During brain ischemia/reperfusion, EGFR could be transactivated, which stimulates these intracellular signaling cascades that either protect cells or potentiate cell injury. In the present study, we investigated the activation of EGFR, PI3K/AKT, and Raf/MAPK/ERK1/2 during ischemia or reperfusion of the brain using the middle cerebral artery occlusion model. We found that EGFR was phosphorylated and transactivated during both ischemia and reperfusion periods. During ischemia, the activity of PI3K/AKT pathway was significantly increased, as judged from the strong phosphorylation of AKT; this activation was suppressed by the inhibitors of EGFR and Zn-dependent metalloproteinase. Ischemia, however, did not induce ERK1/2 phosphorylation, which was dependent on reperfusion. Coimmunoprecipitation of Son of sevenless 1 (SOS1) with EGFR showed increased association between the receptor and SOS1 in ischemia, indicating the inhibitory node downstream of SOS1. The inhibitory phosphorylation site of Raf-1 at Ser259, but not its stimulatory phosphorylation site at Ser338, was phosphorylated during ischemia. Furthermore, ischemia prompted the interaction between Raf-1 and AKT, while both the inhibitors of PI3K and AKT not only abolished AKT phosphorylation but also restored ERK1/2 phosphorylation. All these findings suggest that Raf/MAPK/ERK1/2 signal pathway is inhibited by AKT via direct phosphorylation and inhibition at Raf-1 node during ischemia. During reperfusion, we observed a significant increase of ERK1/2 phosphorylation but no change in AKT phosphorylation. Inhibitors of reactive oxygen species and phosphatase and tensin homolog restored AKT phosphorylation but abolished ERK1/2 phosphorylation, suggesting that the reactive oxygen species-dependent increase in phosphatase and tensin homolog activity in reperfusion period relieves ERK1/2 from inhibition of AKT.The epidermal growth factor receptor (EGFR) is linked to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Raf/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK When members of epidermal growth factor (EGF) family bind to EGF receptor (EGFR), the EGFR becomes phosphorylated (activated) on tyrosine residues. Subsequently, the guanine nucleotide exchange factor Son of sevenless 1 (SOS1) becomes activated, thus leading to stimulation of the small G-protein Ras. The latter actuates serine/threonine-kinase Raf, the first kinase of the three-tiered MAPK/ERK1/2 pathway. There are three Raf isoforms, Raf-1/c-Raf, B-Raf, and A-Raf /extracellular signal-regulated kinase (ERK pathway . PI3K is2+ concentration ([Ca2+]i) -mediated increase of EGFR phosphorylation and its activity in cerebral cortex of newborn piglets   mediatedeceptors  or by otR family . This agpathways . Cardioppathways , 2008. Epathways . In the  piglets . In midd piglets . Inhibit piglets . The PI3 piglets , whereasondition , whereasondition . Cell da all see . Further1/2 represent two parallel signal pathways. Interactions between PI3K/AKT and Raf/MAPK/ERK1/2 pathways may occur at several different stages and could be either positive or negative. In preliminary experiments, we found that ERK activity was dependent on reperfusion. We hypothesized that the PI3K/AKT signal pathway is involved in the regulation of ERK activity during ischemia or reperfusion. Therefore, in the following study, we investigated the activation of EGFR and its two intracellular signal pathways, MAPK/ERK1/2 and PI3K/AKT during ischemia or reperfusion in MCAO model.Conceptually, the PI3K/AKT and Raf/MAPK/ERKAdult male Sprague-Dawley rats, weighing 280 to 400 g, were housed in cages on a 12-hr light/dark cycle in a temperature-controlled (23℃–25℃) colony room with free access to food and water. All experiments were carried out in accordance with the USA National Institute of Health Guide for the Care and Use of Laboratory Animals, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University.Focal brain ischemia was induced unilaterally by occlusion of the right middle cerebral artery (MCAO) as previously described . BrieflyIntraventricular injection was performed according to the rat brain atlas by At the end of experiment, each brain was quickly removed and collected. Brain sectioning was accomplished according to the protocol previously reported by The brains were homogenized with EBC buffer  and centrifuged at 10,000 rpm for 10 min at 4℃. Supernatants were collected and stored at −40℃. The coimmunoprecipitation was