Erianthus, displayed an intermediate ability to use nitrate. We conclude that discrimination against nitrate and a low capacity to store nitrate in shoots prevents commercial sugarcane varieties from taking advantage of the high nitrate concentrations in fertilized soils in the first three months of the growing season, leaving nitrate vulnerable to loss. Our study addresses a major caveat of sugarcane production and affords a strong basis for improvement through breeding cultivars with enhanced capacity to use nitrate as well as through agronomic measures that reduce nitrification in soil.Modern agriculture is based on the notion that nitrate is the main source of nitrogen (N) for crops, but nitrate is also the most mobile form of N and easily lost from soil. Efficient acquisition of nitrate by crops is therefore a prerequisite for avoiding off-site N pollution. Sugarcane is considered the most suitable tropical crop for biofuel production, but surprisingly high N fertilizer applications in main producer countries raise doubt about the sustainability of production and are at odds with a carbon-based crop. Examining reasons for the inefficient use of N fertilizer, we hypothesized that sugarcane resembles other giant tropical grasses which inhibit the production of nitrate in soil and differ from related grain crops with a confirmed ability to use nitrate. The results of our study support the hypothesis that N-replete sugarcane and ancestral species in the Andropogoneae supertribe strongly prefer ammonium over nitrate. Sugarcane differs from grain crops, sorghum and maize, which acquired both N sources equally well, while giant grass,  We examined the validity of the long-held paradigm that nitrate is the main N source for most crops. Nitrate is 5 to 10-times more mobile in soils than alternative N sources ammonium and amino acids versus applied fertilizer-N indicate that N-efficiencies vary considerably across countries. More N-efficient countries, including Brazil and Philippines, have N removal/N-fertilizer input ratios of >0.6 while less efficient countries, including China and Pakistan, have ratios of 0.1 and 0.2  were similar soon after application of 110 kg N ha−1 . In the −1 . In cont−1 . The incThe inherent difficulties associated with quantifying N turnover and interactions between plants, microbes and soil limit our knowledge of which N sources are used by soil-grown plants 15NH4NO3 or NH415NO3 were supplied over 24 h to plants grown at limiting (0.4 mM N), intermediate (1 mM N) and high  N supply in inert substrate. Biomass and N content increased significantly (P<0.001) with N supply rate; low and intermediate N-grown plants produced 26 and 76% of the biomass of N-replete plants (15N-nitrate and 15N-ammonium into shoots and roots (P<0.05) less 15N-nitrate than 15N-ammonium. Incorporation of 15N-nitrate was 5-times lower in roots and 4 to 10-times lower in shoots than 15N-ammonium incorporation  higher than 15N-nitrate incorporation in roots of unfertilized and fertilized plants after 30 minutes  while nitrate contents of shoots were similar (11 and 9 µmol NO3− g−1 dry weight). Nitrogen content of roots and shoots of unfertilized and fertilized plants were similar indicating that differences in N status are only evident in the higher nitrate content of roots of fertilized plants. Increasing nitrate incorporation of unfertilized sugarcane over the 120 minutes incubation period suggests that nitrate transporters were induced in root membranes facilitating transport of nitrate into root cells. Similarly, supply of nitrate to roots of Arabidopsis increased mRNA levels of a dual-affinity nitrate transporter, with Km values of 50 and 4000 µM for high and low-affinity phases, after 30 minutes and nitrate uptake increased in the following 2 to 3 hours Prior to the incubation, roots of fertilized plants contained ∼3-fold more nitrate than unfertilized plants  to minimise the effect of microbial conversion of the supplied N.To provide insight into nitrate use and the regulatory mechanisms involved, nitrate storage and uptake of ammonium and nitrate were assessed in closely related grass species in two experimental comparisons. In the first comparison, 3 genotypes each of the two ancestral species of commercial sugarcane, Saccharum species demonstrated a low capacity to store nitrate in shoots as evidenced by low concentrations of 5 to 13 µmol nitrate gdw−1  in roots and shoots of sugarcane and in shoots of S. spontaneum than 15N-ammonium . The top 20 cm of soil was collected with an auger, kept at stable temperature then analyzed within 24 h. Soil solution containing the dissolved N fraction was collected from approximately 25 g of fresh soil by centrifugation for 45 min at 4500 rpm. Soluble N was extracted using 1.5 M KCl in a 1∶2 soil:solution ratio, shaken for 1 h then centrifuged at 4000 rpm for 10 min. Supernatant containing soluble N and soil solutions containing dissolved N were analysed for nitrate by colorimetric assay 18 1.7µm 2.1×50 mm analytical column and a tuneable UV detector at 254 nm. Extracts were derivatized using the AccQ-Tag™ derivatisation kit .Soil mineral N was sampled from a Q117 plant crop at a subtropical site near Bundaberg Queensland, Australia. The soil was characterized as a yellow to brown dermosol with the top 20 cm a sandy loam. The site was fertilized with ammonium-urea at 110 kg N haSaccharum spp. hybrids, Q138, Q157 and Q179A were grown from setts (stem cuttings) in coarse perlite within 15 cm diameter free-draining pots (2 L), and kept in a naturally lit glasshouse for 3 months. Plants received 200 mL of nutrient solution (pH 5.6) containing 0.2, 0.5 or 5 mM NH4NO3 and other nutrients as; 1 mM K2SO4, 2 mM MgSO4, 2 mM CaSO4, 0.457 mM KH2PO4, 42.5 µM K2HPO4, 100 µM FeEDTA, 20 µM MnSO4, 10 µM H3BO3, 1 µM CuSO4, 2.5 µM ZnSO4, 0.35 µM Na2MoO4 and tap water on alternate days. CaSO4 was added to lower N treatments to maintain equivalent osmolarity. 15N-labelled ammonium or nitrate (98 atom% excess) were added to each plant at 0.2, 0.4, 0.8, 1.6 or 3.2 mM 15N with equimolar concentrations of the unlabelled alternative N form. After 24 h, pots were rinsed with 10 mM KCl and roots cleaned of perlite. Plant tissue was dried at 55°C for 3 days and analyzed for 15N content and nitrate content.Commercial sugarcane cultivars, −1, 100 kg K ha−1 applied 2 weeks prior to sampling) and unfertilized (100 kg K ha−1) 3-month old KQ228A, were excavated approximately 20 cm from the plant base, kept moist and incubated in situ in buffered nutrient solution  with either 1 mM N of NH415NO3 or 15NH4NO3 (98 atom%) for 30 and 120 min at 25°C. Roots of a subset of plants from the fertilized plots were pre-treated with 50 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a protonophore, 30 min prior to 15N addition to indicate passive uptake. Roots were removed from plants, rinsed in 10 mM KCl, then water and dried at 55°C. Separate plants were used for each 15N source, CCCP protonophore treatment and time point combination and all treatments were repeated on 4 consecutive days. Entire aboveground shoot and incubated root material were analyzed for 15N content. No 15N was detected in shoot tissue of any treatment.Attached roots of fertilized ; 3S. officinarum cultivars ; 3 Saccharum-Erianthus hybrids of 3rd generation [Badilla*Erianthus]* CP84-1198]*ROC20, ; and 3 Erianthus spp.  were grown in perlite medium within 2 L, 15 cm diameter pots for 3 months with high N supply . N supply increased from 3 mM N at the start of the experiment to a final concentration of 30 mM N to ensure that plants were well supplied with N. Other nutrients were supplied as 5 mM K2SO4, 2 mM MgSO4, 2 mM CaSO4, 0.457 mM KH2PO4, 42.5 µM K2HPO4, 100 µM FeEDTA, 20 µM MnSO4, 10 µM H3BO3, 1 µM CuSO4, 2.5 µM ZnSO4, 0.35 µM Na2MoO4 with tap water supplied on alternate days. To compare uptake of different N forms 200 ml of solutions containing 15N-labelled ammonium, glycine or nitrate (98 atom%) were added to each plant with the equimolar concentration of the alternative N forms supplied as unlabelled ammonium, glycine and nitrate. After 24 h, pots were thoroughly rinsed with 10 mM KCl prior to harvest. Roots were carefully removed from pots, cleaned of perlite, rinsed with water, then dried and prepared for 15N analysis. Data from the 3 cultivars within each group were pooled for statistical analysis. Saccharum-Erianthus hybrids were generated by the Guangzhou Sugarcane Industry Research Institute at the Hainan Sugarcane Breeding Station, Hainan province, China. S. spontaneum and S. officinarum cultivars were supplied by BSES, Meringa, Australia.Experiment 1: Twelve genotypes including 3Zea mays , Sorghum bicolour , Erianthus arundinaceus (IJ76–394), Saccharum spontaneum (US71-4-1) and commercial sugarcane cultivar Saccharum hybrid (Q232A) were germinated and grown in a sandy loam  within 15 cm diameter free-draining pots (10 L) for 5 weeks in a glasshouse. Plants were supplied with 200 mL 10 mM NH4NO3 and other nutrients, as listed in experiment 1, 3 times a week and supplemented with tap water throughout. In the week preceding 15N application and harvest, stomatal conductance was measured on the youngest fully expanded leaf using a leaf porometer  over 3 days between 8am and 1pm. Plants were supplied with either 10 mM 15NH4NO3 or NH415NO3 (98 atom% excess) for 2 h from 9 to 11 am, after which the above-ground shoots of all plants were immediately removed, separated into leaf blade and stalk and dried. Prior to drying the surface area of leaf blades was measured for each plant with a Licor LI-3100C . Roots were removed from soil, rinsed in 10 mM KCl, then water and dried at 55°C for 3 days. Each of the 4 replicates was harvested on consecutive days. All species were in their vegetative growth stage to avoid phenology-based differences in N relations.Experiment 2: 15N and %N using an Integra-CN  at the UC Davis Stable Isotope Facility . Nitrate content was determined on 20% methanol dried tissue extracts Dried shoot and root material from glasshouse and field experiments were ground to a fine powder  and analyzed for post hoc test and data transformed where indicated throughout text.Data were analyzed using Statistica 9.0 . Significant differences between treatments were determined using (ANOVA) P<0.05 followed by Tukey's


