Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays.The main goal of the present study was to set up an axenic cultivation of Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity (0.5 M and 1 M) of non-nutrient agar plates were performed. One hundred fifty µl of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 µl of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37°C and 42 °C. All plates were monitored for 24 h, 48 h and 72 h.Sixteen Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature (42 °C) and osmolarity (1.5 M) and thus they were classified as weak pathogens.Overall, 16 strains of Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.Most of T4 genotypes are highly pathogenic organisms. This is an important finding since  Acanthamoeba are the ubiquities free-livingamebas with highly resistance to harsh conditions such as high osmolarity, extreme pH and high temperature (Acanthamoeba were divided into 17 different genotypes (T1-T17) based on Diagnostic Fragment 3 of 18S rRNA gene  medium (ATCC 712 PYG). This medium contained 0.4 M MgSO4, 7 H2O (10.0 ml), 0.05 M CaCl2 (8.0 ml), 0.1 M Sodium citrate, 2 H2O (34.0 ml), 0.005 M Fe (NH4)2(SO4)2, 6H2O (10.0 ml), 0.25 M Na2HPO4, 7 H2O (10.0 ml),0.25 M KH2PO4 (10.0 ml) and the pH was adjusted to 6.5 using KOH. Finally, the mentioned medium was supplemented with 2 M glucose (filter-sterilized) and was added aseptically to the solution (50.0 ml). Four isolates were cultivate axenic using TYI-S-33 medium which include glass-distilled water (100 ml), 0.1 g of potassium phosphate, dibasic; 0.06 g of potassium phosphate, monobasic; 0.2 g of sodium chloride; 0.2 g of casein digest peptone; 2 g of yeast extract; 1 g of glucose; 0.1 g of L-cysteine hydrochloride; 0.1 g of ascorbic acid; and 0.0023 g of ferric ammonium citrate  from axenic culture were divided into new culturemediums. Cultures were incubated at 37 °C and 42 °C for 24 h, 48 h and 72 h. Growth of Acanthamoeba isolates previously genotyped as T4 were cultured axenically after several months. Indeed, the amoebas did not adapted to PYG medium readily. After adaptation of amoebas to PYG medium a large amount of trophozoites were achieved. Four isolate adapted to TYI-S-33 medium as mentioned above. Thermotolerance examination revealed that all of clinical isolates can grow at high temperatures including 37 °C and 42 °C. Soil and animal feces strains also were classified as highly pathogen by thermotolerance assays. It should be noted that two dust and five water strains have not grown at 42 °C and these isolates were classified as weakly pathogenic strains . Based on aforementioned describe, Khan et al. set up a simple plating assay for identification of pathogenic Acanthamoeba from non-pathogenic strains (Acanthamoeba strains to variable osmolarity and temperature (Acanthamoeba. These tests were time consuming and expensive (Previous investigations showed that pathogenic stresses , 11. Thieratitis . One exp strains . This plperature , 5. PrevAcanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba in environmental sources may pose a risk within susceptible people.Most of T4 genotypes are highly pathogenic. This is an important finding since


P < 0.001) associated with plant height were detected 13 times in the population with 118 accessions. Three loci  were identified in three, two and two periods of plant height growth, respectively. Markers Xbarc168-2D, Xgwm249-2D, Xwmc349-4B, Xcfd23-4D and Xgwm410-5A located at or near additive QTL regions in the DH population proved to coincide with known Rht loci. The results showed a consistency between linkage analysis and association mapping, and also confirmed the value of fine mapping of QTL through combined linkage and association analyses. For final plant height, the alleles Xgwm11-1B208, Xwmc349-4B103 and Xcfd23-4D202 exhibited negative effects, i.e. reducing plant height; Xwmc349-4B101 and Xcfd23-4D205 showed significant positive effects. A second larger population (262 accessions) was used to validate the effects of these large-effect alleles and the efficacy of pyramiding in eight environments (year × site × water regime combinations). Strong correlations between final plant height and numbers of large-effect alleles indicated that the alleles contributed additively to plant height. The additive effects showed that pyramiding elite alleles for target traits has significant potential for wheat breeding.Plant height is an important botanical feature closely related to yield. Two populations consisting of 118 and 262 accessions respectively were used to identify elite alleles for plant height and to validate their allelic effects. Plant height was measured from the early booting to the flowering stages. Simple sequence repeat markers for candidate quantitative trait locus (QTL) regions with large effects identified in a doubled haploid (DH) population (Hanxuan 10 × Lumai 14) were selected for further verification by association analysis. Nine loci significantly (The online version of this article (doi:10.1007/s11032-013-9873-5) contains supplementary material, which is available to authorized users. Rht genes with major effects have been described  and Rht-D1b (Rht2), carried by Norin 10, and Rht8 are widely used in wheat breeding programs; varieties with one or more of these genes accounted for >70 % of current commercial wheat cultivars worldwide  to t, quantitative genetic effects can be revealed at specific stages excluding the effect of previous times (t − 1). Plant height is not only easily measured, but also undergoes obvious changes along with plant development. It is therefore a popular model trait for the study of quantitative gene expression in developmental genetics  associated with plant height were detected on almost all 21 chromosomes by linkage analysis and association mapping  is a breeding approach increasingly adopted to eliminate defects in elite breeding lines . Plant height was measured from the early booting to the flowering stages and selection of simple sequence repeat (SSR) markers was based on candidate QTL detected in the previous research. The purpose was to (1) verify large-effect QTL previously detected in the DH population and further identify markers closely linked to plant height; (2) identify the large-effect alleles of associated loci; and (3) assess the efficacy of pyramiding large-effect alleles with two populations in nine environments.Two hexaploid winter wheat populations were used as the plant materials. Population 1 (118 entries) was used for conditional and unconditional association mapping of plant height. Population 2 (262 accessions) was used to validate the large-effect alleles of associated loci. All materials were sown at the beginning of October and harvested in the following mid-June. Each experimental unit was a two-row plot of 2 and 0.3 m between two rows, with 40 seeds planted per row.2-3F2-5 progenies of the accessions (Supplementary Table S1). The recurrent parents were Jinmai 47, Lumai 14 and Yumai 18. The accessions were planted in Changping, Beijing  in 2009 (named environment E0). These materials were managed under natural rainfall conditions of 192 mm during the growing season.Population 1 consisted of 50 accessions and 68 homozygous lines with uniform plant height derived from BC3/ha at the pre-overwintering, jointing, flowering and grain filling stages. E1, E2, E3, E4, E5, E6, E7 and E8 indicate the environments of Changping in 2009 under DS, Changping in 2009 under WW, Shunyi in 2009 under DS, Shunyi in 2009 under WW, Changping in 2010 under DS, Changping in 2010 under WW, Shunyi in 2010 under DS, and Shunyi in 2010 under WW, respectively.Population 2 consisted of 262 accessions (Supplementary Table S2), of which 254 were from China, three from USA, two from Australia, two from Italy, and one from Romania, including 209 modern varieties, 43 advanced lines and 10 landraces . For conditional plant heights, 1) revealed the net genetic effects of genes expressed during the first two stages (S1 and S2), and likewise for 2) and 3). All the phenotypic analyses were carried out using SAS V8.1 software.Plant height was measured every 7 days from the early booting stage until flowering, a total of four measurements, designated S Zhu Zhu , conditihttp://www.appliedbiosystems.com.cn/). The allele number, allele frequency and polymorphism information content (PIC) were calculated by PowerMarker V3.25 software  was set from 2 to 9 with a burn-in period length of 50,000 iterations and a run of 500,000 replications of Markov Chain Monte Carlo (MCMC) after burn-in. Each K was duplicated five times. The admixture model of STRUCTURE allowed for a population mixture and correlated allele frequencies. The most appropriate K value was evaluated by lnP(D) in the STRUCTURE output  for significant association between markers and traits was 0.001. The phenotypic variance explained (PVE) for each significantly associated locus was evaluated by R2 values for the markers  in TASSEL 2.1  were 9.2, 6.8 and 10.2 %, respectively (Supplementary Table S5). The high values of CV at various growth periods indicated wide phenotypic variation among accessions, which was suitable for association analysis. Compared to PH1 and PH2, the higher CV of PH3 showed that the third growth period  at the flowering stage in Population 1 was 82.4 cm, ranging from 60.0 to 118.0 cm, with a coefficient of variation (CV) of 16.0 %. The CVs of conditional plant height at the three growth periods (PHP(D) value for each K (from 2 to 9) is visualized in Supplementary Fig. S1a and the inflection point appeared at K = 3 (marked with an asterisk). In addition, the second-order likelihood (ΔK) was also calculated. We found that the highest ΔK value occurred at K = 3, and was much less at K = 4–8 . According to lnP(D) and ΔK, Population 1 was classified into three subpopulations, containing 52, 25 and 41 accessions, respectively .A total of 422 alleles were detected by 58 SSR markers in Population 1. The numbers of alleles per locus varied from 3 to 16, and PIC ranged from 0.033 to 0.818, with averages of 7 and 0.519, respectively. A key issue for association mapping is estimation of population structure, which can result in spurious associations between phenotypes and markers. In previous research, about 60 traits were measured at up to 10 environments in the diverse maize germplasm set of 302 inbred lines. Overall, population structure accounts for an average of 9.3 % of the phenotypic variation  associated with plant height were detected 13 times  were identified in three, two and two periods of plant height growth, respectively. The phenotypic variation explained (PVE) ranged from 14.06 to 28.34 %  was responsible for 14.06 % of the variation in plant height. Two loci on chromosomes 1B and 4B were significantly associated with PH2, with PVEs of 23.55 and 15.55 %, respectively. Seven loci identified in PH3 were distributed on chromosomes 1B, 2D, 4B, 4D, 5A and 7B. Of them, Xgwm213-7B showed the largest effect, explaining 28.34 % of the phenotypic variation. At the flowering stage, Xgwm11-1B, Xwmc349-4B and Xcfd23-4D were significantly associated with the plant height. Of them, Xcfd23-4D, accounting for 25.82 % of the total variation, displayed the largest effect on plant height. Xgwm11-1B was identified in three periods, with PVEs of 23.55, 18.24 and 19.34 %, respectively (Table Marker Xgwm11-1B208 and Xwmc349-4B101 showed the largest negative (−10.7 cm) and positive (15.1 cm) effects on plant height, respectively. Shorter plant height (74.1 cm) was associated with Xgwm11-1B208 (13 accessions), whereas 12 accessions with Xwmc349-4B101 were clearly taller (97.5 cm) at flowering. As with previous reports, alleles at one locus could have opposite functions at the same stage  contributed to