performed with anti-EGFR antibody or with anti-Raf-1 antibody and protein agarose bead. The nitrocellulose membranes were incubated with the anti-SOS1 antibody or anti-EGFR antibody at 1 × 500 or at 1 × 800 dilution, or with the anti-phospho-Akt (p-AKT) antibody or anti-AKT antibody at 1 × 500 or at 1 × 1,000 dilution for 2 hr at 25℃.1/2) at 1 × 1,000 dilution, ERK1/2 at 1 × 3,000 dilution, p-AKT at 1 × 1,000 dilution, AKT at 1 × 1,000 dilution, p-Raf Ser259 at 1 × 1,000 dilution, p-Raf Ser338 at 1 × 1,000 dilution, and Raf-1 at 1 × 1,000 dilution for 2 hr at room temperature. After washing, specific binding was detected by goat-anti-mouse or goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody at 1 × 1,500 dilution or at 1 × 3,000 dilution. Staining was visualized by ECL detection reagents. Digital images obtained using Gel-Imaging System . Optical density for each band was assessed using the Window Alpha-Ease TM FC 32-bit software.The protein content was determined in the homogenates by the Bradford method , using bLY294002 hydrochloride, TCN, and catalase were purchased from Sigma . AG1478, GM6001 (N-[(2 R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide), and U0126 were purchased from Calbiochem . Rapamycin was purchased from Cell Signaling Technology . BPV was purchased from Santa Cruz Biotechnology . Pentobarbital sodium (SCRC 69020180) was obtained from Shanghai Chemical Reagent Co., Ltd . Santa Cruz Biotechnology  supplied first antibodies, rabbit polyclonal antibody raised against ERK (K-23): sc-94, mouse monoclonal antibody against p-ERK (E-4): sc-7383, rabbit polyclonal antibody against SOS1 (C-23): sc-256, rabbit polyclonal antibody against p-AKT1/2/3 (Ser473): sc-33437, rabbit polyclonal antibody against phosphorylated tyrosine of EGFR (pEGFR) (Tyr 1173-R): sc-12351-R, and mouse monoclonal antibody used for immunoprecipitation of Raf-1(E-10): sc-7267. The sheep polyclonal antibody used for immunoprecipitation of EGFR (06–129), as well as Protein G agarose bead slurry (16–266), was purchased from Upstate Biotechnology . The rabbit polyclonal antibody against EGFR (2232), rabbit polyclonal antibody against c-Raf (9422), rabbit polyclonal antibody against p-c-Raf (Ser338) (9427), rabbit polyclonal antibody against p-c-Raf (Ser259) (9421), and rabbit polyclonal antibody against Akt (9272) used for western blotting were purchased from Cell Signaling Technology . ECL detection reagents were purchased from Amersham Biosciences .p < .05.The differences between individual groups were analyzed by one-way analysis of variance followed by Fisher’s least significant difference test. The comparisons were made between the core region on the lesion side with the same region on the contralateral side, and the penumbra on lesion side with the corresponding region on the contralateral side, to identify the significance related to ischemia/reperfusion. The level of significance was set at First, we investigated EGFR status by quantifying its phosphorylation by immunoprecipitation and specific antibodies. EGFR was precipitated with a specific antibody. Western blot showed an increase of EGFR activity during ischemia that was confirmed by an antibody specific to tyrosine phosphorylation sites in both core and penumbra in ischemic hemisphere . With si1/2 and AKT phosphorylation by Western blot. There was no significant difference in ERK1/2 phosphorylation neither in the core nor in the penumbra areas between ischemic and contralateral hemispheres after 2 hr of ischemia  were injected intraventricularly before ischemia. Both drugs abolished phosphorylation of AKT  of contralateral hemisphere, respectively, after 0.5-hr reperfusion  the EGFR transactivation and the activations of PI3K/AKT and Raf/MAPK/ERK1/2 signal pathways during ischemia, (b) the interaction between PI3K/AKT and Raf/MAPK/ERK1/2 signal pathways during ischemia, (c) the EGFR transactivation and the activations of PI3K/AKT and Raf/MAPK/ERK1/2 signal pathways during reperfusion, and (d) the interaction of PI3K/AKT and Raf/MAPK/ERK1/2 signal pathways during reperfusion.Experiments on myocardial ischemia/reperfusion model demonstrated that ERK phosphorylation depends on reperfusion , but the1/2 phosphorylation during ischemia, but phosphorylation significantly increased in reperfusion. This is similar to myocardial ischemia/reperfusion model , an inhibitor of AKT. All these suggest an inhibitory effect of PI3K/AKT on Raf/MAPK/ERK1/2 pathway during