Lactobacillus species on the immune environment in GALT, and the role of dendritic cells and heme oxygenase-1 in mediating these responses. Feeding with a specific strain of Lactobacillus rhamnosus induced a significant increase in CD4+CD25+Foxp3+ functional regulatory T cells in GALT. This increase was greatest in the mesenteric lymph nodes and associated with a marked decrease in TNF and IFNγ production. Dendritic cell regulatory function and HO-1 expression was also increased. The increase in Foxp3+ T cells could be prevented by treatment with a heme oxygenase inhibitor. However, neither inhibition of heme oxygenase nor blockade of IL-10 and TGFβ prevented the inhibition of inflammatory cytokine production. In conclusion Lactobacillus feeding induced a tolerogenic environment in GALT. HO-1 was critical to the enhancement of Foxp3+ regulatory T cells while additional, as yet unknown, pathways were involved in the down-regulation of inflammatory cytokine production by T cells.We investigated the consequences of feeding with a  The gut microbiota impacts various aspects of host physiology including modulation of the immune system Bacteroides fragilis protects animals from experimental colitis through induction of IL-10-producing CD4+ T cells Bifidobacterium infantis induces expression of Foxp3, a key transcription factor in programming Treg, in T cells that protect mice against Salmonella typhimurium infection Lactobacillus rhamnosus JB-1 in mice corresponded with a significant increase in the proportion of functional CD4+CD25+Foxp3+ Treg in the spleen and mediastinal lymph nodes There is evidence linking the immunomodulatory function of certain commensal bacteria, and components thereof, to induction of regulatory T cells (Treg) and their associated cytokines. In vitro, selective bacteria have been shown to induce IL-10 producing Treg L. rhamnousus JB-1, previously demonstrated to have immunomodulatory actions in a number of model systems Dendritic cells (DC) are among the first immune cells to encounter bacteria in the intestine and can directly present antigens from commensals to the mesenteric lymph node (MLN) 4 to 213±4.×104 following 9 days treatment. While the Foxp3+ cell population also increased in the Peyer’s Patches this reached a statistically significant level only after 9 days of treatment  with total Foxp3+ CD4+ cell numbers increasing from 0.37±0.003×104 to 1.5±0.09×104).Feeding with JB-1 lead to a significant increase in the percentage of CD4+ T cells expressing of Foxp3 in both the Peyer’s Patches (PP) and mesenteric lymph nodes (MLN). This increase was greatest in the MLN, where following 3 days of treatment the percentage of Foxp3+ CD4+ T cells increased from 1.9±0.05% to 5.6±0.1% and further increased to 15.6±0.25% and 14.2±0.1% at 5 and 9 days treatment respectively  this refL.salivarius, that we had previously demonstrated to lack some of the immunomodulatory effects of JB-1 When focusing on the expression of Foxp3 in the CD4+CD25+ population there was a small, statistically significant, increase in PP but again changes were most marked in the MLN . The inc+ CD25+ cells in the MLN of mice treated with JB-1 we wanted to assess whether this was associated with enhanced suppressor activity indicative of functional T regulatory cells. CD4+CD25+ cells from the MLN of JB-1 or vehicle-fed mice were freshly isolated ex vivo. These T regulatory cell subsets were co-cultured with CFSE-labeled CD4+ CD25– T cells from DO11.10 transgenic mice under activating conditions. Cells from JB-1 treated animals showed a significant (2 fold) increase in their ability to suppress proliferation of activated CD4+CD25– cells when compared to vehicle treated controls . Adult male Balb/c and DO.11.10 transgenic mice (20–25 g) were maintained in an automatic light/dark cycle (light periods of 12 h) and provided water and chow ad libitum. Mice were acclimatized to the animal facility for 1 week before experimentation. Age-matched (8–9 wk old) animals were used in all experiments.L. rhamnosus (JB-1), is the same as that employed in several published investigations L reuteri. This strain was recently confirmed as a Lactobacillus rhamnosus, by AFLP fingerprinting and full genomic analysis. It was identified as a strain distinct from L rhamnosus GG L. salivarius UCC118 were a gift from Dr. B. Kiely . Both strains were prepared from frozen stocks (–80°C) as described previously 8 bacteria/ml as determined by a Vitek colorimeter . Bacterial suspensions were then centrifuged and resuspended in MRS broth to give a concentration of 5×109/ml24. Suspensions of L. rhamnosus JB-1 were killed by γ-irradiation with Cobalt 60 for 20 hours at 8.05 Gy/minute. The resulting viability was determined by plating on MRS agar plates under anaerobic conditions for 72 hours at 37°C. No bacterial growth was detected in irradiated preparations after 72 hours of culture.Two bacterial strains were used in this study, 9 JB-1 or L.salivarius in 200 µl of MRS liquid medium broth via oral gavage needle daily for 3, 5 or 9 days. Control animals were treated daily with 200 µl of MRS broth alone. Following treatments animals were euthanized and Peyer’s Patches and MLN removed and processed for FACS analysis or cell isolation.Naïve mice received 1×10Heme oxygenase activity was inhibited in vivo using the potent and specific heme oxygenase inhibitor 8 bacteria were washed with PBS and the cell pellet was dissolved in PKH-26 diluent. PKH-26 fluorochrome was diluted in a separate tube in PKH-26 diluent. The bacterial suspension was added to the PKH-26-containing tube and incubated for 7 min at room temp. Staining was stopped by adding PBS containing fetal calf serum for 2 min. Bacteria were then washed three times with PBS and resuspended to the desired concentration in serum- and antibiotic-free medium prior to incubation with DC.Red fluorochrome PKH26  was used to label bacteria according to the manufacturer’s instructions. Briefly, 2×109 bacteria were washed with PBS and CFSE was added to the tube at final concentration of 5 µM and incubated for 10 min under constant shaking at room temperature. Staining was stopped by adding PBS containing FCS for 2 min. Bacteria were then washed three times with PBS, 5% FCS and resuspended in PBS at 5×109/ml prior to gavage.For in vivo studies bacteria were labeled with carboxy fluorescein diacetate succinimidyl ester (CFSE) as described previously Bone marrow cells were cultured in the presence of granulocyte monocyte colony stimulating factor for 7 days as previously described CD4+CD25+ cells were isolated from mesenteric lymph nodes of bacteria or vehicle fed BALB/c mice to 89% purity using MACS bead mouse Treg isolation kit . CD4+CD25– T cells were isolated from DO.11–10 transgenic mice to a purity of 94%. DC were isolated from mesenteric lymph nodes of naïve wild type mice using Miltenyi Biotec CD11c Microbeads.6 cells). This dose was chosen on the basis of previous studies Bacteria were co-cultured with BMDC at a ratio of 10∶1/bacteria:DC, for 24 hr at 37°C. DC were washed three times with PBS and viability assessed with trypan blue stain prior to injection into the footpad (1×103 ) were cultured with CD4+CD25– responder T cells isolated from DO11.10 transgenic mice (5 × 104 ) for 4 days in U-bottomed 96-well plates at a ratio of 1 Treg to 8 CD4+CD25– responder T cells in the presence of soluble anti-CD3 (1 µg/ml) and BM-DC (5 × 103 ). Prior to the co-culture assay, DC were pulsed with 1 mg/ml OVA protein for 16 h followed by a 20 min incubation with 50 µg/ml mitomycin C in a 37°C, 5% CO2 humidified incubator. Responder T cells were labeled with carboxy fluorescein diacetate succinimidyl ester (CFSE). Proliferation of CD4+ T cells from DO11.10 mice, detected by KJ1–26 mAb specific for the transgenic TCR expressed by DO11.10 T cells, was assayed by CFSE dilution using FACS analysis and proliferation calculated using FloJo version 9.5.2 software .To examine the suppressive capacity of JB-1 induced T-regulatory cells, we used the in vitro assay of T-regulatory cell activity as described previously 6 cells/ml with soluble anti-CD28 (2 mg/ml) for 48 hours. Supernatants were collected and stored at −80°C before cytokine quantification.Isolated mesenteric CD11c+ DC (Miltenyi Biotec ) were co-cultured with bacteria at a ratio of 10∶1/bacteria:DC, for 2 h at 37°C and after extensive washing were incubated with purified CD4+CD25– responder T cells from DO11.10 transgenic mice for 5 days in U-bottomed 96-well plates in the presence of soluble anti-CD3 (1 µg/ml) and 1mg/ml OVA protein. Where indicated, anti-IL-10 , anti-TGFβ  or CrMP 5 µM was added to the culture. On day 5 of culture T cells were harvested and replated into anti-CD3 (10 mg/ml) pre-coated 96-well cell culture plates and at a density of 1×106 cells/ml) were stimulated by plate bonded anti-CD3 and soluble anti-CD28 in 96-well cell culture plates and were incubated for 6 h in the presence of the protein transport inhibitor GolgiStop™ (BD Biosciences) prior to the staining Cultured DC or single cell suspensions from PP or MLN were stained as described previously 6 cells/ml with soluble anti-CD28 (2 µg/ml) for 48 h. Supernatants were collected and stored at −20°C prior to cytokine quantification. Cytokines in supernatants were analyzed with the mouse flex set cytokine cytometric bead array kit  and BD FACSArray bioanalyzer.96-well cell culture plates were coated with anti-CD3 (10 µg/ml) overnight. Cells were then plated at a density of 1×10Experimental results are expressed as means ± the standard errors of the means. Statistical analyses were performed by means of one-way analysis of variance (ANOVA), followed by Tukeys test for comparing all pairs of groups. Significant differences between two groups were determined using the unpaired Student's t test. A statistical software package (GraphPad PRISM™ version 5.0) was used for the analysis. A p value of less than 0.05 was considered statistically significant.


While HAART allows for the reconstitution of immune functions in most treated HIV patients, failure to achieve a significant increase in circulating CD4+ T cells despite undetectable viremia occurs.A retrospective study was conducted to evaluate the treatment outcome in a subgroup of 232 patients who after 3.1 years of treatment had not achieved desirable immune reconstitution despite a good virological response to HAART.After a further 3.5±2.7 years of HAART, 41 (17.7%) patients achieved immune reconstitution (681.4±172.7 CD4 cells/µL), while 191 (82.3%) patients did not (306.6±109.16 cells/µL); the difference in the achieved CD4 counts between these subgroups was significant (P<0.01). One patient experienced treatment failure. Eleven patients died to the end of follow-up, of which ten with a continuously dissociated response. Factors associated with immune recovery included, usage of PIs and of drugs from all three classes , and a rise in CD4 count to over 200 cell/µL after the first 3.1 years of treatment . Achievement of a rise in CD4 count to over 200 cell/µL after the first 3.1 years of treatment, and usage of all three drug classes were independent predictor of immune reconstitution in the following period.If patients on HAART reach CD4 cell counts of above 200 cells/µL in the first three years, immune recovery is possible after at least six years of treatment, particularly if treated with drugs from all three classes.