shorter plants (77.4 cm), whereas nine accessions with the 189-bp allele (positive effect 9.5 cm) contributed to taller plants (89.5 cm). At flowering, 55 accessions carrying the allele Xcfd23-4D202 with a negative effect (−4.2 cm) produced shorter plants (78.1 cm), and 50 accessions with a positive effect (5.1 cm) from the 205-bp allele were taller (87.5 cm).Detecting loci associated with dynamic developmental plant height was not the final objective, but was a prerequisite for its application. It is more significant to search for elite alleles, a major advantage of association mapping compared to biparental family-based linkage mapping. Allelic effects were estimated through comparison with the “null allele” for each associated locus  were significantly (P < 0.05) or highly significantly  shorter or taller than those without the large-effect alleles in almost all environments  in the second period (2), but in contrast the effect of the same allele was 1.4 cm in the third period (3). This further indicated that (1) gene expression occurs selectively (even with opposite effects) at different developmental stages; and (2) several genes with opposite genetic effects may express simultaneously in the same or adjacent genomic regions. However, unconditional analysis considering only final plant height in this research perhaps unavoidably excluded some loci associated with plant height development. This may be one reason why ten loci were detected by conditional analysis, whereas only three loci were detected for final plant height , much more than the number of loci found associated with PH1 and PH2, showing that genes controlling plant height were actively expressed in the third period. Thus, final plant height is the accumulation of genetic effects that occur during the whole growth period, and conditional analysis detects the net effects of gene expression at specific stages.There were one, two and seven loci significantly associated with PHQPh.cgb-1B.1, was detected in the DH population. The genetic distance between the flanking markers (Xgwm582–Xgwm273) was 3.9 cM in Ta-SSR-2004  and Xgwm11-1B, in the same region, were detected by association mapping in our research, and the genetic distance between them was only 0.7 cM. Some other additive QTL were mapped at known Rht loci, such as QPh.cgb-2D.1 which maps to Rht8, QPh.cgb-4D.1 to Rht2 or Rht10, and QPh.cgb-5A.6 to Rht12 , Xcfd23-4D (1.6 cM near to one flanking marker of QPh.cgb-4D.1), and Xgwm410-5A (one of the flanking markers of QPh.cgb-5A.6) were detected in our research. Xwmc349-4B is closely linked with Rht-B1 and Xgwm410-5A is linked with Rht12 at a distance of 11.0 cM  can provide high resolution that may contribute to the full use of a potentially large range of allelic variation in natural populations  , but showed extremely tall final plant heights (118 cm and 108 cm) compared to the average of all accessions with Xwmc349-4B103 in Population 1 in E0 (data not provided). Rare individuals like Jimai 32 and Jinguang can hide the effect of Xwmc349-4B103 in small populations, but cannot hide the effect in large populations. Therefore, it is easy for us to understand why no significant difference in plant height was detected between accessions carrying Xwmc349-4B103 and others in E0 in Population 1 (118 accessions), but there was extreme significant difference across E1–E8 in Population 2  how many previously reported QTL could be repeatedly detected or verified, and 2) are there enough elite alleles in germplasm resources? It has been proposed that the narrow genetic base of modern crop cultivars is a major problem for further improvement of crop productivity  in Population 1 (XLS 30 kb)Supplementary Table S2262 accessions of Population 2 and their origins (XLS 53 kb)Supplementary Table S3Candidate SSR markers for association mapping in Population 1 (XLS 26 kb)Supplementary Table S4SSR markers for evaluating population structure of Population 1 (XLS 26 kb)Supplementary Table S5Conditional phenotypic values for plant height of Population 1 during three growth periods and unconditional plant height at flowering (XLS 24 kb)Supplementary Table S6Unconditional plant height of 262 accessions at maturity stage (XLS 24 kb)Supplementary Fig. S1P(D) and ΔK over five repeats of STRUCTURE analysis; c: Structure analysis revealed three sub-populations. Each accession is represented by a vertical bar, and the colored segments within each bar reveal the proportion of each subpopulation (PPT 859 kb)Population structure of Population 1 based on 29 genome-wide SSR markers. a, b: Population structure as determined by lnBelow is the link to the electronic supplementary material.


Inhibition of the activation of the ASK1-MKK4/7-JNK stress signaling pathway and cleaved caspase-3 induced by oxidative stress were involved in the neuroprotection afforded by 20E. In addition, 20E reduced the expression of iNOS protein by inhibition of NF-κB activation. The neuroprotective effect of 20E was also confirmed in vivo. 20E significantly decreased infarct volume and the neurological deficit score, restored antioxidant potential and inhibited the increase in MDA and TUNEL-positive and cleaved caspase-3-positive cells in the cerebral cortex in MCAO rats. Together, these results support that 20E protects against cerebral ischemia injury by inhibiting ROS/RNS production and modulating oxidative stress-induced signal transduction pathways.Oxidative stress plays an important role in the pathological processes of ischemic brain damage. Many antioxidants have been shown to protect against cerebral ischemia injury by inhibiting oxidative stress both in vitro and in vivo. 20-Hydroxyecdysone (20E), an ecdysteroid hormone, exhibits antioxidative effects. For the work described in this paper, we used an in vitro oxidative damage model and an in vivo ischemic model of middle cerebral artery occlusion (MCAO) to investigate the neuroprotective effects of 20E and the mechanisms related to these effects. Treatment of cells with H It is characterised by the disruption of cerebral blood flow and lack of oxygen to the affected area. Brain damage following cerebral ischemia develops from a complex series of pathophysiological events that evolve in time and space. The processes of excitotoxicity, peri-infarct depolarisation, inflammation, and apoptosis within the ischemic penumbra are proposed 2O2) insult and to uncover the mechanisms behind 20E's neuroprotective action. In addition, we observed the neuroprotective effects of 20E in rats with ischemia following middle cerebral artery occlusion (MCAO). Our results demonstrate that 20E protected against oxidative stress-induced neuronal injury in vitro and brain ischemic insult in vivo. Therefore, we suggest that 20E could be considered a potential protective agent for the treatment of brain ischemic insult.20-hydroxyecdysone (20E) is a polyhydroxylated steroid invertebrate hormone found in insects and few plants 2O2), propidium iodide (PI), N-([3-(aminomethyl)phenyl]methyl) ethanimidamide dihydrochloride (1400W), pyrrolidine dithiocarbamate (PDTC), 3--2,5-diphenyltetrazolium bromide (MTT), and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma Chemical Co. . Dichlorofluorescin diacetate (DCF-DA), 5,5′,6,6′-tetrachloro-1, 1′,3,3′-tetraethylbenzimidazol carbocyanine iodide (JC-1) and Fluo-3 AM were purchased from Molecular Probes . The in situ cell death detection kit was purchased from Roche Diagnostics . The reagent kits for determining lactate dehydrogenase (LDH) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Institute of Biological Engineering . The reagent kits for determining total antioxidant capability (T-AOC), superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity level and level of nitric oxide (NO) in the culture media, bicinchoninic acid kit for protein determination (BCA kit), anti-mouse IgG horseradish peroxidase (HRP) and anti-rabbit IgG-HRP were purchased from the Beyotime Institute of Biotechnology . Rabbit monoclonal antibodies against cleaved caspase-3 and rabbit polyclonal antibodies against p-NF-κB (p65 subunit), p-ASK1, MKK4, p-MKK4, MKK7 and p-MKK7 were purchased from Cell Signal Technology, Inc. . Rabbit polyclonal antibodies against ASK1 and iNOS and mouse monoclonal antibodies against p-JNK, JNK and p- IκBα were purchased from Santa Cruz Biotechnology .20-Hydroxyecdysone (20E) was purchased from Tauto Biotech Co., Ltd . Fetal bovine serum and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco . Hydrogen peroxide  were cultured in DMEM containing 10% fetal bovine serum at 37°C in a water-saturated atmosphere with 5% CO2O2 alone for 12 h or different concentrations of 20E alone for 24 h or were pretreated with different concentrations of 20E before the onset of H2O2 treatment. After these treatments, MTT was added to the culture medium at a final concentration of 0.5 mg/mL and incubated at 37°C for 4 h. Then the medium was discarded, and DMSO was added to solubilize the formazan reaction product with shaking for 5 min. The optical density (OD) was spectrophotometrically measured at 490 nm using a microplate reader  with DMSO as the blank. Cell viability of the control group not exposed to either H2O2 or 20E was defined as 100%. Other groups' cell viabilities were expressed as a percentage of the control.An MTT assay was used to estimate cell viability. MTT assays are based on the conversion of MTT to formazan crystals via mitochondrial dehydrogenases in viable cells. In brief, B35 cells were exposed to different concentrations of HIt has been previously established that lactate dehydrogenase (LDH) release correlates linearly with the number of damaged cells after toxic insult A TUNEL assay was used to evaluate cell apoptosis. TUNEL is a common method for detecting the DNA fragmentation that results from cell apoptosis. For the in vitro experiment, the cells were treated as described for measurement of LDH release. Then the cells were fixed in 4.0% paraformaldehyde for 20 min at room temperature and washed three times in PBS buffer. Fixed cells were subjected to a TUNEL assay via an in situ cell death detection kit according to the manufacturer's specifications. Finally, these cells were stained with PI dye at room temperature for 10 min. The stained cells were washed three times with PBS. The results were visualized using confocal laser microscopy (Leica TCS). A standard fluorescein filter set was used to visualize the green fluorescence of fluorescein at 530 nm and the red fluorescence of PI at 620 nm. The cells with green fluorescence and the cells with red fluorescence were counted to quantify the apoptotic process. Data are expressed as the ratio of TUNEL-positive cells to total cells. In the in vivo experiments, the rats were treated as described for the ischemic model and drug administration. The TUNEL assay was performed to reveal apoptosis-induced DNA fragmentation in brain paraffin sections of experimental rats. In brief, the sections were deparaffinized with xylene, hydrated in serially-diluted ethanol, and then treated with protease K (20 µg/mL) for 15 min at room temperature. Reaction buffer containing terminal deoxynucleotidyl transferase was directly applied to tissue sections and incubated for 1 h at 37°C. After being washed with PBS, the sections were incubated with anti-digoxigenin peroxidase conjugate for 30 min at room temperature and subsequently developed color in peroxidase substrate. The nuclei were counterstained with hematoxylin. A TUNEL-positive cell was indicated by brown coloration. The total cell number and the number of TUNEL-positive cells were determined in five areas of the cerebral cortex in every section using a square micrometer eyepiece at 200× magnification. Five sections were selected from every experimental rat. Data are expressed as the ratio of TUNEL-positive cells to total cells.To confirm the results of the TUNEL assay, immunocytochemistry stain for cleaved caspase-3 was used. The cells were treated as described for the TUNEL assay. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed three times in PBS buffer. Fixed cells were blocked by incubation in PBS with 5% normal goat serum and 0.5% triton X-100 at room temperature for 30 min. Then the cells were incubated with anti-cleaved caspase-3 antibody overnight at 4°C. After cells were washed three times with PBS, they were incubated with red fluorescent secondary antibody (cy3) for 1 h at 37°C. Finally, these cells were stained with DAPI dye at room temperature for 10 min. The stained cells were washed three times with PBS. The results were visualized using confocal laser microscopy (Leica TCS). A standard fluorescein filter set was used to visualize the blue fluorescence of DAPI at 488 nm and the red fluorescence of cy3 at 570 nm. The cells with blue fluorescence and the cells with red fluorescence were counted to quantify the apoptotic process. Data are expressed as the ratio of cleaved caspase-3-positive cells to total cells.2O2 for 12 h. The treated cells were loaded with 30 µM DCF-DA. After the cells were incubated at 37°C for 30 min, the dye was removed, and the cells were washed three times with PBS. Finally, the results were visualized using confocal laser microscopy (Leica TCS SP5). Alternatively, fluorescence intensity was monitored using flow cytometry (BD Immunocytometry Systems).The fluorescent probe DCF-DA was used to estimate intracellular ROS accumulation. Cell-permeable DCF-DA is rapidly oxidized to highly fluorescent DCF in the presence of ROS. The DCF fluorescence intensity thus correlates linearly with the amount of intracellular ROS formed. The cells were plated in 6-well plates and glass bottom cell culture dishes and pretreated with 50 or 400 µM 20E for 24 h. Then these cells were treated with 150 µM HA cationic dye, JC-1, was used to monitor the mitochondrial membrane potential. At low mitochondrial membrane potentials, JC-1 continues to exist as a monomer and produces a green fluorescence (emission at 527 nm). At high mitochondrial membrane potentials, JC-1 forms J aggregates (emission at 590 nm) and produces a red fluorescence. JC-1 exhibits potential-dependent accumulation in mitochondria indicated by a fluorescence emission shift from green to red. Thus, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The cells were treated as described for measurement of intracellular ROS, and then JC-1 (10 µM) was loaded. After 30 min incubation at 37°C, the dye was removed, and the cells were washed three times with PBS. Finally, the results were visualized using confocal laser microscopy (Leica TCS SP5). Alternatively, fluorescence intensity was monitored using flow cytometry (BD Immunocytometry Systems).2+ indicator Fura-3/AM was used to monitor the intracellular free calcium concentration ([Ca2+]i). The cells were plated in glass bottom cell culture dishes and then were loaded with the Fluo-3/AM (5 µM) for 40 min at 37°C in Hank's buffer. After loading, cells were washed three times with Hank's buffer to remove excess extracellular Fura-3/AM. Fluorescence measurements were performed using confocal laser microscopy (Leica TCS SP5). Fluo-3 was excited at 488 nm, and fluorescence was measured at wavelengths of 515 nm every 5 s. After the baseline [Ca2+]i was observed for 60 s, 1.5 mM H2O2 was added to the dishes, and changes in [Ca2+]i were measured. Cells were pretreated with 50 or 400 µM 20E for 1 h. All images (about 120 images) from the scanning were processed to analyze changes of [Ca2+]i in a single cell. The change in intracellular calcium was expressed as relative fluorescence intensity (RFI).The fluorescent CaTo protect against oxidative stress, cells usually possess defense mechanisms, including their own intracellular antioxidants (such as antioxidant enzymes and cellular metabolites) 2 standard samples was mixed with 100 µL of Griess reagent at room temperature. The optical density at 540 nm was measured with a microplate reader. The standard curve produced by NaNO2 was generated for quantification.To estimate cellular NO stress, the amount of NO in cellular culture supernatants was determined by measuring the accumulated level of nitrite in the supernatant using a nitrite detection kit. In brief, supernatants were collected after cell treatment. Then 50 µL of cell medium or NaNOThe cells were harvested following treatment. After three washes with cold PBS, cells were lysed directly on culture plates with RIPA lysis buffer  with protease inhibitors  for 30 min on ice. The lysate was centrifuged at 14000×g for 15 min at 4°C. The supernatant consisting of total cellular protein extracts was collected and stored at 70°C until use. The nuclear and cytoplasmic proteins were extracted according to the instructions of the nuclear and cytoplasmic protein extraction kit . In summary, cells were washed in ice-cold PBS and then were gently scraped off the bottom of the flask into the media using a cell scraper. The media were centrifuged for 5 min at 1200 rpm at 4°C, and the pellet was dissolved with cytoplasmic protein extraction agent A supplemented with PMSF. After vortexing for 5 s and incubation on ice for 15 min, the cytoplasmic protein extraction agent B was add into the tubes, which were then vortexed for 5 s and incubated on ice for 5 s. The samples were centrifuged for 5 min at 14000×g at 4°C, and the supernatant, consisting of the cytosolic fraction, was immediately stored at 70°C until use. The pellet was resuspended in nuclear protein extraction agent supplemented with PMSF. After vortexing the tubes 15–20 times for 30 min and centrifuging for 10 min at 14000×g, the supernatants containing nuclear extracts were obtained. These protein extracts were quantified using a BCA Protein Assay kit . The nuclear and cytoplasmic proteins were subjected to immunoblotting to determine the activity of NF-κB in cells.Equal amounts of protein were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane . The membrane was incubated in fresh blocking buffer  at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study were anti-iNOS (1∶500), anti-phosphorylated NF-κB (p65 subunit) (1∶500), anti-phosphorylated IκBα (1∶500), anti-ASK1(1∶500), anti-phosphorylated ASK1(1∶1000), anti-MKK4 (1∶1000), anti-phosphorylated MKK4(1∶1000), anti-MKK7(1∶1000), anti-phosphorylated MKK7(1∶1000), anti-JNK(1∶500), and anti-phosphorylated JNK(1∶500). The blots were washed three times in TBS-T for 5 min and then incubated with specific peroxidase-coupled secondary antibodies (anti-mouse IgGHRP or anti-rabbit IgG-HRP) at room temperature. The bound antibodies were visualized using an enhanced chemiluminescent detection system  and then exposed to X-Ray films . The images were scanned with a GS800 Densitometer Scanner , and OD data were analyzed using Quantity One software. In these analyses, β-actin and Lamin B1 were used as an internal reference.Adult male Sprague-Dawley (SD) rats weighing 250–300 g  were housed in an environmentally controlled room at 22±2°C, with a relative humidity of 55±5%, a 12-h light/ = dark cycle, and food and water ad libitum. All experimental procedures were performed in accordance with the Third Military Medical University Guide for the Care and Use of Laboratory Animals. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Focal cerebral ischemia was produced by a modification of the intraluminal occlusion as described by Feng Zhang et al At 24 h after reperfusion, the neurological behavioral assessment was performed by an observer who was blinded to the experimental conditions in the surviving animals. Neurological deficits were scored on a 0–4 scale: 0 = no apparent deficits; 1 = contralateral forelimb flexion; 2 = decreased grip of contralateral forelimb while the tail was pulled; 3 = spontaneous movement in all directions, contralateral circling if pulled by the tail; 4 = spontaneous contralateral circling Animals were anesthetized with pelltobarbitalum natricum and sacrificed by decapitation 24 h after reperfusion. Brains were quickly removed, and five 2-mm consecutive coronal slices were made beginning from the anterior pole. The sections were stained with 2% TTC in saline for 30 min at 37°C and photographed. Then photographs of the five sections were analyzed for infarct size by a blinded observer using image analysis software (Image-Pro Plus 6.0). Data are expressed as percentage infarct volume. Percentage infarct volume was calculated as described: [(V C–V L)/V C]×100%, V C is the volume of the control hemisphere, and V L is the volume of the non-infarcted tissue in the lesioned hemisphere 2O2, 5% normal goat serum and 0.5% triton X-100 at room temperature for 30 min each. After anti-cleaved caspase-3 antibody was applied to the sections and incubated overnight at 4°C, the sections were incubated with anti-mouse IgG horseradish peroxidase for 1 h at 37°C. The sections were subsequently incubated with diaminobenzidine (DAB) and counter-stained with hematoxylin. A cleaved caspase-3-positive cell was indicated by brown coloration. The total cell number and the number of cleaved caspase-3-positive cells were determined in the five areas of the cerebral cortex in every section using a square micrometer eyepiece at 200× magnification. Five sections were selected from each experimental rat. Data are expressed as the ratio of cleaved caspase-3-positive cells to total cells.At 24 h after MCAO reperfusion, rats were anesthetized with pelltobarbitalum natricum and then perfused transcardially with 200 mL of saline followed by 300 mL of 4% paraformaldehyde solution. Brains were removed and fixed in 4% paraformaldehyde solution at 4°C for three days and then dehydrated and embedded in paraffin blocks. Five-micrometer-thick sections were cut through the dorsal hippocampus. Immunohistochemistry was performed with antibodies for cleaved caspase-3. In brief, tissue sections were deparaffinized and wetted with graded alcohol. The tissue sections were incubated with 3% Hp<0.05 was considered to be significant.Data were expressed as the mean ± S.E.M., and statistical significance was assessed by one-way analysis of variance (ANOVA) and Tukey's test. Neurological behavior scores were analyzed by using a nonparametric Kruskal–Wallis test and Dunn's Multiple Comparison Test. 2O2 decreased cell viability in a concentration- dependent manner. As shown in 2O2 at concentrations of 100 to 500 µM for 12 h decreased to 91.81±2.45%, 49.87%±4.39%, 38.86±3.18%, 35.28±2.22% and 28.60±5.74% of the control value, respectively. Based on this result, a treatment of 200 µM H2O2 for 12 h was used to induce B35 neural cell injury in subsequent experiments. To examine the neuroprotective effects of 20E on H2O2-induced cytotoxicity, B35 neural cells were pretreated with 20E for 24 h before the onset of H2O2 treatment. Application of 20E at 25 µM did not attenuate H2O2-induced cytotoxicity. We therefore increased the amount of 20-Hydroxyecdysone to 50, 100, 200, or 400 µM. The cell viability significantly increased to 60.65±2.15% (p<0.05 in comparison to the H2O2 treatment group), 65.75%±3.53% (p<0.01 in comparison to the H2O2 treatment group), 70.56±4.18% (p<0.01 in comparison to the H2O2 treatment group), and 78.40±5.42% (p<0.01 in comparison to the H2O2 treatment group) of the control value at 50, 100, 200 and 400 µM, respectively , from 28.91±2.52% to 57.37±4.03%. However, pretreatment with 50 or 200 µM 20E for 24 h before H2O2 exposure decreased the LDH release to 45.20±4.82% (p<0.05 in comparison to the H2O2 treatment group) and 39.11±5.95% (p<0.01 in comparison to the H2O2 treatment group), respectively. Similarly, the TUNEL analysis