ischemic period. Similar inhibition has been reported for other tissues and cells, such as AKT-induced phosphorylation of Raf-1 at Ser259 by insulin-like growth factor 1 in MCF-7, a human breast cancer cell line (1/2 phosphorylation in human myotubes (Second, we found that despite the absence of ERKand MAPK , whereasand MAPK . Furtherell line  and by pell line . Neverthmyotubes , that efmyotubes .2+-dependent metalloproteinase, indicating an involvement of EGFR transactivation. The sites of EGFR phosphorylation were the same as ischemic period. As has been reported previously, permanent increase of EGFR and ERK activity during reperfusion and inhibition of EGFR and ERK activity have protective effect (Third, we found no increase of AKT phosphorylation in reperfusion period. However, ERK phosphorylation was significantly enhanced during reperfusion, and it was inhibited by both AG 1478, an inhibitor of EGFR and GM6001, an inhibitor of Zne effect .1/2 pathway during reperfusion. Our data indicate that it is not the case because inhibition of MAPK and ERK1/2 does not reinstate AKT phosphorylation. However, inhibition of catalase activity not only abolishes ERK phosphorylation but also restores AKT phosphorylation. This indicates that both ERK1/2 stimulation and AKT inhibition during reperfusion are mediated by ROS. ROS-induced EGFR transactivation has been repeatedly reported (Fourth, we investigated whether PI3K/AKT pathway is inhibited by Raf/MAPK/ERKreported . More inreported  during rreported .Activity of AKT has been studied in the context of preconditioning in combination with MCAO model . Precond1/2 and PI3K/AKT signal pathways during brain ischemia/reperfusion. Our study provides the information about the underlying mechanisms of this interaction, that is, during ischemia, AKT phosphorylates Raf-1 at its inhibitory phosphorylation site and decreases Raf/MAPK/ERK1/2 activity; during reperfusion, ROS stimulates PTEN that erases the inhibition of AKT on Raf/MAPK/ERK1/2 signaling (1/2 signaling may depend on the type of the cells, the time and extent of ERK activation, and the kind of stimulation/insult. ERK phosphorylation mediated by preconditioning (2-adrenergic agonist, (1/2 and PI3K/AKT signal pathways during brain ischemia/reperfusion may eventually lead to the development of new therapeutic strategy for the disease. Our findings suggest that ROS removes inhibition of AKT on ERK activity during reperfusion. The strong stimulation of ERK activity plays a key role in reperfusion-induced cell damage. Inhibitors of ROS, PTEN, EGFR, and ERK may have therapeutic potential in clinic for brain ischemia.In summary, negative crosstalk exists between Raf/MAPK/ERKignaling . PI3K/AKitioning  and by nagonist,  reduces agonist, . Therefo


Delisea pulchra is a temperate red macroalga that suffers from a bleaching disease that is more frequent during summer, when seawater temperatures are elevated and the alga’s chemical defense is weakened. A bacterial cause for the disease is implied by previous studies showing that some isolated strains can cause bleaching in vitro and that host-associated microbial communities are distinct between diseased and healthy individuals. However, nothing is known about the successional events in the microbial community that occur during the development of the disease. To study this aspect in the future, we aimed here to develop an experimental setup to study the bleaching disease in a controllable aquarium environment. Application of a temperature stress (up to 27°C) did not cause a clear and consistent pattern of bleaching, suggesting that temperature alone might not be the only or main factor to cause the disease. The results also showed that the aquarium conditions alone are sufficient to produce bleaching symptoms. Microbial community analysis based on 16S rRNA gene fingerprinting and sequencing showed significant changes after 15 days in the aquarium, indicating that the native microbial associates of D. pulchra are not stably maintained. Microbial taxa that were enriched in the aquarium-held D. pulchra thalli, however, did not match on a taxonomic level those that have been found to be enriched in natural bleaching events. Together our observations indicate that environmental factors, other than the ones investigated here, might drive the bleaching disease in D. pulchra and that the aquarium conditions have substantial impact on the alga-associated microbiome.In recent years, there has been an increase in the rate and severity of diseases affecting habitat-forming marine organisms, such as corals, sponges, and macroalgae.  