± 7 nmol/109 erythrocytes/min at baseline and 124 ± 7 nmol/109 erythrocytes/min immediately before the LPS experiment (P = 0.86).Uridine uptake into the erythrocyte via the ENT was profoundly inhibited by dipyridamole: from 113 ± 9 nmol/10P < 0.01). The adenosine concentration further increased with 2.1 ± 2.8 and 2.1 ± 0.9 nmol/ml after administration of LPS in both groups . Dipyridamole concentrations correlated strongly with peak adenosine concentrations . Dipyridamole-treated subjects had significantly higher amounts of circulating monocytes in the period of 4 to 8 hours after LPS, with a peak at 8 hours after LPS administration . The increase in body temperature after administration of LPS was similar in the dipyridamole and placebo groups; from 36.5°C ± 0.1°C to 38.0°C ± 0.2°C and from 36.4°C ± 0.1°C to 38.2°C ± 0.1°C, respectively (P = 0.76 between groups).During the first hour after LPS administration, the total white blood cell count decreased from 6.2 ± 0.3 to 2.2 ± 0.3 × 10P < 0.0001 compared with the placebo group; Figure r = 0.88; P < 0.0001), as illustrated in Figure P < 0.05 and < 0.01, respectively; Figure r = -0.54; P = 0.02), but not with that of IL-6 , Figure Dipyridamole treatment augmented the IL-10 response during endotoxemia . This baseline difference disappeared after LPS administration: 7.3 ± 0.8 in the dipyridamole group versus 8.6 ± 1.2 ml/min/dl in the placebo group. No significant changes in FBF were found in the noninfused forearm during the intrabrachial infusions of acetylcholine, nitroprusside, and norepinephrine, excluding systemic hemodynamic effects of these drugs. As shown in Figure Subjects treated with dipyridamole had a lower baseline FBF ; VCAM: dipyridamole, 19.1 ± 1.2 × 104 pg/ml versus placebo, 16.5 ± 0.7 × 104 pg/ml (P = 0.08)). Both ICAM and VCAM levels increased after LPS administration (P < 0.0001), but dipyridamole treatment did not affect the endotoxemia-induced increase in ICAM and VCAM levels .Baseline plasma levels of ICAM and VCAM tended to be higher in dipyridamole-treated subjects . No significant difference in FRAP was found between both groups .Endotoxemia resulted in a cumulative GSTA1-1 excretion of 11.2 (6.2 to 13.0) μg compared with 5.1 (3.9 to 9.4) μg 12 hours after LPS administration in dipyridamole- and placebo-treated subjects, respectively. Cumulative GSTP1-1excretion was 6.4 (5.4 to 7.8) μg and 6.3 (4.5 to 8.0) μg, respectively. No differences were seen between the LPS-induced increase between both groups .