An increasing number of animal and human studies are indicating that inflammation is associated with behavioral disorders including aggression. This study investigates the association between chronic physical aggression during childhood and plasma cytokine levels in early adulthood.T(28.7) = 3.48, P = 0.002) and IL-6 (T(26.9) = 3.76, P = 0.001), lower anti-inflammatory interleukin IL-4 (T(27.1) = 4.91, P = 0.00004) and IL-10 (T(29.8) = 2.84, P = 0.008) and lower chemokine IL-8 (T(26) = 3.69, P = 0.001). The plasma levels of four cytokines accurately predicted aggressive and control group membership for all subjects.Two longitudinal studies were used to select males on a chronic physical aggression trajectory from childhood to adolescence (n = 7) and a control group from the same background (n = 25). Physical aggression was assessed yearly by teachers from childhood to adolescence and plasma levels of 10 inflammatory cytokines were assessed at age 26 and 28 years. Compared to the control group, males on a chronic physical aggression trajectory from childhood to adolescence had consistently lower plasma levels of five cytokines: lower pro-inflammatory interleukins IL-1α (Physical aggression of boys during childhood is a strong predictor of reduced plasma levels of cytokines in early adulthood. The causal and physiological relations underlying this association should be further investigated since animal data suggest that some cytokines such as IL-6 and IL-1β play a causal role in aggression. Physical violence is an important health problem, especially among males A growing body of research suggests that inflammatory cytokines might have systemic effects in addition to their traditional roles in the immune response. Recent studies have shown that cytokines are associated with various behavioral disorders such as anxiety, depression, suicide, childhood mood disorder and post-traumatic stress disorder (PTSD) Although alterations in the levels of various cytokines have been shown to be associated with human disease and behavioral disorders, the association between cytokine levels and long-term regulation of physical aggression in humans is still unknown. We therefore used two longitudinal cohorts of children to examine whether cytokine levels in blood of young adults associate with their history of childhood physical aggression. To test this hypothesis, we measured an array of 10 inflammatory cytokines in a sample of males who had a history of chronic physical aggression between 6 and 15 years of age and compared them with boys from the same background who followed a normal physical aggression trajectory We first compared the two groups, controls and chronic physical aggressive (CPA) to identify possible confounders . They diR = 0.554, P = 1.48E-17). Using repeated measure ANOVA, no significant mean differences between times and no interactions with the aggression groups for all the 10 cytokines were revealed after correcting for multiple testing (Bonferoni correction with α ≤0.005). The analyses were therefore performed on the average cytokine concentration observed at time 1 and time 2.We measured the plasma concentration of 10 cytokines using multiplex array-based ELISA at two time points (age 26 and 28) to obtain a reliable cytokine concentration for each subject. As expected, the cytokine concentrations at time 1 were significantly correlated with their concentrations 2 years later  = 2.9, P = 0.019). Student T-test analysis with Bonferroni correction (α ≤0.005) showed that the CPA group compared to the control group had lower pro-inflammatory interleukins: IL-1α (T(28.7) = 3.48, P = 0.002) and IL-6 (T(26.9) = 3.76, P = 0.001); lower chemokine: IL-8 (T(26) = 3.69, P = 0.001); and lower anti-inflammatory interleukin: IL-4 (T(27.1) = 4.91, P = 0.00004) concentration. A trend was also observed for the other anti-inflammatory interleukin interrogated, IL-10 (T(29.8) = 2.84, P = 0.008), going in the same direction with lower concentration in the CPA group. There were no other significant differences between the groups for the other cytokines analyzed (IL-1β (T(30) = 0.38, P = 0.71), IL-13 (T(30) = 1.08, P = 0.29), CCL2 (T(30) = −1.66, P = 0.87), IFNγ (T(30) = 0.75, P = 0.46) and TNF-α (T(30) = 1.46, P = 0.15)). Interestingly, Levene’s test of equality of variances indicated higher variance in basal cytokine concentration for IL-1α, IL-4, IL-6, IL-8 and IL-10 for the control group compared to the CPA group. These results show a tighter and lower basal concentration of pro and anti-inflammatory cytokines in the CPA group.Results obtained from the multiplex ELISA for 10 cytokines concentration in plasma analyzed for the CPA group and control group are displayed in P = 0.0004), IL-4 (P<0.0001), IL-6 (P = 0.0005) and IL-8 (P = 0.001) levels and marginally associated with IL-10 (P = 0.008) levels after Bonferroni correction (α <0.005). We then tested the association between cytokine levels and childhood hyperactivity as a possible confounder since the CPA group had a higher hyperactivity average score than the control group and hyperactivity is associated with CPA from childhood to adolescence P<0.05. Adjusting for both, family adversity and childhood hyperactivity in our regression analysis showed that the CPA group was still significantly associated with lower level of IL-4 (P = 0.023) and IL-8 (P = 0.017) and marginally associated with lower level of IL-1α (P = 0.060), IL-6 (P = 0.082) and TNF-α (P = 0.091). In addition, cytokine levels may vary in response to external stimuli such as infections. CRP, a well-known marker of infection, was also shown to associate with the hostility trait in humans P = 0.025) and IL-8 (P = 0.019) and marginally associated with lower level of IL-1α (P = 0.062), IL-6 (P = 0.088), IFNγ (P = 0.099) and TNF-α (P = 0.098).To further test the hypothesis that cytokines are associated with CPA rather than a confounder, we conducted regression analyses using linear mixed model on each cytokine adjusting for familial adversity. Familial adversity was chosen since it is known to predict CPA trajectory membership We used logistic regression to test how well the cytokine concentrations could classify the subjects in the two aggression groups (CPA-CG). Two cytokines were sufficient to classify all but 5 subjects in the appropriate aggression group. Cytokine pairs consisted of IL-4 and either CCL2, IL-13, IL-8 or IL-1β. All misclassifications were eliminated when we included 4 cytokines: IL-1α combined with two of the four other differentiating cytokines (IL-6/IL-4/IL-10/IL-8) and with one non-differentiating cytokine (IL-1β or CCL2). According to the Akaike Information Criterion (AIC), these 4-cytokine classifiers were the strongest of all possible cytokine classifiers.P = 0.036), IL-4 (P = 0.002), IL-6 (P = 0.007), IL-8 (P = 0.010). Bootstrap analysis done on 1000 permutations found the same cytokines to be associated with the aggression groups (IL-1α (P = 0.011), IL-4 (P = 0.002), IL-6 (P = 0.018), IL-8 (P = 0.006) and IL-10 (P = 0.036)).As the sample number was small and there was unequal variance in our sample we also applied Mann-Whitney and bootstrap nonparametric tests. Mann-Whitney analyses for each of the cytokines show similar results to the one obtained by Student t-test with significant group difference at α ≤0.05 seen for IL-1α  in mice resulted in increased aggression compared to control mice, which is consistent with our data showing reduced IL-6 in the CPA group The main limitation of the present study is the small sample size of the chronic aggression group. The two longitudinal studies we used to recruit subjects had followed more than 1000 males from childhood to adolescence. Unfortunately, young adult Caucasian males with a history of chronic physical aggression during childhood are relatively rare R = 0.554, P = 1.48E-17) suggests that one daytime sample is a relatively robust assessment.Diurnal variation has been reported for IL-6 This study has several implications. The results suggest that cytokines may be involved in chronic physical aggression, hence that a peripheral immune component may play a key role in regulating these behavioral states. We also showed that measuring the levels of a panel of 4 cytokines in plasma could accurately discriminate adult males with and without a history of childhood chronic physical aggression. This raises the possibility that cytokines could become peripheral biomarkers of risk for chronic physical aggression and related serious behavioral problems such as hyperactivity. New longitudinal studies that repeatedly assess cytokine, cortisol and physical aggression from early childhood onwards are needed to define the temporal relationship between changes in basal cytokine levels, cortisol and appearance of aggressive behaviors.The subjects were recruited from participants in two longitudinal studies of child development A total of 65 eligible subjects accepted to participate (8 CPA and 57 CG). One of the 8 CPA subjects had to be discarded because of data quality and for economic reasons we randomly reduced the CG group to 25. Characteristics of the 2 groups are presented in After complete description of the study to the subject, all participants provided written informed consent. The study was carried out in accordance with the Declaration of Helsinki, and was approved by the research ethics committee of the University of Montreal pediatric hospital .Blood samples (8 to 10 ml) were drawn in EDTA coated-tubes and shipped on ice to the laboratory within 24 hours. Blood samples were immediately resuspended in equal volume of PBS (1X) and plasma isolation was performed by centrifugation on a Ficoll-Paque gradient . The samples were then frozen and stored at −80°C in aliquots of 1 ml. Quantification of 10 cytokines in plasma was done by ELISA using the Quantibody® Human Inflammation Array 1 from RayBiotech following the manufacturer instructions for both time points C-reactive protein levels in plasma were quantified by Bio-Medic Laboratories using a particle enhanced immunoturbidimetric assay. Briefly, human CRP agglutinates with latex particles coated with monoclonal anti-CRP antibodies. The precipitate causes an increase in the intensity of scattered light and is proportional to the amount of CRP in the sample. All values were below the reference value for plasma (≤10.00 mg/L).The index of family adversity is a composite score of the degree of adversity in families ranging from 0 to 1 which has been used regularly with these cohorts. The index includes parent’s level of education, type of employment, age at the birth of their first child and marital status when the subjects were age 6 Self reported violence. During the data collection at 21 years, subjects were asked how often in the past year they had been implicated in physical fights and how often they had physically attacked someone.In the course