Anthropogenic environmental stressors  are increasing the rate and severity of marine diseases worldwide . ParticuDelisea pulchra is arguably one of the best-studied models for macroalgal disease  suffer from microbial diseases, but there is a general lack of understanding for the mechanistic processes and ecological factors that determine disease . The ble disease . D. pulcnization  and invenization  based onnization , when ulnization . Experimnization . The invncreased . Furtherncreased  and an ong event . These oRhopaloides odorabile controlled aquarium experiments have shown that an increase in temperature resulted in reduced expression of symbiotic functions in both the host and its microbial community, which was then followed by the proliferation of opportunistic, scavenging bacteria that ultimately led to tissue necrosis  and sequencing of the 16S rRNA gene.Time-series experiment under controlled conditions can provide valuable insight into processes of disease development and specific environmental and biological factors that drive it. For example, for the sponge necrosis . In ordeTwo criteria were set to be met for an aquarium model to be suitable to study the effect of environmental stressors on the occurrence of bleaching and its associated microbial community shifts: (1) the algae should remain healthy under control conditions for the duration of the experiment and (2) an environmentally relevant stressor should cause bleaching symptoms in a reproducible and predictable manner. To address these two points, three separate temperature-stress experiments were conducted in an aquarium-laboratory at the Sydney Institute of Marine Science (SIMS).D. pulchra samples were collected by SCUBA at depths of around 9 m at Long Bay, Sydney, Australia, during the austral spring and summer . All collected individuals were healthy juveniles  that presented neither visible signs of bleaching nor any atypical phenotypes . Immediately after collection, thalli were placed in 20 L buckets with fresh seawater for transport to the aquarium-laboratory facilities of SIMS. The aquarium constantly uses fresh seawater that is filtered through a set of hydrocyclones, a set of 100 μm disk filters and a series of bag filters of 50, 25, and 10 μm pore size. The aquarium system is fed by hot- and cold-water sumps (200 L), which take fresh seawater at a rate of approximately 180 L/h and from which the water is mixed for temperature-adjusted fed into individual containers.For these experiments, D. pulchra  were placed into separate 4 L containers and letting them freely sink to the bottom. Flow-through seawater was delivered at an average constant rate per container of 55 L/h ± 25. Fluorescent light tubes (Sylvania Premium Extra FL36W Cool) were used to supply the algae with an average photosynthetically active radiation (PAR) of 16 μmol photons m-2 s-1 in a 12 h light:dark cycle. This light intensity was below the average of 103.94 ± 6.96 μmol photons m-2 s-1 that the algae receive at the collection site at the depth of sampling. All containers were kept at a temperature of 19°C (approximate in situ temperature during collection) for one day to let the algae acclimatize. High- (HT) or low-temperature (LT) treatments were randomly assigned to half of the individuals. For the HT treatment during the first two trials, the water was raised to 22°C on the second day and to 25°C on the third day (ramping time of 3 h). This temperature corresponds to the peak temperature found in Sydney waters during summer  was measured with a portable pulse amplitude-modulated fluorometer . As defined in Figure 1). An alga was considered damaged-by-bleaching, if bleaching observed at a previous time point caused part of the thallus to break off.At time 0 and thereafter every 7 days, each individual was carefully taken out of the container and placed on a light-colored surface to carefully inspect for the appearance of any signs of bleaching. In addition, the algae were photographed and the effective quantum yield of photosynthetic energy conversion  were collected on the 18th of September of 2013 from Long Bay, Sydney, and surface-associated microbial DNA was extracted from half of these (field t0), while the other half was placed in the SIMS aquarium (same set-up and conditions as above). The temperature was kept constant