showed that H2O2 stimulation increased the percentage of TUNEL-positive cells from 8.17±2.32% to 45.30±5.78%, which was significantly reduced to 32.99±4.51% (p<0.05 in comparison to the H2O2 treatment group) and 22.97±4.40% (p<0.01 in comparison to the H2O2 treatment group), respectively, by pretreatment with 50 or 400 µM 20E  and 33.93±6.34% (p<0.01 in comparison to the H2O2 treatment group), respectively, when the cells were pretreated with 50 or 400 µM 20E for 24 h before H2O2 exposure . As shown in 0 µM 20E . Consistexposure .2O2 exposure. In this study, a fluorescent probe, DCF-DA, was used as a specific marker for quantitative intracellular ROS formation. T-AOC, SOD activity and GPX activity level were used to evaluate cellular antioxidant potential. After B35 neural cells were exposed to 200 µM H2O2 for 12 h, the intracellular DCF-fluorescence intensity significantly increased (p<0.01 in comparison to the control group)  , and thel group) . Howeverntensity  and incrntensity .2O2 for 12 h rapidly caused MMP dissipation, as shown by the increase in green fluorescence and the concomitant disappearance of red fluorescence, which decreased the red/green fluorescence intensity ratio (13.31±2.80% of control). Pretreatment with either 50 or 400 µM 20E for 24 h before H2O2 exposure attenuated the changes in MMP as indicated by repression of green fluorescence and restoration of red fluorescence. The red/green fluorescence intensity ratio was increased to 39.26±9.46% of control and 78.33±9.60% of control, respectively.Mitochondria are an important cellular target of oxidative stress in neurons under ischemic conditions 2O2 for 12 h caused an increase in the levels of intracellular MDA from 2.49±0.47 to 8.75±0.75 nmol/mg protein. However, pretreatment with either 50 or 400 µM 20E for 24 h before H2O2 exposure dramatically decreased the levels of intracellular MDA to 7.49±0.7 and 3.56±0.61 nmol/mg protein, respectively phenyl]methyl) ethanimidamide dihydrochloride (1400W), a selective iNOS inhibitor, significantly decreased the nitrite content to 22.55±4.04 µM (p<0.01 in comparison to the H2O2 treatment group) and 17.1±2.22 µM (p<0.01 in comparison to the H2O2 treatment group), respectively  (NO and iNOS have been implicated in oxidative stress apoptotic cell death ectively . In addit group) .2O2. The transcriptional activity of NF-κB can be regulated by the phosphorylation of IκB as well as the NF-κB p65 subunit. In our study, we observed that H2O2 elevated the expression of phosphorylated IκBα in the cytosol and phosphorylated NF-κB p65 in the nucleus. However, 20E was as effective in preventing the phosphorylation of IκBα and NF-κB p65 as pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor (p<0.01 in comparison to the H2O2 treatment group)  .2O2 alone for different times or pretreatment with different concentrations of 20E before the onset of H2O2 treatment to investigate whether the ASK1-MKK4/7-JNK signaling pathway is involved in the neuroprotective mechanisms of 20E. Western blot analysis showed that at 1, 6 h and 12 h after exposure to H2O2, there was a significant increase in the phosphorylation of ASK1 compared with the control group. Pretreatment with either 50 or 400 µM 20E for 24 h before H2O2 exposure inhibited the phosphorylation of ASK1 induced by H2O2. Total ASK1 did not change in any group  and 27.75±5.22% (p<0.01 in comparison to the MCAO group) of the coronal sections, respectively, from 42.53±2.14% of the coronal sections. Furthermore, 20E  also reduced the neurological deficit scores to 1.88±0.31 (p<0.05 in comparison to the MCAO group) and 1.52±0.35 (p<0.01 in comparison to the MCAO group), respectively, compared to sham-operated rats that scored 2.43±0.15  at the onset of reperfusion significantly reduced the cerebral infarction area induced by MCAO . Fig. 8B.43±0.15 .p<0.05 in comparison to the MCAO group) and 40.37±9.15% (p<0.01 in comparison to the MCAO group), respectively, compared with the MCAO group. Consistent with the TUNEL analysis data, the percentage of cleaved caspase-3-positive cells increased from 4.83±1.17% to 64.00±8.70% in the infarct region in the MCAO group. However, the percentage of cleaved caspase-3-positive cells was significantly reduced to 51.14±6.04% (p<0.05 in comparison to the H2O2 treatment group) and 33.83±7.52% (p<0.01 in comparison to the H2O2 treatment group), respectively, when rats were treated by 20E  (Apoptotic cell death was determined by TUNEL staining and immunohistochemistry staining for cleaved caspase-3. As shown in 0 mg/kg) . To ruleTo evaluate oxidative damage following cerebral ischemia and the anti-oxidative effects of 20E in rats, we measured T-AOC, SOD activity, GPX activity level and MDA level in the rat cortex. As shown in 2O2 has been extensively used as an inducer of oxidative stress 2O2-induced injury in B35 neuroblastoma cells in vitro to simulate the oxidative damage following cerebral ischemia, attempting to search for a naturally occurring drug with neuroprotective effects. 20E, an insect steroid hormone, exhibits a potent antioxidative effect. This study demonstrates that 20E reversed the H2O2-induced cell injury, which was characterized by a decrease in cell viability, release of LDH, and mitochondrial membrane potential dissipation and increases in the number of TUNEL-positive and cleaved caspase-3-positive cells. Four specific actions by 20E may contribute to this neuroprotective effect: first, 20E inhibits the generation of ROS and restores cellular antioxidant potential; second, 20E blocks oxidative stress-induced increases of [Ca2+]i; third, 20E reduces the production of NO and the expression of iNOS protein, which may be mediated by inhibition of NF-κB activation; fourth, 20E suppresses the activation of the ASK1-MKK4/7-JNK stress signaling pathway induced by oxidative stress. Finally, we establish a rat MCAO model to further confirm the neuroprotective effect of 20E. The results of our in vivo studies showed that 20E significantly attenuated ischemia injury caused by MCAO in rats. These findings indicate that 20E is useful as a potential protective agent against cerebral ischemic injury.Oxidative stress is a major cause of neuronal injuries induced by cerebral ischemia 2O2-induced intracellular ROS production, as revealed by reduced distribution of the DCF-DA fluorescent dye in cells pretreated with 20E. On the other hand, we also observed that H2O2 decreased intracellular T-AOC, SOD activity and GPX activity level and that treatment with 20E markedly attenuated the above changes, which suggested that 20E enhanced cellular antioxidant defense activities. Accumulating evidence demonstrates that mitochondria are an important cellular target of oxidative stress 2O2 could cause the increases of MDA, the end product of lipid peroxidation, and depolarization of mitochondrial membrane potential in cells, both of which were alleviated significantly by pretreatment with 20E. Together, these results support that 20E could reduce ROS accumulation, protect antioxidant enzymes, prevent lipid peroxidation and preserve the structural and functional integrity of mitochondria in H2O2-treated B35 neural cells.Ischemia could increase production of ROS and undermine antioxidant defense in brain tissues, which causes an imbalance of pro-oxidants and antioxidants 2+]i 2+]i. Moreover, the elevation in [Ca2+]i causes ROS generation by activating enzymes. Conversely, ROS generation can facilitate [Ca2+]i increases by damaging the [Ca2+]i regulatory mechanism and activating Ca2+ release from intracellular Ca2+ stores 2+]i in the skeletal muscles of rats suffered from D-hypovitaminosis 2O2-induced [Ca2+]i elevation, suggesting that 20E can also attenuate H2O2-induced B35 neural cell injury by preventing intracellular calcium overload.There is increasing evidence that cerebral ischemia leads to an increase in i, reduceing the production of NO and the expression of iNOS protein and suppressing the activation of the ASK1-MKK4/7-JNK stress signaling pathway, as shown in In summary, this study demonstrates that 20E is effective in reducing oxidative stress-induced cytotoxicity in B35 neural cells by inhibiting the generation of ROS and mitochondrial membrane potential dissipation, restoring cellular antioxidant potential, blocking the increases of [Ca


Low-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto- and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes. HMGB1 is a non-histone nuclear protein which is a highly conserved, ubiquitous and is comprised of 215 amino acids that are organized in two globular DNA-binding domains, box A and box B, and with an acidic C-terminal tail Once in the extracellular space, HMGB1 interacts with the receptor for advanced glycation end products (RAGE) and/or members of the Toll-like family of receptors (TLRs) including TLR2 and TLR4. Activation of these receptors results in the activation of NF-κB, which influences the production of pro-inflammatory cytokines The role of HMGB1 on macrophages is clearly described and stimulation of macrophages with HMGB1 induces a production of pro-inflammatory cytokines Obese adipose tissue is characterized by low grade inflammation (LGI) .However long before the onset of obesity and macrophages infiltration, adipose tissue is able to implement and maintain LGI. Indeed it has been recognized that adipocytes also demonstrate significant inflammatory properties like IL-6 or TNFα production Moreover, pre-adipocytes contribute significantly to inflammation of the adipose tissue in obesity A number of studies have demonstrated an autocrine and paracrine effect of HMGB1 on macrophages which can potent LGI. However, it is not known whether preadipocytes and adipocytes may secrete and respond to HMGB1. There is currently no published report regarding HMGB1 expression and functions in fat cells and possible links to LGI in obesity. In this study we investigated the implication of HMGB1 in the regulation of inflammation in preadipocytes using the human preadipocyte cell line SW872 as a model. We showed that HMGB1 is released actively by SW872 and that extracellular HMGB1 contributes to IL-6 secretion through RAGE. These results suggest that HMGB1 and associated-signaling pathways could be used as targets for the development of new therapeutics against pathologies related to adipose tissue inflammation, insulin resistance and diabetes.Except when indicated, all drugs and reagents were from Sigma Aldrich .5′-AGGCAAAATGTCATCTTATGCATTTTTTGTG-3′ and 5′-CACAAAAAATGCATAAGATGACATTTTGCCT-3′. It was then amplified by PCR using primers 5′-AACCATGGGCAAAGGAGATCCTAA-3′ and 5′-TTTGAATTCTTGCTGCCGCGCGGCACCAGTTCATCATCATCATCTTCTT-3′ and subsequently cloned in pET28 vector using NcoI and EcoRI sites. This lead to a sequence encoding HMGB1 – thrombin site – C-terminal his-tag. After transformation of BL21-codon plus E. Coli bacteria (Agilent), culture and induction were done as described −1 Hepes . Cells were lyzed by sonification  at 80% of amplitude for 2 min with 10 sec of burst and 10 sec of pause in the ice. Lysate was centrifuged at 15,000 g for 10 min at 4°C, the supernatant was submitted to the action of DNAse I at 80 U/mL for 30 min at 25°C, then diluted to a final volume of 10 mL and mixed gently with 0.5 mL of agarose Ni-NTA . After 30 min, the column was washed with 10 mL of 25 mmol.L−1 Hepes, 500 mmol.L−1 NaCl, then with 10 mL of 25 mmol.L−1 Hepes, 20 mmol.L−1 imidazole, then with 25 mL of 25 mmol.L−1 Hepes, 0.1% (v/v) Triton X-114, 20 mmol.L−1 imidazole, then a final washing step with 10 mL of 25 mmol.L−1 Hepes, 20 mmol.L−1 Imidazole. HMGB1-His was eluted with 5 mL of 300 mmol.L−1 imidazole in 25 mmol.L−1 Hepes. The elute (1 mL) was dialyzed overnight at 4°C against 25 mmol.L−1 Hepes. Levels of LPS and DNA in the preparation were evaluated