of the longitudinal studies, teachers annually rated the frequency of boys’ physical aggression, opposition, hyperactivity, inattention and anxiety from kindergarten to secondary school with the Social Behavior Questionnaire Canadian youth between 13 and 17 years who commit delinquent acts are referred to the juvenile courts. From official records we identified subjects who were found guilty by a juvenile court. From official records we also identified subjects in each group who had been convicted of a criminal offence between 18 and 24 years.When the subjects were 15 years structured psychiatric interviews were used with the mothers and the subjects to assess the following psychiatric disorders: simple phobia, anxiety of separation, generalized anxiety, hyper anxiety, major depression, dysthymia, oppositional disorder and conduct disorder P<0.1. Linear regression analyses were performed using statistical analyses software (SAS 9) version 9.2 . The cytokine classifications were assessed using logistic regressions and the Akaike Information Criterion (AIC) values in R software environment for statistical computing.The normalized cytokine concentrations and CRP values in plasma showed a right-skewed distribution. In order to achieve normality, the data were transformed using log based 2. The correlation between cytokine concentrations at time 1 and time 2 was calculated using linear mixed effects model with concentrations grouped by subject and cytokine in the R software environment for statistical computing P = 0.004) than by including them (P = 0.008). Since for 9 out of 10 cytokines the results were not affected, the analysis presented here included these 4 values of 0 in all the analysis to allow equal number of subject for each cytokine. Nine of the 10 cytokines analyzed (excluding CCL-2) had at least one or more values outside of the distribution (> upper quartile +1.5 times the interquartile range (IQ)) that could be considered as possible outliers. Comparing the groups with these data brought into range  Excluding the 4 undetectable values, mentioned above, in the final analysis did not affect the significant group differences observed for all the cytokines except for IL-10. IL-10 group difference observed without the undetectable values gives a slightly lower P value (


Daphne genkwa Sieb. et Zucc. (Thymelaeaceae) are a Chinese herbal medicine used as an abortifacient with purgative, diuretic and anti-inflammatory activities. However, the activity of this medicine against enteroviral infections has not been investigated. The water-extract of dried buds of D. genkwa Sieb. et Zucc. (DGFW) was examined against various strains of enterovirus 71 (EV71) by neutralization assay, and its initial mode of action was characterized by time-of-addition assay followed by attachment and penetration assays. Pretreatment of DGFW with virus abolished viral replication, indicating that DGFW inhibits EV71 by targeting the virus. GFW exerts its anti-EV71 effects by inhibiting viral entry without producing cytotoxic side effects and thus provides a potential agent for antiviral chemotherapeutics. Dried flowers of  Enterovirus genus of the family Picornaviridae. It is a member of the Picornaviridae family and has a (+)RNA genome 7.5 kb in length that encodes 2193 amino acids . In. In2 in Daphne genkwa were supplied and authenticated by the Department of Pharmacy, Chang Gung Memorial Hospital at Chiayi, Taiwan. A voucher specimen (No. CGU-DG-1) was deposited in the herbarium of Chang Gung University, Taoyuan, Taiwan. Dry flower buds (5.0 kg) were extracted with methanol seven times (30 L each) under reflux for 8 h and concentrated to give a brown syrup (745.75 g) .Dry flower buds of 45.75 g) . The syr4 per well) were cultured in 96-well plates overnight before use. EV71 TW/2231/98 was used hereafter if not mentioned otherwise. A virus suspension with threefold serial dilution in DMEM containing 2% FBS (E2) was added to the cells and incubated at 37 °C for 72 h and subsequently fixed with 100 μL of 4% paraformaldehyde (PFA) for 2 h. The attached cells were stained with 0.1% crystal violet at room temperature, and the density of staining was measured in a microplate reader . The dilution causing cytopathology in half the cultures  was then calculated using the Reed–Muench method. The TCID50 determinations for the attachment and penetration assays were performed similarly except that preadsorption was carried out for 3 h on ice. A neutralization assay (EC50) was used to test the antiviral efficacy of extracts or compounds by measuring the inhibition of CPE induced by enterovirus on RD cells. RD cells (2 × 104 cells/well) were seeded into 96-well plates and incubated for 16–20 h at 37 °C in a 5% CO2/air incubator. After infection, cells were overlaid with 50 μL of different concentrations of extracts and incubated at 37 °C in a 5% CO2/air incubator for 72 h. At the end of incubation, cells were fixed with PFA, followed by 0.1% crystal violet staining. The concentration required for extracts to reduce the virus-induced CPE by 50% relative to the virus control was defined as the EC50.Monolayers of RD cells -2,5- diphenyltetrazolium bromide; 0.5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) was added at 50 μL/well and incubated for 3 h. Dimethylsulfoxide  was added to dissolve the crystals, and the absorbance at 570 nm of each well was measured using a microtiter plate reader . The concentration of DGFW that caused the death of 50% of the cells was defined as the 50% cytotoxic concentration (CC50).Aliquots of 2 × 105 per well) were seeded in 6-well plates. After an 18 h culture, the cells were inoculated with serially diluted viral suspensions for 1 h. The cells were washed with DPBS and overlaid with warm 0.3% agarose in E2. After incubation for 4 days, the cells were fixed with 4% PFA for 2 h. The agarose was carefully removed and subsequently stained with 0.1% crystal violet. The titer of the virus was expressed as plaque-forming units (PFU) per mL.Monolayers of RD cells . The proteins of interest were incubated with primary antibodies for 1 h and then incubated with appropriate secondary antibodies. Proteins were detected by using an enhanced chemiluminescence western blotting detection system (Millipore). Polyclonal antibodies against 3A and a monoclonal antibody against 3C were as described ,32.5 cells/mL) were seeded into 6-well plates and then challenged with viruses at an m.o.i. of 1. The cells were pretreated with viruses for 1 h on ice and washed with DPBS, and then E2 was added. After 20 h incubation at 37 °C, the cells were stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid, and CPE was observed using a microscope. For DGFW treatment, the cells were pretreated with 0.5 mg/mL extracts and virus simultaneously for 1 h and washed with DPBS, and then E2 containing 0.5 mg/mL DGFW was added. The protective effect exerted by DGFWwas also observed after incubation for 20 h at 37 °C.RD cells  × 6.022 × 1023 = Y molecules/μL. The viral RNA was then mixed with 50 nM forward and reverse primers and SYBR green PCR Master Mix  followed by qPCR analysis using an ABI StepOne Plus Sequence Detector System (Applied Biosystems). The primers used were: EV71 VP2 forward, 5´–CTG ATG GCT TCG AAT TGC AA–3´, and reverse, 5´–GCG TTT ATG TAC GGC ACT ATT ATT GT–3´; GAPDH forward, 5´–GGT GGT CTC CTC TGA CTT CAA CA–3´, and reverse, 5´–GCG TCA AAG GTG GAG GAG TG–3´. A standard curve  was thus generated using different dilutions of the standard. RD cells (4 × 105 cells/well) were seeded in 6-well plates overnight. The cells were then preadsorbed with EV71 at an m.o.i. of 5 for 1 h in the presence of 0.5 mg/mL DGFW. The culture medium was then replaced with E2 containing the same concentration of DGFW, and the cells were incubated at 37 °C and harvested at 0, 3, 6 and 9 h post infection (p.i.) for total RNA extraction using TRIzol. cDNA was prepared by reverse transcription with M-MLV RT kits  for qPCR analysis using specific viral primers: forward, 5´–CTG ATG GCT TCG AAT TGC AA–3´, and reverse, 5´–GCG TTT ATG TAC GGC ACT ATT ATT GT–3´. Amplification was achieved using the following universal thermal cycling protocol: one cycle at 95 °C for 15 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The absolute amounts of viral RNA were then determined using an external standard curve.The 5 cells/well), and incubated at 37 °C for 16–20 h under 5% CO2 in air. The cells were infected with EV71 at an m.o.i. of 5 on ice. After viral adsorption, the cells were washed with Hank’s buffered salt solution to remove any unbound virus. DGFW (0.5 mg/mL) in E2 was added at −9 to −1 h, −6 to −1 h and −3 to −1 h (preadsorption), −1 to 0 h (adsorption), 0 to 10 h, 3 to 10 h and 6 to 10 h (postadsorption). The supernatants were harvested at 10 h p.i., and the viral titers were determined by plaque-forming assays.A 6-well tissue culture plate was seeded with RD cells (4 × 104 cells/well), which were then incubated overnight at 37 °C under 5% CO2 in air. The cells were chilled on ice for 1 h, and the medium was removed. The cells were infected with 100 TCID50 of EV71 in the presence of increasing concentrations of DGFW on ice for 3 h. The medium containing the unadsorbed virus was removed, and the cells were then washed three times with DPBS. After incubation for 72 h in E2 at 37 °C, cells were fixed with 4% PFA and stained with crystal violet. Cell viability was determined by measuring the cell density in a microplate reader . A Pueraria lobata extract at 3.125 mg/mL served as a positive control [A 96-well tissue culture plate was seeded with HFF3 cells (2 × 10 control . This at control .4 cells/well), which were then incubated overnight at 37 °C under 5% CO2. The cells were chilled on ice for 1 h, and the medium was removed. The cells were infected with 100 TCID50 of EV71 on ice for 3 h. The medium containing unbound virus was then removed; various concentrations of DGFW in E2 were added, and the cells were incubated at 37 °C for 1 h to trigger endocytosis of the virus. The infected cells were then treated with alkaline phosphate-buffered saline  for 1 min to inactivate any viruses that had not penetrated the cells, and then acidic PBS (pH 3) was immediately added to neutralize the mix. The neutralized medium was removed, E2 was then replaced, and the cells were incubated at 37 °C for 72 h. The cells were fixed with PFA and subjected to 0.1% crystal violet staining. A Pueraria lobata extract at 3.125 mg/mL served as a positive control [A 96-well tissue culture plate was seeded with HFF3 cells (2 × 10 control . This pe control ,36.t-tests with p < 0.05 taken as significant.Data are expressed as the mean ± standard error of the mean  and analGFW exerts its anti-EV71 effects by inhibiting viral entry without producing cytotoxic side effects and thus provides a potential agent for antiviral chemotherapeutics.


Evidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs) to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function.in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O2) or hypoxic (2% O2) conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM) had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist) had no affects on heart function, whereas DPCPX (A1AR-specific antagonist) had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR−/−) had elevated heart rates compared to A1AR+/− littermates, A1AR−/− heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR−/− embryos.Effects of caffeine and adenosine receptor-selective antagonists on heart rate were studied These data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic hypoxia. Mature and developing mammals modulate cardiac output through alteration of heart rate and cardiac contractility In the embryo and fetus, hypoxia is a stressor with multiple etiologies that include placental insufficiency, maternal smoking, anemia, umbilical cord compression, pre-eclampsia, and living at high altitudes Adenosine is a crucial humoral mediator of functional changes induced by hypoxia Recent studies show that adenosine acts via A1ARs in the heart to protect embryos against hypoxia Caffeine is a multifunctional compound that influences intracellular calcium levels, inhibits phosphodiesterase activity, and blocks GABA receptors Considering that adenosine protects the embryo against hypoxia and that A1ARs are the dominant regulator of embryonic heart rate, we postulated that caffeine alters the normal embryonic cardiac response to hypoxia. To test this hypothesis, we examined responses to hypoxia and adenosine receptor blockade on embryonic cardiac activity at two ages spanning the critical period of cardiovascular morphogenesis, E9.5 and E12.5 To assess the effects of caffeine on embryonic heart function, whole embryos at E9.5 or isolated hearts at E12.5 were studied. At E9.5, the heart is at an early stage of cardiogenesis, corresponding to only one and a half days after the start of cardiac contractility in vivo using Doppler ultrasonography, or in situ via hysterectomy Before treatments, baseline heart rates were measured following a 1–2 h period. Mean baseline heart rates in the C57Bl/6 embryos were 109.2 beats per minute (BPM) at E9.5  and 124.2 BPM at E12.5 . There was a significant age-dependent increase in mean baseline heart rates (p = 0.0001). These values are similar to those observed for murine embryos studied in culture max and EC50. In the E12.5 hearts, caffeine treatment increased heart rates to an Emax of 38% above baseline levels (400 µM), with an EC50 of 118.4 µM , DPCPX caused significant elevations in heart rates that were more pronounced at E12.5 (67% increase) than at E9.5  of Yale University approved all experiments conducted on animals (protocol #2007-11136). The inbred mouse strain C57Bl/6  and A1AR-deficient mice (A1AR −/−) were studied 2. Embryonic age was determined by morphology and somite number 2 –room air incubator for 1–2 h prior to conducting tissue hypoxia studies and heart rate analyses.Embryos and hearts were cultured as described 2 exposure, C57Bl/6 embryonic tissues were cultured with 200 µM Hypoxyprobe-1 . Hypoxyprobe-1 forms protein adducts in O2 levels <10 mm Hg that can be detected by immunostaining 2 in a Plexiglass chamber  for 1 h or left in the incubator for 1 h for room air controls. O2 levels were maintained at 2% with a regulator (Biospherix) by introducing 95% nitrogen with 5% CO2 into the chamber. Chamber temperature was maintained between 35°C and 37°C with a heating cord. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Conjugates were visualized with a monoclonal antibody to Hypoxyprobe-1  followed by a goat anti-mouse secondary antibody conjugated to Alexa488 , as described To assess if there was embryonic tissue hypoxia associated with 2% OUsing the chamber described above, heart rate measurements were performed through direct visualization with a dissecting microscope over 15-second intervals by a single individual who was blinded to treatment conditions. Measurements were performed in triplicate per sample at each time point.To generate concentration-response curves at E9.5 and E12.5 under normoxic conditions, individual specimens of the C57Bl/6 strain were treated with vehicle or increasing concentrations of caffeine . Six to 12 specimens were analyzed per experiment. Following a 1–2 h initial incubation period, the baseline heart rate for each specimen was measured. Individual specimens were treated with increasing concentrations of caffeine  or vehicle without changing the media, and heart rates were reassessed after 2 min. In addition, at E12.5, individual hearts were treated with vehicle or a single concentration of caffeine  to refine the concentration-response curves.Heart rates of C57Bl/6 embryos were assessed following hypoxia exposure and/or treatment with adenosine receptor antagonists. Cultures were treated with 200 µM caffeine. This concentration was selected based on our caffeine concentration-response data see . In addi2 level was selected based on our Hypoxyprobe-1 studies that showed increased tissue hypoxia while embryonic cardiac function was preserved. Heart rates were measured after 5 min in 2.0% O2 (t = 50 min) and 20 min in 2.0% O2 (t = 65 min). Finally, heart rates were reassessed after a 10 min recovery period in room air O2 (t = 100 min). In control experiments to show that embryonic heart function in culture is stable over time, heart rates were assessed at these time points without hypoxia or caffeine treatment.Heart rate experiments began after an initial 1–2 h incubation period following dissections. Plates containing 6–12 specimens were placed in the chamber and baseline heart rates were measured (time [t] = 0 min). Drugs or vehicle were added to each well in a blinded fashion and heart rates were reassessed after 5 min (t = 20 min). The chamber oxygen levels were lowered to 2.0%. This OEffects of caffeine and hypoxia on A1AR knockout mice was determined as described above for C57Bl/6 mice.5′- ATGACAAGAAAAAGCGGATG – 3′, reverse 5′ – CTTTTCTGCCTGTTTCCGTA – 3′; A1AR: forward 5′ – GTCCTGGTATGTGACCAAGC – 3′, reverse 5′ – CTGAGGCTCTGGGTGAACTA 3′; A2aAR: forward 5′ – GCAGAACGTCACCAACTTCT – 3′, reverse 5′ – ATGGCGATGTATCTGTCGAT – 3′; A2bAR: forward 5′ TGCATGCCATCAACTGTATC – 3′, reverse 5′ – TGGAAACTGTAGCGGAAGTC; A3AR: forward 5′ – AGCGAGCTCTCAAAATAGCA 3′, reverse 5′ – ATAAAATGCACGCGTCTCTC 3′) and were obtained from RealTimePrimers.com . Each real-time PCR experiment was performed in triplicate. PCRs were analyzed by mean normalized expression using Microsoft Excel, with Rp113a as the control gene. DNA contamination was assessed by a “no RT control” real-time PCR reaction, no DNA contamination was observed.Gene expression was determined for all four adenosine receptor subtypes in isolated hearts from E9.5 and E12.5 C57Bl/6 embryos, as described 50, defined as the drug concentration that caused heart rates to increase to levels halfway between baseline and maximum levels, was obtained from these curves where the lowest concentration corresponded to the addition of vehicle. The maximal mean increase in heart rate from basal levels was defined as Emax. Differences in response profiles over time between treatment or genotype groups were analyzed with repeated measures two-way ANOVA, differences within groups at different time points were analyzed with repeated measures one-way ANOVA, and differences in mean baseline heart rates were analyzed with unpaired Student's t-test assuming equal variances or one-way ANOVA. Bonferroni's tests were used for post hoc analyses. Data are presented as mean +/− the standard error of the mean (SEM). Two-tailed p<0.05 was considered to be statistically significant.Heart rates were measured in triplicate and recorded in a spreadsheet (Microsoft Excel) where average heart rate in beats-per-minute (BPM) were calculated. To standardize heart rates from all studies, heart rates were recorded as percent change from baseline using the formula: percent change = heart Rate/baseline×100. Descriptive statistics including 95% confidence intervals (CI) were calculated using Excel . Concentration-response curves were generated using GraphPad Prism version 5 . The EC


PDYN (prodynorphin) fragments were used as model molecules. The presence of secondary DNA structures was evident from differences in oligonucleotide mobilities on PAGE, from CD spectra, and from formation of A-T, G-C, and non-canonical G-T base pairs observed by NMR spectroscopy. The oligonucleotides displayed secondary structures at 4°C, and some also at 37°C. Methylation at CpG sites prompted sequence-dependent formation of novel conformations, or shifted the equilibrium between different existing ssDNA conformations. The effects of methylation on gel mobility and base pairing were comparable in strength to the effects induced by point mutations in the DNA sequences. The conformational effects of methylation may be relevant for epigenetic regulatory events in a chromatin context, including DNA-protein or DNA-DNA recognition in the course of gene transcription, and DNA replication and recombination when double-stranded DNA is unwinded to ssDNA.Single-stranded DNA (ssDNA) is characterized by high conformational flexibility that allows these molecules to adopt a variety of conformations. Here we used native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy to show that cytosine methylation at CpG sites affects the conformational flexibility of short ssDNA molecules. The CpG containing 37-nucleotide  In the cell nucleus, DNA may be present in single-stranded form as an intermediate in gene transcription, and also during DNA replication, repair and recombination Cytosine methylation at CpG and non-CpG sites is a covalent DNA modification that plays an essential role in controlling gene transcription by turning off a specific gene or by inactivating an entire X chromosome PDYN) gene were used as model molecules because they a) contain CpG sites; and b) may be involved in DNA metabolic processes due to their potential ability to form ssDNA in the cell  spectroscopy, we analyzed a set of 37-mer ssDNA oligonucleotides with two or four CpG sites in each molecule. Effects of cytosine methylation of one or two of these CpG sites were examined. Because DNA secondary structures typically are most stable at low temperatures, the experiments were performed at 4°C, and for comparison at 37°C where non-canonical DNA secondary structures usually have melted the cell . These Psitivity  suggestisitivity , Table 1sitivity , Table 15mC1 (5-methylcytosine), Dyn (dynorphin) A, Dyn A (AS (antisense)), and Dyn A5mC2) was performed using PAGE on 15% denaturating gels in 10 M urea, followed by desalting on NAP 5 and NAP 10 columns . Oligonucleotide (20 ng) labeling was performed at the 5′-end using T4 polynucleotide kinase with [γ-32P]-ATP as a substrate, and the labeled oligonucleotides were precipitated by 66% ethanol and 1.2 M ammonium acetate in the presence of glycogen (0.01 µg/µl), followed by washing with 80% ethanol. Prior to labeling the oligonucleotides were heated for 10 min at 95°C. Native and denaturating PAGE analysis showed no differences in the behavior and purity between the HPLC purified and the double HPLC and PAGE purified oligonucleotides. The HPLC purified oligonucleotides were used in the CD and NMR experiments.The