at 17°C, which corresponded to the sea temperature at the time of sampling. After 15 days, six thalli were collected from Long Bay (field t1) and DNA was extracted from these as well as from the thalli that were kept in the aquarium (aquarium t1) following the methods described by An experiment was carried to assess the possible impact of aquarium-maintenance on the microbial community associated with HaeIII. Reactions were incubated at 37°C for 4 h followed by 80°C for 20 min. Digested PCR products were analyzed on an ABI3730 DNA analyzer  using the LIZ600 size standard. The raw electropherogram data was processed using the Peak Scanner Software v1.0 (Applied Biosystems) with the peak detection range set to correspond to the size standard used and the minimum peak threshold for the FAM dye, the standard dye and the other dye options set to 1, 500, and 5000, respectively. The resulting table of electropherogram peak information was further processed using T-Rex , 2 μl of each of the primers 27F-FAM and 1492R for a final concentration of 2 ng/μl each and 4 μl of DNA template (20 pg/μl). The PCR cycling conditions consisted of: 3 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, and a final 10 min at 72°C. PCR products were purified using a DNA Clean and Concentrator Kit (Zymo Research) following the manufacturer’s protocol and the DNA was eluted in 30 μl of sterile, de-ionized water. Purified PCR products were digested in 20 μl reactions containing 17 μl purified PCR product, 2 μl 10X restriction buffer and 1 μl ng T-Rex  to filte1 using the primers 515F and 806R at the Ramaciotti Centre for Genomics. The MiSeq platform was used to generate 151 nucleotide reads from both ends of the amplified products. The sequencing data have been deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) under the study accession SRP065248.Three DNA samples from each of the three groups  were randomly picked for sequencing of the 16S rRNA gene. The V4 region of the 16S rRNA gene was amplified following standard protocols of the Earth Microbiome Project2, which can be found in the Supplementary Material. Briefly, paired-end raw reads were merged into contigs of around 250 bp, which were then quality filtered, taxonomically classified and clustered at a sequence identity cutoff of 97% to obtain a ‘sample by OTU’ abundance matrix. In order to reduce the number of spurious OTUs that can pass quality-filtering, OTUs with an overall abundance across samples below 0.0009% (which corresponds to eight sequences per OTU) were removed. Due to the difference in the number of 16S rRNA gene copies that exists between different taxa, the Copyrighter software (Supplementary Table S2). The average of the subsamples was used for statistical analysis.Sequence processing was carried out using the Mothur software v.1.34  and a mosoftware  was usedThe most abundant sequence for each OTU was used as reference for taxonomic classification. Sequences were classified using the ‘classify.otu’ command in Mothur with default parameters based on three different bacterial 16S rRNA taxonomic outlines, including Silva (release 119), RDP (PDS version 10) and Greengenes (release of August 2013). For the OTUs selected from the statistical analysis, a consensus of the three classifications was manually built by choosing the deepest taxonomic assignment . When different classifications were obtained, alternative taxa are reported.Tables 1 and 2). Pairwise tests were also carried out. The data matrices (TRFLP and OTU) were square-root and presence/absence transformed using Primer-E  and diversity (InvSimpson) between sample groups, Analysis of Variance (ANOVA) was calculated using a one-factor design with three levels: aquarium t1, field t0, and field t1  that contributed to the top 50% of the overall deviance and that had a p-valueunadjusted < 0.01 were selected as the highest contributors to the difference between communities. These OTUs were then classified as “unique” , if they were consistently present in all replicates of a group and consistently absent in the other group. The rest of the selected OTUs were classified as “abundant”  as they could be present in both groups, but were significantly more abundant in one of them.Operational taxonomic units that contributed most to the difference between sample types were identified by adjusting the OTU matrix to a multivariate generalized linear model using the Mvabund R package . Here, eFigure 1; for bleaching