respectively by LAL assay  and ethidium bromide (EtBr) coloration of DNA after gel electrophoresis. Level of LPS was under 1ng.L−1 and no DNA was detected by EtBr coloration. Biochemical evaluation of rHMGB1 was conducted by SDS-PAGE with coomassie-blue staining and western blot  was modified by directed mutagenesis to remove the endogenous NdeI restriction site using ern blot .2 in DMEM medium, supplemented with 10% heat inactivated fetal bovine serum and 2 mmol.L−1 L-Glutamine, 1 mmol.L−1 sodium pyruvate, 100 U.mL−1 of penicillin, 0.1 mg.mL−1 of streptomycin and 0.5 µg.mL−1 of fungizone . All experiments were done between passage 2 and 8 at 80% of confluence. For treatment, the media was replaced by fresh complete media (see above) containing drugs or rHMGB1 for 5 h or indicated time. Cells were harvested and kept as frozen pellet for further protein or mRNA analysis. The media were centrifuged for 5 min at 800 g and supernatants were kept at −20°C until use for quantification by ELISA or Dot-Blot.SW872 liposarcoma cells line  were cultured at 37°C with 5% CO−1) as selection agent for twenty days 29mer shRNA constructs (TR316576) in retroviral untagged pRS vector, against HMGB1 were purchased from Origene . Retroviral particule were produced in PT-67 packaging cell lines as described by manufacturer’s instructions . SW872 were stably infected using the tissue cell supernantant of transfected PT67 cells as before using puromycin (1 µg.mL−1) and incubated with control or blocking antibodies for 5 h at 1∶20 dilution. Blocking antibodies against TLR2  and TLR4  −1 .SW872 cells were treated with rHMGB1 . Protein extracts (cytoplasmic or nuclear fractions) were separated by SDS-PAGE and transferred to nitrocellulose membranes . The Western blot was performed as described Secretion of cytokines  was measured by ELISA kit  according to manufacturer’s instructions. Of note, no exogenous HMGB1, LPS and IL-6 were detected in commercial cell culture media or complete media by Dot-blot, LAL-assay and ELISA, respectively (data not shown). Secretion of HMGB1 was measured by ELISA kit from IBL international according to manufacturer’s instructions . 100 µL of SW872 cell culture supernatant were spotted on nitrocellulose membrane  with a Dot-blot apparatus (Biorad). Membranes were processed as for the Western-blot technique.−1 DTT (Invitrogen), 3 µL of 10 mmol.L−1 dNTP mix , 2.5 µL of 5 U.mL−1 of random hexamers (pdN6), 1.5 µL of 40 U.µL−1 RNAse Inhibitor (Promega). After a denaturation at 65°C for 5 min, 2 µL of RTase 200 U.µL−1 (Invitrogen) were distributed. The RT steps are the followings, 37°C for 1 h, 95°C for 5 min. cDNAs were then kept at 4°C. The PCR was done in a final volume of 20 µL containing 4 µL of 5X PCR buffer (Promega), 2 µL of 25 mmol.L−1 MgCl2 (Promega), 0.4 µL of 10 mmol.L−1 dNTP mix, 0.1 µL of 5U.µL−1 Taq polymerase (Promega), 11.5 µL of water DNase RNase free, 1 µL of cDNA and 1 µL of 10 mmol.L−1 primer mix  according to the manufacturer’s specifications. The reverse transcription (RT) was done with 2 µg of total extracted RNA and 14.5 µL of RT mix containing: 6 µL of 5X RT buffer (Invitrogen), 1.5 µL of 0.1 mol.Limer mix . 30 cycl−1) in PBS-BSA 1% (w/v) and incubated with the secondary antibody Alexa Fluor 594-conjugated anti-mouse IgG (1∶1000 dilution). Nuclei were stained with DAPI  at a final concentration of 0.1 ng.mL−1). Coverslips were mounted in Vectashield , and fluorescence was observed using a Nikon Eclipse E2000-U microscope . Images were obtained using the Nikon Digital sight PS-U1 camera system and the imaging software NIS-Element AR .SW872 cells were grown on glass coverslips, washed twice with PBS and then fixed for 5 min in fixation buffer  and washed twice with PBS. Coverslips were conserved at −20°C. Cells were incubated in the following primary antibody, anti-HMGB1  1∶200 dilution , anti-TLR4 directly conjugated with R-Phycoerythrin  or anti-RAGE  at 1∶100 dilution. When required, we used anti-goat or anti-mouse conjugated to R-Phycoerythrin  at 1∶500 dilution as secondary antibodies. After two washes with PBS, cells were harvested in PBS. For each experiment 10000 cells were counted and were analyzed by flow cytometry .Cells were harvested after two washes with PBS, then pelleted at 800 g for 5 min. Cells were incubated with 5% (w/v) BSA in PBS for 10 min at room temperature under agitation. Primary and secondary antibodies were diluted in PBS-BSA and incubated at room temperature with agitation for 45 min. Primary antibodies were anti-TLR2 . Degrees of significance are indicated in the figure captions as follow: * p<0.05; ** p<0.01; *** p<0.001, ns  =  not significant.All values are expressed as mean +/− SD and are representative at least of 3 independent experiments. Comparisons between different treatments have been analyzed by a one-way ANOVA test. Values of p<0.05 were considered statistically significant for a post-hoc Tukey-Kramer test in order to compare treated In this study we wanted to test the secretion of HMGB1 and the contribution to fat inflammation of preadipocyte. We used the human liposarcoma cell line SW872 as a model. We first tested the phenotype of SW872 considered as a preadipocyte model. Using RT-PCR we found expression of preadipocyte markers Pref1, GATA2 and AEBP in SW872 as in 3T3L1 preadipocyte . We wereWe analyzed the basal expression of HMGB1 in the human preadipocyte SW872 cells by RT-PCR. The transcript of HMGB1 was detected in SW872 cells in basal culture conditions . We next−1 ±24.57) by SW872 cells in normal cell culture conditions  . These r−1 of rHMGB1, higher concentrations of rHMGB1 significantly enhanced the release of IL-6 (p<0.01) and in a dose dependent manner (We produced a recombinant form of human HMGB1 (rHMGB1) to investigate the direct effects of HMGB1 on SW872 adipocytes . SW872 ct manner . IL-6 set manner . Moreovet manner . These r−1 of IL-6 secretion ± 73.88 after treatment with 10 µmol.L−1 of Ethyl-pyruvate, 323.99 pg.mL−1 ±88.64 after treatment with 20 µmol.L−1 281.54 pg.mL−1±237.78 after treatment with 30 µmol.L−1) compared to untreated cells (1220 pg.mL−1±88.68) (−1 (615.79 pg.mL−1 of IL-6 secretion ± 257.01 or 1000 ng.mL−1of glycyrrhizin (574.95 pg.mL−1±196.24) compared to untreated cells (1300 pg.mL−1±202.32)  . Similar±202.32) . To vali±202.32) . HMGB1 d±202.32) .It is well documented that HMGB1 can bind to the cellular receptors TLR2, TLR4 and RAGE In LGI other inflammatory markers were also important like TNFα and MCP1 (CCL2). We investigated basal expression of TNFα and MCP1 in our SW872 preadipocyte model, as previously observed To validate HMGB1 action, we analyzed MCP1 secretion after treatment of SW872 with plasmid encoding shRNA against HMGB1 as described above for IL6. Down regulation of HMGB1 levels significantly (p<0.01) reduced MCP1 secretion . All togThe secretion of HMGB1 by macrophages is well characterized and essentially through the active release from cytoplasmic stores. Moreover, secreted HMGB1 can induce a signaling cascade leading to the production and release of a plethora of pro-inflammatory molecules and leading to LGI. It is recognized that adipose tissue contributes to LGI but the role of HMGB1 specifically on preadipocytes has remained largely unknown.To test the hypothesis that HMGB1could participate to LGI in fat tissues, we used the human liposarcoma cell line SW872 as a preadipocyte model Interestingly, we found that SW872 cells released constitutively high levels of IL-6 and through a direct action of the endogenous HMGB1. The levels of secreted IL6 correlated positively with the level of HMGB1 and, more importantly, we found that two inhibitors of HMGB1 release impaired IL6 production by SW872. Thus, our data show strong inhibitory activities either with ethyl-pyruvate When SW872 cells were exposed to rHMGB1, we observed an increased secretion of IL-6 in a dose dependent manner further suggesting that HMGB1 can act in a direct manner on fat cells to promote the release of IL-6.In a previous study, IL6 secretion was significantly increased in culture medium of adipocytes in presence of LPS The strong association between high levels of secreted HMGB1 by SW872 and strong levels of IL-6 is interesting to shed new light on the mechanisms of LGI and possible therapeutic avenues. It has also been shown a strong link between IL-6 serum levels and insulin resistance Using our preadipocyte SW872 cell model, we showed that HMGB1 behave as a positive regulator of MCP1 secretion. As MCP1 act as a chemokine which attract macrophages, HMGB1 may contribute to immune cells infiltration in adipose tissue and instigating LGI. The presence of macrophages with potent pro-inflammatory activities and in response to HMGB1 released in the vicinity of the adipocytes may contribute further to insulin resistance  Several key receptors have been implicated in HMGB1 signaling, including RAGE, TLR2 and TLR4. We showed that RAGE, TLR2 and TLR4 are transcribed and expressed at the surface of SW872 cells. However, only RAGE blocking antibody induced a strong inhibition of IL-6 secretion by preadipocytes SW872 stimulated with rHMGB1.RAGE is known to be implicated in obesity, diabetes and their complications Our results argue in favor of a central effect of RAGE in fat tissue inflammation. Administration of soluble RAGE or blocking antibody can induce a protective effect against inflammation and diabetes In summary, our study is the first report ascribing a novel proinflammatory activity of HMGB1 on adipose tissue and particularly in preadipocyte. We can consider that soluble HMGB1 is a novel adipokine secreted by preadipocyte and controlling inflammation through the binding to RAGE receptors Figure S1Preadipocyte markers expression in SW872 and 3T3L1 cell lines. Expression of pref-1, GATA2, AEBP by RT-PCR from total RNA obtained from SW872 cell after 12 h in culture (A) or from 3T3L1 after 16h in culture, with (+) and without (−) Reverse Transcriptase (RT). GAPDH served as housekeeping gene.(TIF)Click here for additional data file.Figure S2HMGB1 expression and localization in the human preadipocyte cell line 3T3L1. A) Expression of HMGB1 by RT-PCR from total RNA obtained from 3T3L1 cell culture after 16h in culture, with (+) and without (−) Reverse Transcriptase (RT). B) Detection of HMGB1 protein in different cell fractions by Western blot analysis. HSP60 is a cytoplasmic protein (Cy)  and H4 a nuclear protein (Nu) (Antibody from Santa-Cruz Biotechnology sc-10810).(TIF)Click here for additional data file.Figure S3SW872 and 3T3L1 releases actively HMGB1 in cell culture media. A) Quantification of HMGB1 secretion after renewal of cell culture media from SW872 cells in time dependent manner by Dot-Blot analysis. B) LDH assay on cell culture media from SW872 from A) according to manufacturer instructions . C) Quantification of HMGB1 and IL6 secretion after renewal of cell culture media from 3T3L1 cells in time dependent manner by respectively by Dot-Blot and ELISA assays.(TIF)Click here for additional data file.Figure S4HMGB1 inhibition downregulates IL6 release. A) Biochemical evaluation of HMGB1 purity by SDS-PAGE followed by Coomassie Blue staining or Western blotting with HMGB1 antibody . B) Cytotoxicity evaluation of rHMGB1 on SW872 cell line using MTT assay according to manufacturer’s instructions . C) Impact of ethylpyruvate on HMGB1 secretion by Dot-Blot analysis.(TIF)Click here for additional data file.Figure S5MCP1 (CCL2) and TNFα mRNA expression. MCP1 and TNFα mRNA expression were assessed by RT-PCR from total RNA obtained from 3T3L1 cells after 12h of culture, with (+) and without (−) Reverse Transcriptase (RT).(TIF)Click here for additional data file.Table S1List of primers used in this study for RT-PCR on 3T3L1 and SW872 RNA.(TIF)Click here for additional data file.