studied oligonucleotides and their abbreviations are shown in A Premier qTOF mass spectrometer  using negative-mode electrospray ionization was employed to study the homogeneity of selected methylated oligonucleotide samples. The oligonucleotide samples were dissolved to a concentration of 8 g/L in 1∶1 (v/v) water-acetonitrile solution to which 3% piperidine was added to assist with deprotonation and salt adduct removal. Each mass spectrum was accumulated for 5 minutes and the data were integrated. Neutral mass deconvolution was performed using commercial software . In addition, MALDI spectra provided by Eurofins MWG Operon GmbH  for showing the quality of some of the oligonucleotides analyzed in this study are presented in 2. To examine effects of ion composition, NaCl and MgCl2 were omitted from the loading buffer, or substituted with 0.5 mM ZnCl2 or 100 mM KCl or 50 mM NH4SO4. The samples were analyzed on a 15% polyacrylamide (29∶1 acrylamide∶bisacrylamide) gel with 0.5× TGE  as running buffer. Four single-stranded oligonucleotides were used as reference oligomers (RO) for migration, i.e. a 26-mer (26RO), two 37-mers with (+) and (−) strands (37RO(+), 37RO(−)), and a 54-mer (54RO)  was calculated as the ratio of the distance migrated by an oligonucleotide to the distance migrated by the pair of 37-nt reference oligonucleotides, i.e. 37RO(+) and 37RO(−), which demonstrated virtually identical mobility on native PAGE.Native PAGE was carried out at 4°C for 5 hours or 37°C for 3 hours. Prior to the analysis, the oligonucleotides (approximately 0.5 ng per cell) were annealed at 95°C for 10 min, mixed with loading buffer, and incubated for 30 minutes or overnight at 4°C or 37°C. The loading buffer consisted of 20 mM Tris/HCl pH 7.5, 37.5% glycerol, 50 mM NaCl and 15 mM MgClr (54RO) . Relativ2, 50 mM NaCl and 50% formamide; and run at 37°C on a 7.5 M urea, 15% polyacrylamide (19∶1 acrylamide∶bisacrylamide) gel in 1× TBE .For denaturing PAGE, samples were prepared in loading buffer consisting of 20 mM Tris/HCl pH 7.5, 15 mM MgClt−S60°C)/(S5°C−S60°C), where St, S5°C and S60°C are the 275 nm signal intensities at a given temperature, at 5°C, and at 60°C, respectively.A Chirascan CD unit  was used to record CD data of the oligonucleotides dissolved in 10 mM sodium phosphate buffer at pH 7.3. A 2 mm quartz cuvette was used to hold 400 µl samples of oligonucleotides in the concentration range 20–30 µM. CD spectra between 220 and 340 nm were recorded at 4°C and at 60°C for all samples, and melting profiles between 5°C and 60°C were recorded at 275 nm at a rate of 0.2°C/min. Before the measurements all samples were annealed at 95°C for 3–5 min. The CD melting profiles were normalized using the formula (S2O/D2O). The sample concentrations were in the range 100–300 µM, with 30 µM tetramethylsilane added for reference purposes. The water signal was suppressed using the excitation sculpting method. Before the measurements all samples were annealed at 95°C for 3–5 min.A Bruker Avance 500 MHz NMR spectrometer equipped with a cryogenic probehead was used to record 1D spectra at 4°C and at 37°C of unlabelled oligonucleotides dissolved in 10 mM sodium phosphate buffer at pH 7.3  resulted in formation of a dominant slowly migrating conformer at 4°C, while methylation of the second CpG site (Dyn A5mC2) produced no changes in mobility , or the first and third sites  resulted in patterns similar to that produced by the Dyn A5mC1 oligomer , a G to C substitution in the middle of the G run (M4), and a T to A substitution producing an in-frame stop codon (M5)  in the Dyn A oligonucleotide sequence, including the three human pathogenic mutations causing SCA23 (Mdon (M5)  and 2. T2, 100 mM KCl, 50 mM NH4SO4 or 0.5 mM ZnCl2 in the loading buffer during the pre-incubation period, nor on whether this period was 10 or 30 min, or 18 h, before loading on the gel (data not shown). All analyzed 37-mer oligonucleotides demonstrated virtually identical mobility on the denaturing PAGE , nine (Dyn A5mC2), eleven  and ten  imino proton resonances/hydrogen bonds , four (Dyn A M1), seven (Dyn A M5), eight (Dyn A M4) and ten (Dyn A M3) imino proton resonances/hydrogen bonds  are stabilized by canonical A-T and G-C and non-canonical G-T base pairing, b) display characteristic CD melting profiles, and c) show differential mobility on a native gel. Cytosine methylation appears to induce the formation of novel conformers or shift the equilibrium between existing conformational variants. The effects of CpG methylation are nucleotide sequence dependent, comparable in strength with those induced by point mutations, and are manifested at 4°C but not at 37°C.The results obtained from native PAGE, CD and NMR spectroscopy are in general agreement and demonstrate that the 5mC1) substantially decreased the mobility at 4°C or at both temperatures, respectively. In contrast, methylation of the α-NE oligomer and the Dyn A oligonucleotide at the second CpG site (Dyn A5mC2) did not affect the native gel mobility. Although methylation- and structure-dependent base stacking interactions of the oligonucleotides with the polyacrylamide matrix can not be completely ruled out as a cause for the mobility differences, it appeared more likely that the methylation-induced mobility differences arise from conformational differences between the un-methylated and methylated molecules.PAGE analysis demonstrated that Dyn A, Dyn B and Dyn B (AS) oligonucleotides may adopt compact conformations characterized by higher mobility on a native gel, that are evident at 4°C (Dyn A), or at both 4°C and 37°C (Dyn B and Dyn B (AS)). The α-NE oligomer may be present either in an unstructured form, or in a structured form that does not differ in mobility from those of the reference oligonucleotides at 4°C and 37°C. Methylation of the Dyn B oligonucleotide and of the Dyn A oligomer at the first CpG site  paired either with bases 25–27 (GCG) in structures A and B, or with bases 28–30 (GCG) in structure C . The existence of multiple conformations in the Dyn B5mC1 NMR sample provides a simple explanation for the two bands observed on the PAGE gel for the same sequence  Dyn A and (B) Dyn A(TIF)Click here for additional data file.Figure S2CD spectra between 220 and 340 nm for selected Dyn oligonucleotides in 10 mM sodium phosphate buffer, pH 7.3, at 4°C (solid lines) and at 60°C (dashed lines). The oligonucleotide abbreviations used in the figure are presented in.(TIF)Click here for additional data file.Figure S3Seven Dyn A secondary structures, which thermodynamic properties were calculated using mFold software  and listed in (TIF)Click here for additional data file.Figure S4Six Dyn B secondary structures, which thermodynamic properties were calculated using the mFold software  and listed in (TIF)Click here for additional data file.Table S1Mass of oligonucleotides used in the study determined by mass spectrometry.(DOCX)Click here for additional data file.Table S2Melting temperature (Tm), number of G-C, A-T, and G-T base pairs, and folding energies (ΔG) between 5 and 37°C for seven different secondary structures of the Dyn A-coding sequence.(DOC)Click here for additional data file.Table S3Melting temperature (Tm), number of G-C, A-T, and G-T base pairs, and folding energies (ΔG) between 5 and 55°C for seven different secondary structures of the Dyn B-coding sequence.(DOC)Click here for additional data file.


A fragment of genomic DNA containing dmaW, easF, easE, and easC was amplified from the human-pathogenic, ergot alkaloid-producing fungus Aspergillusfumigatus and transformed into Aspergillusnidulans, a model fungus that does not contain any of the ergot alkaloid synthesis genes. HPLC and LC-MS analyses demonstrated that transformed A. nidulans strains produced chanoclavine-I and an earlier pathway intermediate. Aspergillusnidulans transformants containing dmaW, easF, and either easE or easC did not produce chanoclavine-I but did produce an early pathway intermediate and, in the case of the easC transformant, an additional ergot alkaloid-like compound. We conclude that dmaW, easF, easE, and easC are sufficient for the synthesis of chanoclavine-I in A. nidulans and expressing ergot alkaloid pathway genes in A. nidulans provides a novel approach to understanding the early steps in ergot alkaloid synthesis.Ergot alkaloids are pharmaceutically and agriculturally important secondary metabolites produced by several species of fungi. Ergot alkaloid pathways vary among different fungal lineages, but the pathway intermediate chanoclavine-I is evolutionarily conserved among ergot alkaloid producers. At least four genes,  Claviceps, Neotyphodium, and Epichloë species of the order Hypocreales produce a variety of complex ergot alkaloids + of chanoclavine-I  and by co-elution with authentic standard  and WFE . The presence of each gene in the genomes of the different mutants was verified by PCR  overnight at 37 °C. The uridine auxotrophic A. nidulans isolate FGSC A767  was grown at 37 °C on SYE medium . To test alkaloid accumulation, cultures were grown on SYE agar medium at 37 °C for one week. solution , 2.2 g/LA. fumigatus according to the protocol described by Richards et al. [dmaW, easF, easC, and easE was amplified by PCR primed with oligonucleotides primer 1 (5'-TACCTATACCTAATCGAAGCCGCACGCAGTGCACC-3') and primer 2 (5'-GCCGCCCATTCACCAAGATTTTTGCACAAATCTGCG-3'). A 25 μL reaction contained 1× LongAmp Taq reaction buffer, 200 μM of each deoxynucleotide triphosphate, 1 μM of each primer, and 2.5 units LongAmp Taq DNA polymerase . The reaction began with an initial denaturing step of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C and 10 min at 65 °C, with a final extension of 5 min at 65 °C. DNA was extracted from s et al. . An 8774dmaW, easF, and easE was amplified by PCR primed with oligonucleotides primer 3 (5'-GAGAGCTACTTGACATATTGTGTCGGCAGGTGCGCA-3') and primer 2 (5'-GCCGCCCATTCACCAAGATTTTTGCACAAATCTGCG-3'). The PCR conditions were the same as stated above except the elongation time was 7 min. A 6123-bp fragment containing dmaW, easF, and easC), two separate DNA fragments were amplified for co-transformation. The first fragment containing dmaW and easC was primed with primer 1 (5'-TACCTATACCTAATCGAAGCCGCACGCAGTGCACC-3') and primer 5 (5'-CTACTCCATTGTCCTGTGAGTTG-3') which primed amplification of a 4846-bp fragment. The PCR conditions were the same as stated above except the elongation time was 5 min. The second fragment (2943 bp) contained only easF and was amplified by PCR primed with oligonucleotides easFF (5'-TCCATTCTTCGCTCGTTCAACCAGCAGG-3') and easFR (5'-CAGGACCTGTACCTAAAGCCTGGTAACC-3') in a 25 μL reaction containing 1× GoTaq Flexi buffer, 200 μM each deoxynucleotide triphosphate, 1.5 mM MgCl2, 1 μM of each primer, and 2.5 units of Taq DNA polymerase . The reaction began with an initial denaturing step of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 3 min at 72 °C, with a final extension of 5 min at 72 °C.In order to generate transformant WFC  and purified with QIAquick gel extraction kit prior to transformation.PCR products were purified with QIAquick gel extraction kits  prior to their inclusion in fungal transformations. The selectable marker pPyrG was obtained from the Fungal Genetics Stock Center and was digested with A. nidulans FGSC A767 were prepared by incubating overnight cultures with 50 mg driselase , which was filter sterilized with a 0.22 μm filter, and 75 mg lysing enzyme  in 15 mL of 0.7 M sodium