definition see “Materials and Methods”). Consistent with the visual identification of the bleaching phenotype, the photosynthetic yield of bleached algae was always lower in the bleached tissue  compared to healthy tissue  and this difference was statistically significant when assessed for trial 1 .After 1 week of maintenance in the aquarium, we could detect algal thalli that show signs of bleaching . The number of bleached (including damaged-by-bleaching) individuals in the LT (control) group also increased over time, reaching proportions above the HT group in some of the time points of trials 1 and 3. In addition, the HT treatment had no apparent effect on the photosynthetic yield of healthy tissue of D. pulchra compared to the control in any of the trials .In all three trials the proportion of bleached  individuals in the HT treatment increased with time, but never increased above a 40% frequency . The results also show that high temperatures alone do not result in a reproducible increase in the incidence of bleaching disease above the control level. Therefore the criteria evaluated in the present experiments  were not met, which made the current aquarium model not suitable to test the effect of temperature on the incidence of bleaching and the associated changes of the microbial community.This experiment showed that the aquarium conditions alone can produce bleaching in Figure 3) and composition  showed that all field samples (field t0 and field t1) clustered together regardless of the sampling time point and that the field samples clearly separated from the aquarium t1 samples. PERMANOVA hypothesis testing showed that the community composition and structure were significantly different across the three sample types , but in pairwise comparisons, only between field and aquarium samples (Tables 1 and 2). This shows that aquarium maintenance for 15 days can produce significant changes in the microbial communities of D. pulchra.The transfer of marine organisms from their natural environment to an aquarium can have an impact on their associated microbial communities . To inveSupplementary Table S2). Rarefaction analysis indicated that the sequencing effort covered most of the bacterial diversity present in the samples, which was further supported by Good’s coverage estimates of 99% . Most field samples showed a greater OTU richness estimate (Chao1) than aquarium samples and ANOVA showed this to be statistically significant in the overall test and between field t0 and aquarium t1 samples (Supplementary Table S2 and Table 2). No statistically significant differences were observed for the inverse Simpson diversity index (Table 2).From the sequencing of the 16S rRNA gene we obtained between 25,297 (F1-3) and 66,948 (F0-3) quality-filtered sequences, which clustered into OTUs (at 97% sequence identity) ranging between 566 (A1-3) and 1,068 (F0-3) per sample  and identified 95 OTUs that showed a significant individual difference . These OTUs represent 5% of the total OTUs and contribute together to the top 21% of the overall community difference. Of these top contributors, 30 were only found in aquarium samples and could represent potential laboratory contamination . These OTUs represented only 1.6% of the total community abundance . In contrast, the remaining 65 OTUs were present in field samples and thus are considered part of the algae’s natural community. Here, in particular, several OTUs were enriched in aquarium samples to achieve an abundance of 28% or were reduced to levels of 4% (from 16% in filed samples). In summary, these results show that most of the difference between field- and aquarium-held algae was produced by shifts of naturally occurring bacteria and that potential contaminants represent only a small fraction of the community in the aquarium.We then asked if the differences between field- and aquarium-held thalli were due to the colonization and proliferation of non-native bacteria  or due to a general shift of the natural microbial community of Supplementary Figure S4). Analysis of the novel/enriched OTUs at the order level, where most (87%) of the OTUs could still be taxonomically classified, showed that cultivation of D. pulchra under aquarium conditions preferentially enriches for certain taxonomic groups of microbial associates . For example, OTUs assigned to the orders Kordiimonadales, Alteromonadales, and Cytophagales were frequently enriched or novel in microbial communities in the