Furthermore, the recently proposed combination of bSSFP imaging and T2Prep for T2-mapping at 1.5T has enabled a rapid quantitative cardiac T2 estimation . However, the accuracy of this method may still be limited due to the complex T2/T1 signal weighting. Especially for longitudinal studies designed for monitoring and/or guiding therapy, accurate and reproducible T2 measurements will be critical. A novel quantitative 3T T2-mapping protocol was therefore developed and tested in both healthy volunteers and patients.Recently, T2prep with 3 incremental TE values, affine coregistration, a navigator and 2D radial gradient echo imaging were combined for free-breathing T2-mapping at 3T with a spatial resolution of 1.25mm. Bloch equation simulations of this sequence were used to optimize scan parameters and to determine an empirical equation that compensates for T1 relaxation and which returns the “true” T2. The T2-mapping sequence and empirical equation were then validated in a series of 15 phantoms in which the true T2 was determined with a 9-TE spin-echo sequence. Next, the myocardial short axis T2 of 8 healthy volunteers was mapped in two different scan sessions while a reference phantom (T2=43.1±0.7ms) was placed next to the thorax. The average myocardial T2 for both sessions was computed with and without correction with the “true” reference phantom T2. Finally, this validated protocol was used in 5 patients in the subacute phase after revascularization of acute ST-elevation myocardial infarctions and compared to T2-weighted TSE imaging.An adiabatic TT2prep=60/30/0ms, TRR=3 heartbeats, TR/TE=5.3/2.4ms. The empirical equation to determine T2 was S=S0[exp(-TET2prep/T2)+0.06], where S and S0 are the measured and steady-state signal  improvement in T2 estimation with the empirical equation as compared to the standard T2 decay measurements  and on average showed a 5±2% difference between the two scan sessions. When compensated with the T2 from the reference phantom, this difference decreased to 2±1% (p=0.02). In all patients, T2maps could successfully be obtained and a clear demarcation of zones with elevated T2 values was consistent with the findings on T2-weighted MRI and X-ray coronary angiography as shown in the example in Fig. As a result of both the simulations and phantom scans, optimized sequence parameters included: TEnal Fig. . Scans onts Fig. . The myo2-mapping at 3T, while the addition of a reference phantom improves reproducibility. Therefore, it may be well-suited for longitudinal studies in patients with ischemic heart disease.The methodology presented in this study enables robust and accurate cardiac TN/A.


Medication errors are frequent in the ICU and may occur during medical ordering, transcription or administration of drugs. A system consisting of a computerized physician order entry (CPOE) with bar-code verification of medications  has been described as a tool to improve medication safety , but fewn = 42), nurses (n = 58), nurses technicians (n = 84) and other professionals (n = 66).We conducted a survey to evaluate the satisfaction of healthcare workers when using a CPOE with bar-code verification of medications in a tertiary 40-bed adult ICU in Sao Paolo, Brazil 6 months after implementing the system. A satisfaction questionnaire which consisted of items in a numeric scale type from 1 (low satisfaction) to 10 (high satisfaction) was filled out by physicians (P < 0.0001). The ease to place the first medical order and to copy the order form the previous day scored 5.41 ± 2.05 and 6.39 ± 1.93. The visualization of the medical order with the bar-code verification of drugs administration scored 5.95 ± 2.51 by the nurses. On average, physicians found the system less user-friendly (score 3.88 ± 1.85) than other professionals .Most subjects were female (66%), below 36 years of age (69%) and used the computer daily at home (81%). On average, respondents were satisfied with the CPOE system (score 5.74 ± 2.14) and believed it improved safety (score 7.64 ± 2.42). Satisfaction was lower among physicians (score 4.62 ± 1.79) when compared to other professionals (score 5.97 ± 2.14; Although most of the ICU staff believe that the CPOE and bar-code has the potential to improve medication safety and the quality of care for critically ill patients, our survey showed a low level of satisfaction 6 months after implementing the system, particularly for physicians who consider the system unfriendly.


As a proof of principle, transgenic plants expressing nitrous oxide reductase (N2OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N2OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N2OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N2O reduced min−1 g−1 root protein. Another event, plant line 1.9, also demonstrated high specific activity of N2OR, 13.2 µmol N2O reduced min−1 g−1 root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops  in this way, it may be possible to reduce the atmospheric concentration of N2O that has continued to increase linearly (about 0.26% year−1) over the past half-century.The nitrous oxide (N With a global warming potential 310 times that of carbon dioxide, N2O contributes to the rising of atmospheric temperature . This reaction often does not occur if ideal metabolic conditions are not met, and results in emission of N2O  cells were plated on Luria broth (LB)-agar medium and grown at 30°C for 48 h. A single colony was used to inoculate 5 mL of liquid LB medium, and mixture was incubated at 30°C overnight with shaking. The bacterial culture was added to 100 mL liquid LB medium and incubated for 3 h. Methods of g for 15 min at 4°C, the pellet was washed in phosphate buffered saline (PBS) buffer, and resuspended in 5 mL SET . Lysozyme was added to a concentration of 1 mg mL−1, and the resulting suspension incubated at 37°C for 30 min, mixing occasionally by inversion. To inactivate DNases, 0.5 mL of proteinase K (1 mg mL−1) was added along with 0.5 mL 10% sodium dodecyl sulfate (SDS), and mixture was incubated at 55°C for 2 h with occasional inversion.g for 10 min. The aqueous upper phase was transferred to a new tube. Introduction of 20 mL isopropanol, mixing by inversion, and incubation at –20°C for 15 min were sufficient to precipitate DNA. The DNA was wound onto a pipette tip, washed twice with 70% ethanol in a new 1.5-mL tube, placed in a centrifugal evaporator to dry, and resuspended in 500 µL deionized water.To precipitate proteins out of solution, 2.5 mL of 5 M NaCl was added and gently mixed. Adding 10 mL chloroform, the tube was mixed at low speed for 1 h, and then centrifuged at 3000 Pseudomonas stutzeri genomic DNA was used as a template for polymerase chain reaction (PCR) amplification of the nosZ gene or nosFLZDY genes. Primers were gene-specific, and were designed to introduce restriction sites so the resulting segments could be fused (Table S1). The root-specific promoter of the rolD gene from Agrobacterium rhizogenes was chosen to direct root-specific expression of N2OR. The promoter sequence 426D . The leader sequence of the Daucus carota (carrot) extensin (ex) gene was amplified from the pHBV-CO plasmid using forward primer, ExSigZF/ExSigDF, and reverse primer, ExSigZR/ExSigDR (Phaseolus vulgaris (Pv) alpha amylase inhibitor-1 signal sequence was amplified using forward and reverse primers PvSigF and PvSigR, respectively  gene, which confers resistance to the antibiotic kanamycin. The resulting prolD-nosZ plasmid was sequenced at StemCore Laboratories  to verify accuracy of the DNA sequence. The megacassette prolD-nosFLZDY was constructed in a similar fashion, ligating individual expression constructs for nosF, nosD, nosY, and nosL and cloning the resulting sequence into pRD400 .nce 426D , contain/ExSigDR . The Phaectively . Purifier pRD400   seeds were sterilized with a 1-min wash in 70% ethanol, 10-min wash in 10% bleach containing 1–2 drops Tween-20, and three rinses in sterile distilled water. Sterilized seeds were grown on germination medium  in petri dishes. Tobacco leaf transformation followed the general method of Agrobacterium tumefaciens harboring either prolD-nosZ or prolD-nosFLZDY plasmid was used to infect leaf sections for 48 h on cocultivation medium .−1 6-benzyladenine, 0.1 mg L−1α-naphthalene acetic acid, pH 5.8, 200 mg L−1 of ticarcillin, and 300 mg L−1 of kanamycin). Calli began to appear after two weeks. When the shoots had reached a height of 1 cm, they were excised and transferred to root generating medium . When the roots had reached 5 cm long, the plantlets were transferred to potted soil and grew in the greenhouse where they were maintained through seed set with 16 h daylight (400 W/m2) at 25°C and 8 h dark at 21°C, with watering as necessary. Seeds were collected once seedpods had matured and planted in hydroponic medium to perform the rhizosecreted protein experiments described below.Transformed leaf cells were grown on regeneration medium , 250 µM of each dNTP, 0.5 unit Taq DNA polymerase, with the following cycling condition: initial denaturation at 94°C for 4 min; 30 cycles with denaturation at 94°C for 30 sec, annealing at 55°C for 1 min, and elongation at 72°C for 2 min; followed by a final elongation at 72°C for 10 min. DNA from nontransgenic (NT) plant roots was used as negative control, while either the prolD-nosZ or prolD-nosFLZDY plasmid was used as positive control. The following primers were used to detect the nosZ gene in genomic DNA from prolD-nosZ transgenic plants: forward primer (ExSigZF) 5′-TAGGTACCTACTCGAGATGGGAAGAATTGCTAGAGG-3′ and reverse primer (NosZR) 5′-TAGGATCCAACATATGTTAGGCCGGCTCGACCATC-3′. For prolD-nosFLZDY transgenic plants, the specific primers used to detect the nosZ gene were forward primer (NosZF) 5′-GACTAGTCAGGCCGTCAAGGAGTCCAAG-3′ and reverse primer (NosZR) 5′-TAGGATCCAACATATGTTAGGCCGGCTCGACCATC-3′.A portion of the root tissue (100 mg) was collected from each 6-week-old plant and genomic DNA was extracted using the DNeasy Plant Mini Kit . PCR was performed in a 25 µL reaction mixture containing 50 ng of tobacco genomic DNA, 25 pmol of each primer, 2.5 µL of 10 × PCR buffer  5′-TAGGTACCTACTCGAGATGGGAAGAATTGCTAGAGG-3′ and reverse primer (NosZR) 5′-TAGGATCCAACATATGTTAGGCCGGCTCGACCATC-3′. For prolD-nosFLZDY transgenic plants, the following primers were used to detect the nosZ gene: forward primer (NosZF) 5′-GACTAGTCAGGCCGTCAAGGAGTCCAAG-3′ and reverse primer (NosZR) 5′-TAGGATCCAACATATGTTAGGCCGGCTCGACCATC-3′. A control PCR, with total RNA not treated with reverse transcriptase, was