chloride. Protoplasts were purified and co-transformed with PCR product and selectable marker (pPyrG) as described by Coyle et al. [Protoplasts of e et al. . ProtopldmaW, easF, easC, and easE. An internal region of dmaW was primed with dmaWF (5'-TTGATCTGGAGTGGTTCCGC-3') and dmaWR (5'-CGTTCATGCCGAAGGTTGTG-3') yielding a 651-bp fragment. Primers for amplifying an internal region of easE were easEF (5'-CCAGATACATTGCCATCGCATG-3') and easER (5'-TGTTCCAACTGCTTGGCCAGAT-3'), which primed amplification of a 897-bp fragment. The presence or absence of easC was tested by PCR with primers easCF (5'-GAATTCGAGGTATTGATCTCC-3') and easCR (5'-AGCCAGGCAAAGATCCATAGTT-3') which flank a 1036-bp fragment. An internal region of easF was primed with easFF (5'-AAGTTGTCGAAGGTCTCACGAA-3') and easFR (5'-GTGATTAGAGATGCTTCTGTC-3'), yielding an 820-bp fragment. Each of these PCRs were 25 μL reactions containing 1× GoTaq Flexi buffer, 200 μM deoxynucleotide triphosphate, 1.5 mM MgCl2, 1 μM of each primer, and 2.5 units of Taq DNA polymerase . PCR conditions began with an initial denaturing step of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C followed by 30 s at 55 °C, and 1 min and 30 s at 72 °C, followed by a final extension of 5 min at 72 °C.Each transformant was screened by PCR for the presence or absence of 2 of culture surface area, in 400 μL of methanol for 30 min and clarifying the extract by centrifugation. Extracts were injected into a C18 column (Prodigy 5-μm ODS3 (150 mm by 4.6 mm); Phenomenex, Torrance, CA, USA) and subjected to a multilinear binary gradient from 5% (v/v) acetonitrile plus 95% (v/v) aqueous 50 mM ammonium acetate to 75% acetonitrile plus 25% aqueous 50 mM aqueous ammonium acetate at a flow rate 1 mL/min, as previously described [N-Me-DMAT  were run as standards. Peaks of interest were isolated from the HPLC by collecting the runoff from the discard tube for 30 s after the peak was visualized on the detector. Transformants positive for the presence of the target genes were analyzed by HPLC for the accumulation of chanoclavine-I. Ergot alkaloids were extracted by incubating a spore-rich sample of the colony, containing approximately 1 cm18 column (Phenomenex 4-μm polar-RP (150 mm by 2 mm)), maintained at 30 °C, by combining mobile phases A  and B . Initial conditions were 86% A + 14% B, which ramped linearly to 73% A + 27% B at 25 min, 25% A + 75% B at 40 min, and 0% A + 100% B at 42 min. The flow rate was 200 μL/min. Analytes were ionized by electrospray ionization in positive mode and were detected by scanning for ions with a m/z between 200–400. Ergot alkaloid extracts were prepared by washing the surface of a conidiating colony with 2 mL of methanol, rotating the extract at 30 rpm for 30 min, and then clarifying the extract by centrifugation. Clarified extracts were then concentrated to a volume of 50 μL in a speed-vac apparatus, and 10 μL of concentrated extract were injected into the LC-MS. To isolate individual analytes, concentrated extracts were analyzed by fluorescence HPLC, peaks were collected manually, and concentrated to a volume of 50 μL in a speed-vac apparatus prior to LC-MS analysis. Concentrated samples were analyzed on a Finnigan LCQDecaXP plus mass spectrometer equipped with a Surveyor HPLC system. Analytes were separated on a CdmaW, easF, easE, and easC—are sufficient to direct synthesis of chanoclavine-I in A. nidulans. Both easC and easE are required for the synthesis of chanoclavine-I from N-Me-DMAT, but neither alone is sufficient. Aspergillus nidulans is a suitable heterologous host for expressing ergot alkaloids synthesis genes. Four genes common to all ergot alkaloid synthesis gene clusters—


Drosophila melanogaster, and it regulates the stability of prolyl hydroxylase domains (PHDs), with a concomitant effect on HIF-1α availability in the hypoxia response pathway. We previously reported that the hypoxia response pathway contributes to eye development during the embryonic development of Xenopus laevis. In this paper, the role of Siah2-mediated hypoxia response pathway in eye development of X. laevis embryos was further characterized. Xenopus Siah2 (xSiah2) mRNA was detected in lens tissue and xSiah2 overexpression caused a thickened lens placode, leading to loss of the optic lens. In embryos overexpressing xSiah2, lens marker gene transcription was reduced, suggesting that xSiah2 contributes to lens formation. xSiah2 overexpression decreased Xenopus PHD accumulation and increased Xenopus HIF-1α (xHIF-1α) accumulation. xHIF-1α degeneration with resveratrol restored the optical abnormality caused by xSiah2 overexpression, suggesting that the xSiah2-mediated hypoxia response pathway contributes to lens formation. Moreover, xSiah2 overexpression decreased endothelial–mesenchymal transition (EMT)-related Notch signaling-responsive genes transcription during the invasion of the lens placode. Our results suggest that the hypoxia response pathway plays an important role in the regulation of the EMT via the Notch signaling pathway during lens formation.The RING finger ubiquitin ligase seven in absentia homolog 2 (Siah2) was identified in the R7 photoreceptor cells of  •The ubiquitin lyase Siah2 regulates HIF-1α availability in the hypoxia response pathway.•Xenopus laevis.xSiah2 overexpression causes loss of the lens of •xSiah2 mRNA is detected in the lens placode from embryonic stage 30.•Xenopus laevis.xHIF-1α overexpression at stage 38 causes loss of the lens of •xSiah2 overexpression suppresses endothelial–mesenchymal transition-related genes. Siah2 fnogaster . PHD pronogaster . Availabnogaster , is a trnogaster .Xenopus Siah2 (xSiah2) in Xenopus laevis causes the small eye phenotype [The possible involvement of the hypoxia response pathway in the neurogenesis of vertebrates such as mice and frogs has recently been reported. HIF-1α knockout mice show defective angiogenesis as well as abnormal neurogenesis. Overexpression of henotype . This opXenopus development. There are four phases of lens formation: (1) presumptive lens ectoderm (PLE) is formed in the surficial layer of the embryo during the neurula stages; (2) interaction between the PLE and anterior neural tube results in PLE thickening and development into a lens placode during the early tailbud stage; (3) the lens placode invaginates and develops into a vesicle through the endothelial mesenchymal transition (EMT): and (4) differentiation into cellular layers occurs [Lens tissue is formed during the neurula and tailbud stages of s occurs .Xenopus PHD (xPHD) proteins, xPHD45 and xPHD28, and characterized them during the embryonic development of X. Laevis [xPHD28 mRNA restores the small eye phenotype caused by xSiah2 overexpression, suggesting that xSiah2 contributes to eye development via xPHD. However, the function of the hypoxia response pathway in embryonic sensory organogenesis, including the lens, remains unclear. Given the importance of xSiah2 in the stability of xPHD and consequent Xenopus HIF-1α (xHIF-1α) levels, we asked whether the hypoxia response pathway plays a potential role in lens formation.We previously isolated two . Laevis . In the 22.1Escherichia coli (E. coli) strains. xSiah2 peptide was then expressed in E. coli DH5α and purified using Ni-NTA agarose (QIAGEN). Antibodies were then raised against human PHD3, xSiah2, and human HIF-1α in rabbits using a previous described method [Xenopus β-actin, respectively, was confirmed. All experiments were conducted in accordance with guidelines on the welfare of experimental animals and with the approval of the Ethics Committee on the use of animals of Kwansei Gakuin University.Resveratrol was purchased from Sigma ; MMLV reverse transcriptase from Fermentas ; KOD plus DNA polymerase from TOYOBO ; and T3, T7, and SP6 RNA polymerases and Go taq polymerase from Promega . Antihuman β-actin antibody was purchased from Sigma and horseradish peroxidase-conjugated antirabbit IgG antibody was purchased from Bio-Rad . Antixenopus Siah2 antibody was prepared as follows. The first half of xSiah2 were ligated into pQE80L vector , which allows protein expression in d method ,10. Reac2.2M21286).Total RNA extracted from 5 embryos was prepared with Isogen  according to the manufacturer's instructions. cDNA was synthesized using total RNA (1 μg) in a total volume of 10 μL with MMLV reverse transcriptase according to the manufacturer's instructions as follows: incubation at 25 °C for 15 min and at 42 °C for 60 min followed by heating at 70 °C for 10 min. Polymerase chain reaction (PCR) was performed at 94 °C for 2 min and then for a particular number of cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s in a reaction mixture containing 10 pmol of each primer, Go taq polymerase, and cDNA (100 ng). Primers, GenBank accession numbers, cycles, and sequences for PCR are shown in 2.3AF155509) was amplified by PCR. Thirty-five cycles of PCR  were performed using the cDNA obtained from reverse transcription of total RNA from embryos as the template, KOD plus DNA polymerase and corresponding primer pairs. Primer pairs are shown in cDNA of xSiah2  according to the manufacturer's instructions. Synthesized mRNAs  were injected into each dorsal blastomere at the two-cell stage.2.5X. laevis  were obtained by injecting a female with 120 units of human chorionic gonadotropin . The eggs were fertilized with the chestnuts suspended in 1.0× Modified Birth's Solution (MBS) containing 0.5 mM HEPES (pH 7.5), 10 mM NaCl, 0.2 mM KCl, 0.1 mM MgCl2, and 0.2 mM CaCl2. The chestnuts were surgically isolated from a male. The fertilized embryos were dejellied with 1% sodium thioglycollate and washed with 0.1× MBS several times. The developmental stage of embryos was determined according to Nieuwkoop and Faber's normal table of X. laevis [Unfertilized eggs of wild type and albino . laevis .2.6p-toluidine salt (BCIP) (Roche) were added to 1 mL of alkaline phosphatase buffer containing 100 mM Tris–HCl (pH 9.5), 100 mM NaCl, 50 mM MgSO4, 0.1% Tween 20, and 25 mM levamisole.Thirty albino embryos were fixed in fully dehydrated ethanol. Sense and antisense probes for xSiah2 were prepared from xSiah2/pBS and then linearized with SpeI or BamHI, respectively, and transcribed with T3 or T7 RNA polymerase, respectively, in the presence of digoxygenin UTP (Roche). Hybridized probes were visualized according to the Roche DIG protocol with a minor alteration that 0.45 mL of nitro-blue tetrazolium chloride (NBT) (75 mg/mL in dimethyl formamide) and 3.5 mL of 5-bromo-4-chloro-3′-indolylphosphatase 2.7Twenty embryos were homogenized in buffer containing 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 mM DTT, 150 mM KCl, and 100 mM PMSF, and then solubilized with sodium dodecylsulfate (SDS). The resulting solution was subjected to SDS–polyacrylamide gel electrophoresis. Proteins were blotted onto a nitrocellulose membrane and reacted with antibodies against human β-actin, human HIF-1α, human PHD, and xSiah2.2.8Twenty embryos were fixed in fully dehydrated ethanol and embedded in paraffin. Sagittal sections were cut 10 μm thick and stained with hematoxylin and eosin.2.9All data are reported as mean ± SD. Statistical analysis of the data was performed by one-way ANOVA. Significance was determined by ANOVA followed by Fisher's protected least significant difference.33.1X. laevis [X. laevis were grown until st. 24 (early tailbud stage), 30 (middle tailbud stage), and 38 (later tailbud stage) and harvested at each stage. The accumulation pattern of