aquarium.Most of differential OTUs were assigned to the phyla Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria and Verrucomicrobia  between bleaching incidence and water temperature for D. pulchra, but deeper (7–10 m) populations had no significant correlation , this suggests that handling and/or the aquarium conditions impose a stress with consequences for the algae’s health. Transfers to an aquarium have previously been observed to cause stress in other marine sessile organisms, including corals, sponges and macroalgae , Mycale laxissima and Ircinia strobilina after 6 and 3 months in a flow-through and recirculating aquarium, respectively. Also, Fungia granulosa after being held for just 3 weeks in an aquarium with artificial seawater. However, some host-associated communities have also been reported to experience little or no noticeable change when moved from the field to aquaria conditions. For example, DGGE and metagenomic data found that the microbial communities of wild specimens of the sponge Rhopaloides odorabile were highly similar to those kept in a flow-through aquarium for up to 4 weeks . Bacteria that were enriched under aquarium conditions belong to orders that are commonly found in the marine environment, including those that are in association with sediments and multicellular marine eukaryotes. The Kordiimonadales, for example, are globally distributed alphaproteobacteria, which have been isolated from coastal (Kordiimonas) had an average relative abundance of 12%, which was the highest for any OTU significantly enriched in algae held under aquarium conditions . The second most abundant aquarium-enriched OTU  belonged to the genus Glaciecola , which contains species that have been isolated from diverse marine environments, such as coastal surface seawater  are members of the Cytophagales (phylum Bacteroidetes), which are found in coastal environments rich in organic material, such as living or dead macroalgae, aerobic and anaerobic seafloor sediments and decaying marine animals  microorganisms. If this is true, then the new microbial community, although being different from the ones in the field, could obtain or preserve functions to sustain the health of the host. Future studies therefore need to define the functionality of aquarium-based microbiomes associated with these marine hosts and how they relate to host function in natural situations.Secondly, microbial changes could represent an adaptive response of the holobiont to the aquarium environment as has been suggested for corals . This ouEZ-V and TT designed and conceived the research. EZ-V and AJR-S performed the experiments. EZ-V and AJR-S analysed the data. EZ-V and TT wrote the manuscript with input from AJR-S.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. The serine-threonine protein phosphatase, protein phosphatase 2A (PP2A), is constitutively expressed in all eukaryotes. This phosphatase is implicated in diverse functions such as metabolism, DNA replication, transcription, RNA splicing, translation, cell-cycle progression, morphogenesis, development, and transformation ,2. Protea, a substrate which was used to measure its catalytic activity [v-scr transfected fibroblasts, p56lck from transformed T cells, and the receptor kinases, for epidermal growth factor and insulin [The catalytic subunit of PP2A (PP2A-C) can be covalently modified at the C-terminus by carboxymethylation  and tyroactivity . Howeveractivity . Phosphoactivity ,14. Ther insulin . Reversi insulin ,16.in vitro. Sperm motility initiation and fertilizing ability develop during their passage through the epididymis [Testicular spermatozoa in mammals are immotile and lack the ability to fertilize eggs ididymis . Despiteididymis ,19. It iididymis –22. A roididymis ,24. Inclididymis .We have shown that the testis isoform of protein phosphatase 1, PP1γ2, is essential for spermatogenesis and is also involved in sperm motility regulation ,24. Immo4) [8 sperm/ml by resuspension in homogenization buffer with protease inhibitors  β-mercaptoethanol and 1 mM sodium orthovanadate). The sperm suspension was sonicated on ice with three 10-sec bursts of a Microson ultrasonic cell disruptor (Misonix Inc.) set at level three. The suspension was then centrifuged at 16,000 x g for 20 min. at 4°C. The supernatants were supplemented with 5% glycerol and stored at -20°C.Mature bull testis were obtained from Duma Meats, a local commercial slaughter house within couple of hours of