performed to ensure that no DNA was present. Expression of other genes in the nos cassette  was also analyzed using RT-PCR in the same way, with gene-specific primers.Root tissue (100 mg) was collected from each 6-week-old plant and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen). After treatment with RNase-free DNase , RT-PCR was performed according to the SuperScript II Reverse Transcriptase system protocol . In a 10 µL reaction mixture, 250 ng of total RNA was reverse transcribed at 70°C for 10 min with an oligo (dT) primer. The conditions for cDNA synthesis were 10 min at 25°C, 60 min at 37°C, and 5 min at 95°C. Each PCR reaction used 2 µL cDNA as a template and nosZ or the rolD-FLZDY expression cassette were surface-sterilized using 20% v/v bleach, washed in sterile distilled water, and sown onto a 9-cm petri dish containing 0.7% w/v agar-solidified MS medium . Total protein extract was quantified using the BCA Protein Assay Kit .Root tissue was harvested, washed three times in ddH2OR activity analysis.Rhizosecreted protein from each plant culture was collected over a period of 25 days. Aliquots of MS medium (500 µL) from both rolD-transgenic and NT plant cultures were collected at regular intervals. Samples were concentrated to a volume of 20 µL by means of YM50 Microcon filters  and stored at –20°C. Protein samples were pooled together prior to western blotting and NPseudomonas stutzeri Zobell (ATCC 14405) cells were plated on LB-agar medium and grown at 30°C for 48 h. A single colony was used to inoculate 200 mL of synthetic medium , purged with argon, and stored at –70°C. After dissolving the pellet in 25 mM Tris buffer (pH 7.5), cells were disrupted by sonication.c medium , and cul−1 using 25 mM Tris (pH 7.5) for buffer A, and 25 mM Tris (pH 7.5), 0.3 M NaCl for buffer B. Protein was eluted at 4°C over five column volumes with a linear gradient of 0–100% buffer B. Collected 5 mL fractions were stored at 4°C after purging with argon. Those containing N2OR and nitrite reductase were identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), pooled and then dialyzed using a membrane with a 14-kDa cutoff. Fractions were subjected to hydroxyapatite chromatography using 10 mM NaH2PO4 (pH 7.2) for buffer A and 400 mM NaH2PO4 (pH 7.2) for buffer B. Protein was eluted over 10 column volumes with a linear gradient of 0–80% buffer B. Collected 2 mL fractions were purged with argon and stored at 4°C prior to SDS-PAGE analysis . Chromatography was performed under anaerobic conditions, with all reagents degassed by vacuum and procedures carried out under 100% argon. Concentration of purified N2OR was determined using the The supernatant was applied to a DEAE Sepharose ion-exchange chromatography column and eluted at 2 mL min2OR purified from P. stutzeri, and the negative control was protein extract from a nontransformed plant. Samples were run on a 10% acrylamide gel with a 5% stacking gel, then transferred onto a nitrocellulose membrane using the Trans-Blot transfer cell . To prevent nonspecific binding, membrane was blocked with Tris-buffered saline with Tween (TBST) plus 5% skim milk before incubation with rabbit anti-N2OR serum  and subsequently antirabbit biotin horseradish peroxidase-linked antibody (1:1000 in TBST) . Immunoblotted N2OR was visualized using enhanced chemiluminescence (ECL) western blotting chemiluminescent reagents .Samples were prepared by heating 40 µg protein extract for 10 min at 95°C in 2× protein sample buffer . The positive control was N2OR activity was determined using a modified protocol from 2O to be monitored spectrophotometrically. Assays were carried out in an anaerobic chamber  at 37°C, and all reagents were degassed by vacuum and purged with 100% argon gas. For the positive control, the reaction mixture comprised 5 µL purified N2OR (0.956 µg/µL), 0.05 mL of 10 mM methyl viologen, and 0.05 mL of 5 mM sodium dithionite in 2 mL of 10 mM KH2PO4 (pH 7.1). For transgenic root samples, the mixture comprised 50 µL root extract, 0.2 mL of 10 mM methyl viologen, and 0.2 mL of 5 mM sodium dithionite in 1.5 mL of 10 mM KH2PO4 (pH 7.1). For all samples, reactions took place in a stoppered 3.5-mL cuvette (light-path length 1 cm).In vitro N2O, was added in one 25-µL injection of N2O-saturated ddH2O. By monitoring the change in absorbance at 600 nm of the reaction mixture, the specific activity of the N2OR therein was determined . The specific activity was expressed as micromoles of N2O reduced per min per milligram of N2OR protein.Absorbance at 600 nm was monitored for 1 min to quantify the background oxidation rate. The substrate, NN. tabacum cv. Xanthi nc. plants expressing the nosZ gene under the transcriptional control of the root-specific A. rhizogenes rolD promoter  plant lines were grown from the seeds of the T0 generation, with no apparent phenotypic differences between transgenic and nontransgenic plants.We generated transgenic promoter . FourteenosZ transgene under the control of the rolD promoter, transgenic plants were screened by PCR using sequence-specific primers. NT plants were used as negative controls, while the plasmid prolD-nosZ or prolD-nosFLZDY was used as a positive control. PCR analysis of the putative prolD-nosZ transformed lines showed the presence of a 1869-bp band . PCR analysis confirmed the presence of nosZ in all transgenic lines, whereas no amplification was observed in NT lines.To confirm the presence of the -bp band , top rowsequence , top rownosZ transgene under the control of the rolD promoter, RT-PCR was conducted to amplify the nosZ fragment of the transgene with gene-specific primer sets. RT-PCR products with the expected size of 1869 bp for nosZ mRNA were observed in all nosZ-transformed and 1767 bp for nosZ mRNA were observed in nosFLZDY-transformed transgenic lines . NT controls did not exhibit any bands corresponding to nosZ transcripts. There were notable differences in transcript abundance among the nosZ and nosFLZDY transgenic lines. Transcript abundance was much higher in nosZ-transgenic plants than in nosFLZDY-transgenic plants. PCR was also conducted with total RNA not treated with reverse transcriptase to verify that there was no DNA contamination (data not shown).To confirm the transcription of the 2OR in tobacco root from T0 transgenic plants, an immunoblot assay of total soluble protein extracts was performed using a polyclonal antibody against the N2OR antigen. nosZ and rolD-nosFLZDY samples, showing a single band of the expected molecular mass of 72 kDa. The positive control, N2OR obtained from P. stutzeri, also yielded a signal band at 72 kDa. There was no signal for the presence of N2OR in nontransformed plant extracts. The images were scanned using ImageJ software (http://rsbweb.nih.gov/ij/) to semiquantify the expression of N2OR. The corresponding histogram shows yield of recombinant N2OR in µg N2OR/100 µg crude root protein. Recombinant protein yield varied among rolD-nosZ samples, with the lowest nosZ yield of 0.032 µg N2OR/100 µg crude root protein and the highest 0.079 µg N2OR/100 µg crude root protein. On average, the rolD-nosZ plants expressed N2OR at levels 10-fold higher than rolD-nosFLZDY plants, which varied from 0.003 µg N2OR/100 µg crude root protein to 0.009 µg N2OR/100 µg crude root protein.To demonstrate the expression of the N2OR in four transgenic tobacco lines was assessed using a spectrophotometric methyl viologen-linked assay. A600 was plotted against time for the slope-based activity calculations, and the calculated specific activity of the recombinant enzyme (expressed as µmol N2O reduced min−1 g−1 root protein) is presented in 2O reduced min−1 g−1 root protein). The positive control, N2OR from P. stutzeri, gave a specific activity of 454 µmol N2O reduced min−1 mg−1 protein. N2OR from the four samples assayed showed considerable variability in their specific activity. Activity was assayed for one individual plant per transgenic line, for the same lines assayed in The specific activity of N2OR into the rhizosphere. Fractions of hydroponic medium collected over a 25-day period from individual plants were pooled, and the crude protein extracted. The resulting immunoblot is shown in P. stutzeri is present in the exudates from all transgenic lines. The nontransformed control did not exhibit a signal for the presence of N2OR. Yield of recombinant N2OR was determined based on the chemiluminescent signal and ranged from 0.016 µg N2OR/100 µg crude root secreted protein (rolD:nosZ-5.8) to 0.044 µg N2OR/100 µg crude root secreted protein (rolD:nosZ-1.7).A Western immunoblot was performed to test for the expression and subsequent excretion of recombinant N2O reduced min−1 g−1 root secreted protein. The nontransformed control showed a background specific activity (1.72 µmol N2O reduced min−1 g−1 root secreted protein). The purified enzyme itself serving as a positive control, N2OR from P. stutzeri, gave a specific activity of 454 µmol N2O reduced min−1 mg−1 protein. N2OR from the rolD:nosZ-1.7, rolD:nosZ-1.9, and rolD:nosZ-1.10 transgenic lines showed the highest specific activities of 7.03, 6.27, and 5.66 µmol N2O reduced min−1 g−1 root secreted protein, respectively. This is less than half the specific activities of N2OR located in the root tissue of the same transgenic lines .The crude protein extracted from the hydroponic culture medium was assayed for specific activity using the methyl viologen-linked assay. ines see . In cont2O emission rates have been exacerbated with the increasing use of synthetic nitrogen-based fertilizers whereby annual N2O emissions from global soils now exceed 2.6 Tg (1 Tg = 1 million tons) of N2O-N , roots might be exposed to sufficient levels of N2O to induce higher production of recombinant N2OR  that are engineered to protect soil and groundwater by reducing the spraying of insecticides and herbicides. Such gene pyramiding is growing rapidly in genetic engineering, delivering many input and output advantages to producers and consumers, for example, enhanced water-use efficiency, enhanced cold tolerance, and increased yield. Extending the remit of biotech crops to include soil gas flux, namely, reduction of fertilizer-induced N2O emission, should encounter positive regulatory approval given the potential contribution to purge those same soils of this deleterious gas . If N2OR was expressed in maize, the conversion of N2O by the roots of maize could be as high as 2.11 × 103 g N2O ha−1 day−1 (Supplementary Methods), more than 50 times greater than N2O emissions from agricultural soil. By accumulating sufficient levels of N2OR, soils growing these transgenic crops could capture and alleviate N2O pollution.Since “arable lands” produce two-thirds of the world's N2O , field cious gas . Agricul


Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4β2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4β2 nicotinic acetylcholine receptors. The IC50 obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4β2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4β2 subtype.Crude extracts and three isolated alkaloids from  Erythrina mulungu (Papilionaceae) is a native tree from Southern Brazil, known as mulungu or coral tree due to its reddish flowers E. mulungu are often used in Brazilian traditional medicine as mild sedatives and to treat insomnia and depression E. mulungu produce anxiolytic-like effects in rats submitted to elevated T-maze and light/dark transition tests E. mulungu reproduced some of the CNS effects of the polar extracts E. americana is widely used as selective antagonist of neuronal α4β2 nicotinic receptors at nanomolar concentrations E. mulungu alkaloids are structurally related to DHβE and other erythrinian alkaloids known to act on nicotinic receptors E. mulungu alkaloids  with Earle's salts  supplemented with 5 g/l of d-glucose, 2 mM glutamine, 3.7 g/l NaHCO3, and 10% horse serum . Plating medium further contained 10% fetal bovine serum  and 20 µg/ml deoxyribonucleotidase type II and was progressively replaced by maintenance medium starting 1 or 2 days after plating. Cell proliferation was inhibited 6 to 7 days after plating, when the glial monolayer was confluent, by the addition of 14 µg/ml 5-fluoro-2'-deoxyuridine and 7 µg/ml uridine. Half of the medium was changed twice a week, and cultures were used 14 to 40 days after plating. Culture medium components were purchased from Invitrogen or Sigma .Pregnant Wistar rats at 18 to 20 days of gestation were sacrificed under CO2 in RPMI 1640 medium with 5% FBS, 10% HS, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were plated in collagen-coated Petri dishes and maintained for at least three days before experiments.Wild-type rat pheochromocytoma PC12 cells  provided by Dr. S. S. Smaili  were grown at 37°C in a humidified incubator under 10% CO2 in minimal essential medium with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were plated in Petri dishes and maintained for at least three days before experiments. Genes for the rat α4 and β2  nicotinic acetylcholine receptor subunits were kindly donated by J. Boulter  HEK 293 cells  provided by Dr. J. P. B. Viola  were grown at 37°C in a humidified incubator under 5% CO2 2, MgCl2 1, D-glucose 10, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) 5, and NaOH ∼2, pH 7.3, with added tetrodotoxin , when recording in neurons, and atropine sulfate (0.5 µM). This solution replaced the culture medium approximately 20 min before the recordings and was continuously perfused at a rate of ∼1 ml/min throughout the experiments. A U-tube system controlled by the pClamp software was used to deliver fast pulses of drug solutions onto the patch-clamped cells. Unless otherwise noted, fast nicotinic responses were evoked by pulses of acetylcholine chloride dissolved in extracellular solution. Pulse duration was 0.5 s for neurons and PC12 cells, and 2 s for HEK 293 cells. In screening experiments, putative antagonists were applied with acetylcholine, through the U-tube only, but for the inhibition curves they were also pre-applied before acetylcholine via bath solution so that they were in equilibrium at the time of the agonist pulse. Patch micropipettes were made from borosilicate glass capillaries  in a P-97 horizontal puller . The intracellular solution was (in mM) CsCl 80, CsF 80, glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) 10, HEPES 10, and CsOH ∼6, pH 7.3. The filled patch microelectrodes had resistances of 2 to 5 MΩ in the bath; the access resistance was left uncompensated. Recordings were made at room temperature (23°C).Whole-cell membrane currents were recorded at a membrane potential of –67 mV for neurons and –87 mV for PC12 and HEK 293 cells. Values are corrected for measured liquid junction potentials. The recordings were made with an EPC-7 patch-clamp system . Currents were low-pass filtered at 3 kHz (8-pole Bessel) and digitized with a LabMaster interface under the control of pClamp software (Axon Instruments). The standard extracellular solution was (in mM) NaCl 165, KCl 5, CaCl50) are reported with their 95% confidence interval.Peak amplitude and area (charge transfer) were measured for each current trace after baseline subtraction, using pClamp software. These values were then expressed as percent of the controls measured in the same cell. A minimum of three cells were assayed per condition and data are reported as mean ± SEM. Concentration-response data were analyzed by nonlinear regression with the Hill equation, constraining the maxima and minima to 100% and 0%, respectively. The estimated mean inhibitory concentrations  and a Hill coefficient of 1.0 (data not shown). As expected, currents elicited by 2-s pulses of acetylcholine 100 µM were completely blocked by 100 nM DHβE, when the cells were pre-exposed to the inhibitor (data not shown). To screen for inhibition, cells were stimulated by 50 µM acetylcholine pulses and then concomitantly by acetylcholine and one of the three alkaloids at 10 µM (Inward currents were evoked by acetylcholine in HEK 293 cells transiently expressing α4β2 nicotinic acetylcholine receptor with an ECat 10 µM . (+)-Ery50 of 46 µM (38 to 57 µM) and were blocked by low concentrations of mecamylamine (1 µM) in a use-dependent manner. Furthermore, neither 100 nM DHβE nor 1 nM methyllycaconitine (an α7* nicotinic acetylcholine receptor antagonist) affected these currents and choline acted as partial agonist (data not shown). These data are typical of responses mediated by ganglionic (α3*) nicotinic acetylcholine receptor with negligible contribution of α4 or α7-containing receptors We next tested whether the alkaloids were able to block nicotinic responses in PC12 cells, a widely used model for the study of neuronal nicotinic acetylcholine receptors. We have not induced further neuronal differentiation of the cells, which were round, adhered lightly to the collagen substrate and presented 2–3 short processes (< 3 µm). In these cells, whole-cell currents elicited by acetylcholine showed an ECIn single application experiments, all three alkaloids (at 50 µM) were able to reduce currents elicited by acetylcholine 100 µM when co-applied with this agonist . Again, 50 value of 5.9 µM (3.9 to 9.0 µM), with a Hill coefficient of 0.50. In HEK 293 cells expressing the α4β2 nicotinic acetylcholine receptor, (+)-erythravine potently inhibited the currents evoked by 50 µM acetylcholine. Inhibition was nearly complete at 1 µM and the IC50 value was 4.4 nM (2.1 to 9.4 nM), with a Hill coefficient of 0.61.The two most potent compounds were then chosen for further quantitative evaluation of their interaction with the α7* and α4β2 nicotinic acetylcholine receptor. For the inhibition curves, each concentration of the alkaloid was applied to the cell by bath perfusion before the agonist pulse, which also contained the alkaloid. Responses were measured as the areas under the current trace. (+)-Erythravine at increasing concentrations progressively inhibited the response to 300 µM acetylcholine in α7-expressing neurons . Non-lin50 value of 4.8 µM (3.4 to 6.7 µM), and a Hill coefficient of 0.53. In HEK 293 cells expressing the α4β2 nicotinic acetylcholine receptor, (+)-11α-hydroxyerythravine also potently inhibited acetylcholine-stimulated currents, with an IC50 value of 11.9 nM (7.7 to 18.3 nM) and Hill coefficient of 0.58.The hydroxylated analogue (+)-11α-hydroxyerythravine inhibited the response to acetylcholine in α7-expressing neurons  with a sE. mulungu previously shown to have anxiolytic-like effects in rodents inhibit neuronal nicotinic acetylcholine receptors. Although the two main receptor subtypes expressed in the mammalian brain were sensitive to the alkaloids, the α4β2 receptor was more potently inhibited. Because low nanomolar concentrations of (+)-erythravine and (+)-11α-hydroxyerythravine markedly inhibited the α4β2 receptor function, this receptor must be considered a possible target of phytotherapeutic preparations of E. mulungu.Our data demonstrate that the alkaloids from E. mulungu extracts. Santos Rosa and collaborators E. mulungu and demonstrated a marked anticonvulsant and a limited anxiolytic-like effect of this alkaloid (i.c.v.) in rats. Based on negative results in binding and uptake assays in synaptosomes, the authors excluded mechanisms involving GABA or glutamate signaling. In fact, erysotrine inhibits currents mediated by α4β2 and α7 nicotinic receptors expressed in Xenopus oocytes with IC50 of 0.37 µM and 17 µM, respectively E. mulungu.In spite of the high potency of (+)-erythravine and (+)-11α-hydroxyerythravine, other compounds may contribute to the α4β2 receptor-dependent effects of Several erythrinic alkaloids are known to be competitive antagonists of CNS nicotinic receptors Erythrina species have been known to induce neuromuscular blockade for a long time, at least since the report by Dominguez and Altamirano in 1877  nicotinic receptors, they are likely to be much less potent than what we have found for α4β2 receptors.Extracts of cited in  and manyThe actual mechanism by which nicotinic acetylcholine receptors affect anxiety is controversial but available evidence suggests the involvement of 5-hydroxytryptamine receptors S-(+)-mecamylamine (TC-5214) has reached phase III clinical trial for concomitant use with citalopram in patients unresponsive to citalopram alone E. mulungu alkaloids are potent antagonists of α4β2 nicotinic receptors provides a strong working hypothesis for the mechanism of action of the plant extracts, traditionally used to treat insomnia and agitation. Our data also imply that bioassays for neuronal nicotinic receptor antagonist activity could be used to assess the pharmacological activity of the extracts, providing a rational basis for product standardization and for dosing recommendations.The mounting evidence of the involvement of nicotinic receptors in anxiety and depression pathways has prompted the search for novel tools to investigate them, as well as for new compounds with potential clinical use. The non-selective stereoisomer