xSiah2 mRNA in these embryos was investigated by whole in situ hybridization  or both dorsal blastomeres (S2 treatment group). The effect of xSiah2 overexpression on the level of xPHD and xHIF-1α accumulations at st. 30 and 38 was then investigated by western blotting  and β-crystallin (Genbank: BC084735) mRNAs, at st. 30 and 38 were then investigated by RT-PCR , Snail-1 (Genbank: BC056857), and ESR-1 (Genbank: AF383157) were investigated at st. 30 using RT-PCR . At low oxygen concentrations in vitro, PHDs exhibit 10% and 50% of their maximum hydroxylase activity toward their substrates, including HIF-1α [xSiah2 overexpression induced a decrease in xPHD expression. In addition, xPHD overexpression by co-injection with xSiah2 restored the absence of lens formation caused by xSiah2 overexpression, suggesting that xPHD is the substrate of xSiah2 and that xSiah2 participates in lens formation via xPHD g HIF-1α . During Snail-1, N-cadherin and ESR-1. This result suggests that the disruption of EMT by xSiah2 overexpression inhibited lens vesicle formation, leading to the lens defect. The xSiah2-mediated hypoxia response pathway thus participates in the EMT. Snail-1, ESR-1 and FoxE3 are directly up-regulated by Notch signaling. However, xSiah2 overexpression suppressed the transcription of Snail-1 and ESR-1 mRNAs at st. 30, but not that of FoxE3 mRNA at st. 24. Notch signaling is the communication system between cells. During lens formation, some Notch signaling pathways are activated independently. Between retinal progenitor cells in the optic cup and lens epithelial cells, Notch ligands interact with the Notch receptor. This interaction is necessary for the transcription of FoxE3 mRNA in lens epithelial cells, which maintains the undifferentiated state, and is located in the embryonic surface ectoderm, leading to the induction of PLE thickening. xSiah2 overexpression did not alter the transcription of FoxE3 mRNA at st. 24, suggesting that xSiah2 did not affect Notch signaling between retinal progenitor cells and lens epithelial cells. xSiah2 overexpression suppressed the transcription of Snail-1 and ESR-1 mRNAs at st. 30. These genes are necessary for the invasion of lens placode through the EMT and the differentiation into lens endothelial cells, which serves as the progenitors for lens fibers, and is located in the anterior portion of the lens between the lens capsule and the lens fibers. Accordingly, xSiah2 might affect Notch signaling in endothelial cells.In this study, xSiah2 overexpression caused a decrease in the expression of EMT-related genes such as


One strategy to reduce the pathophysiology associated with CF is to increase Cl− transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl− channels to mediate Cl− transport across lipid bilayer membranes is capable of restoring Cl− permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl− channel dysfunction.Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl Great efforts have been made to develop gene transfer of the CFTR to epithelia − channels 2 receptor opens up an alternative chloride channel resulting in Cl− ion and liquid secretion, and partly compensates for effects caused by the mutant CFTR − channels − secretion for the treatment of CF.Chloride  solutions  used in the standard bath and pipette solutions were replaced by the larger NMDG cation (Erev) shifted from 0 to 26.99±1.38 (n = 5) mV, which is quite close to the value of the Cl− reverse potential (ECl) (27.76 mV) calculated from the Nernst equation. Taken together, these results indicate that the ion channels formed by compound 1 are permeable to Cl− ions only, and not to NMDG ions, and the increased whole-cell currents observed in previous experiments can therefore be attributed to the Cl− currents induced by synthetic Cl− channels rather than to a non-specific membrane leak.We then used the whole-cell configuration of patch clamp technique to examine the electrophysiological properties of the ionic currents induced by compound ions see . While tions see , compoun− channel by comparing cell reverse potentials when replacing the extracellular Cl− ions with equimolar F−, Br−, I−, and NO3− ions. The relative permeability ratios against Cl− (F− ∶ Cl− ∶ Br− ∶ I− ∶ NO3−) were determined to be 0.673 ∶ 1 ∶ 1.121 ∶ 1.251 ∶ 1.358  failed to block the whole-cell currents induced by compound 1 , whereas the CF cells (CuFi-8) exhibited little response to forskolin. In contrast, the addition of 1 µM compound 1 resulted in an even larger increase in the whole-cell currents of the CuFi-8 cells than that recorded in those of the NuLi-1 cells treated with 2 µM forskolin, indicating that compound 1 is capable of increasing Cl− conductance in these CF cells via a novel pathway. Similarly, compound 1 also increased Cl− conductance in NuLi-1 cells. The compound 1-induced Cl− current was a little smaller than that recorded in CuFi-8 cells. Because compound 1 is generated from an α-aminoxy acid unit, in considering the possibility of applying compound 1 in future, we also examined its toxicity in, for example, immune response in animals. The data showed that compound 1 did not induce mouse immune response, especially in lung tissue , were derived from the human bronchial epithelia of a patient without CF  and a subject with CF , respectively. These cell lines were grown on human placental collagen type VI (Sigma)–coated flasks in BEGM  medium, which includes BEGM basal medium and eight SingleQuots of supplements. Cells were grown at 37°C in a 5% CO− channels. After the inside-out recording was achieved without any channel opening, 1 µM compound 1 was perfused into the bath. The single channel current was recorded continuously and sampled at 4 kHz, and was typically low-pass filtered at 0.4 kHz with an 8-pole Bessel filter.The single channel current was recorded using an EPC 9 patch clamp amplifier  in voltage-clamp mode, controlled by Pulse/PulseFit 8.7 software (HEKA) with inside-out patch configuration. Patch pipettes  were filled with internal pipette solution containing 150 mM NMDG-Cl and 10 mM HEPES (pH 7.2). The bath was composed of symmetric 150 mM NMDG-Cl and 10 mM HEPES (pH 7.2) with 200 µM diphenylamine-2-carboxylic acid that blocks native Cl2 1, MgCl2 1 and HEPES 10 in mM, pH 7.4 with CsOH) and pipette  solutions, the cells were held at 0 mV and voltage steps ranging from −100 to +100 mV were applied for 800 ms in 20-mV step increments. In these experiments, the native K+ currents were eliminated by using K+-free intra- and extracellular solutions containing Cs+, and any low threshold native Ca2+ or Na+ channels were inactivated by using a holding potential of 0 mV. Internal free Ca2+ was chelated by EGTA in pipette solution to prevent Ca2+-dependent channels from being activated. Changes in the whole-cell currents were detected from the same cells before and after exposure to the bath solution containing compound 1  or forskolin . Unless indicated otherwise, all control data compared with compound 1 were from the 0.1% DMSO treatment. In ion selectivity experiments, the symmetric solution was first used in both intra- and extracellular sides. The cells were held at 0 mV and whole-cell currents were recorded with a ramp from −100 mV to +100 mV for 500 ms. Liquid junction potential was calculated by pClampfit 9.0 software  and the corresponding reverse potential was determined. After observing a stable whole-cell current induced by the application of 1 µM compound 1, the bath solution was replaced by isotonic (adjusted by sucrose) desired extracellular solution also containing 1 µM compound 1. In these experiments, all bath solutions containing 200 µM diphenylamine-2-carboxylic acid were used to suppress native Cl− channels other than those conducted to investigate the effect of Cl− channel inhibitors. All patch-clamp experiments were performed at room temperature (22–25°C).In the whole-cell current recording of the HEK 293, NuLi-1 and CuFi-8 cells in the standard bath  was added through an injection port, followed by the injection of 20 µL of 0.5 M aqueous NaOH. The addition of NaOH caused in increase of ca. 1 pH unit in the extravesicular buffer. The maximal change in dye emission was obtained at the end of each experiment after lysis of the liposomes with a detergent (40 µL of 5% aqueous Triton X-100). The final transport trace was obtained from the ratio of the emission intensities monitored at 460 and 403 nm and was normalized to 100% of transport.Typically, 100 µL of the HPTS-loaded liposomes (stock solution) was suspended in 1.9 mL of the corresponding buffer and placed into a fluorimetric cell. HPTS emission at 510 nm was monitored during simultaneous excitation at wavelengths of 403 and 460 nm. During each experiment, 20 µL of a 1 mM THF solution of compound Figure S1Current–voltage relationships obtained in the absence (•) and presence (○) of 0.1% DMSO in HEK 293 cells.(PDF)Click here for additional data file.Figure S2pH-Stat ion transport assays illustrating the ion selectivity of compound 1. All experiments employed suspensions of EYPC liposomes containing the pH-sensitive dye HPTS in a HEPES buffer. The intravesicular solutions were 10 mM HEPES (pH 6.8) and 100 mM NaCl and extravesicular solutions were 10 mM HEPES (pH 6.8) and 100 mM MCl  in a. Both the intra- and extravesicular solutions contained 10 mM HEPES (pH 6.8) and 100 or 75 mM NanX  in b. At t = 100 s, a THF solution (20 µL) of the testing compound at 10 µM final concentration was added to the extravesicular solution; 0.5 M NaOH solution (20 µL) was then added. At t = 700 s, 5% Triton X-100 (40 µL) was added to lyse the liposomes.(PDF)Click here for additional data file.Figure S3The data summary of 1 µM compound 1-increased whole-cell current densities (pA/pF) in the absence (control) and presence of 100 µM 4,4′-dithiocyanatostilbene -2,2′- disulfonic acid (DIDS), 100 µM 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), 200 µM diphenylamine-2-carboxylic acid (DPC), 100 µM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and 100 µM niflumic acid (NFA), respectively, at ±80 mV in HEK 293 cells. The cells were pretreated with the inhibitors, respectively, for 10 min before the application of 1 µM compound 1. All data are mean ± s.e. n = 3–8, P>0.05 compared to control group.(PDF)Click here for additional data file.Figure S4The data summary of serum TNF-α concentration of mice in the first 24 hrs after intraperitoneal injection with DMSO, lipopolysaccharide (LPS) or compound 1 (Comp 1). DMSO is a negative control because it is the solvent of compound 1. LPS is a well-known endotoxin and capably of eliciting strong immune responses in animals. Here, LPS is a positive control. All data are mean ± s.e. n = 4–6 mice, P>0.05 compared to DMSO group. P<0.01 compared to DMSO group.(PDF)Click here for additional data file.Figure S5The H & E staining images of mice lung sections in the first 24 hrs after intraperitoneal injection with DMSO, lipopolysaccharide (LPS) or compound 1 (Comp 1). DMSO is a negative control because it is the solvent of compound 1. LPS is a well-known endotoxin and capably of eliciting strong immune responses in animals. Here, LPS is a positive control. The images show inflammatory cells (labeled by blue arrow) infiltration in LPS treatment, but no inflammatory cells can be found in DMSO and compound 1 treatment.(PDF)Click here for additional data file.Methods S1Immune response test in mouse.(DOC)Click here for additional data file.