sacrifice. Spermatozoa were isolated from proximal caput, distal caput and caudal epididymis and washed twice in Hepes buffer, pH 7.0  . Sperm pMicrocystin-agarose beads (Upstate) were washed two times with an equal volume of HB+ buffer supplemented with 5% BSA to prevent nonspecific binding. The Microcystin-agarose beads were mixed with 100 μl sperm extract at 4°C with rotation overnight. Following this incubation the flow-through (unbound) fraction was collected by centrifugation. The bead pellet (bound PP2A) was washed by centrifugation twice with TTBS  and two times with HB+ buffer. The flow-through and pellet fractions were boiled with Laemmli sample buffer and stored at -20°C for Western blot analysis.For alkali mediated PP2A demethylation, PP2A in sperm extracts was first bound to microcystin. A 10 μl volume of microcystin-agarose bead slurry (prepared as described above) was incubated with sperm extracts prepared from the proximal caput, distal caput and caudal regions of the epididymis. The extracts were incubated for 150 min, washed in TTBS wash buffer  twice and in HB+ once. The washed microcystin-agarose beads were then resuspended in 30μl of HB+. Microcystin bound PP2A from each of these three regions was divided into two equal aliquots; one part was treated with 0.2 M NaOH while the other was left untreated as control. After 8 minutes at 4°C the alkali was neutralized with 0.2 M Tris-HCl (pH 6.8). An equal volume of neutralized alkali was added to the control suspension . MicrocySperm extracts, denatured by boiling for 3 min in Laemmli sample buffer, were separated by SDS-gel electrophoresis using 12% polyacrylamide slab gels in a Mini-Protean II system (Bio-Rad Laboratories). After electrophoresis, proteins were electrophoretically transferred to Immobilon-P, PVDF membrane (Millipore Corp). Nonspecific protein binding sites on the membrane were blocked with 5% nonfat dry milk in TTBS . The blots were washed with TTBS and then incubated overnight with primary antibody at 4°C. The following primary antibodies were used during this study: PP2A-C demethyl-sensitive antibody 4b7  (1:2000), anti-PP2A-C  (1:2000), anti-PP2A-C  (1:1000), anti-phospho-PP2A-C Y307  (1:500), anti-PP1γ2  (1:5000) , anti-PMa was used as a substrate to measure catalytic activity of PP2A [32P released from phosphorylase a. Enzyme activity is expressed in mmol of PO4 released/minute/2x105 sperm.Radio-labeled phosphorylase  of PP2A . The sub7 sperm/ml. Motility was analyzed on 20 μm capillary microscope slides by the computer assisted analyzer (CASA) of Hamilton Thorne Biosciences installed with the CEROS version 12.2g software. Three random fields were chosen for each sample and motility was recorded for 90 frames at 60 frames per second. The data was analyzed with the following settings: minimum contrast 30, minimum cell size 4 pixels, static cell size 8 pixels, static cell intensity 60, low size gate 0.17, high size gate 2.26, low intensity gate 0.35, high intensity gate 1.84, minimum static elongation gate 0, maximum static elongation gate 90, minimum VAP 50μ/s, minimum STR 50%, VAP cut off 10μ/s and VSL cut off 10μ/s.Sperm from the caudal epididymis were isolated and washed twice by centrifugation with HEPES buffer. Sperm were then incubated at 37°C in HEPES containing 20mM glucose and 20% BSA for control measurements and along with1 mM L-homocysteine in addition to 1 mM adenosine to elevate intracellular S-adenosylhomocysteine (SAH). For motility determination, samples were taken from sperm preparations at a concentration of 2X10Western blot analysis of soluble extracts of sperm isolated from proximal caput, distal caput, and caudal regions of the epididymis when probed with anti-PP2A antibody each showed one immunoreactive band at ~36 kDa, presumably due to PP2A. There was equal PP2A immunoreactivity in the three extracts obtained from each region . Little Since PP2A, in somatic cells is known to undergo tyrosine phosphorylation , we nextData in Though demethyl PP2A antibodies are well characterized, we sought to determine by an independent method that sperm PP2A methylation is different in the three regions of the epididymis. Carboxy-methyl groups in proteins can be removed by sodium hydroxide treatment ,31. PP2AThe enzyme PP2A methyl transferase, LCMT1 responsible for transferring the methyl group from S-adenosyl methionine specifically to PP2A is detected by Western blot analysis of mouse testis and sperm extracts probed with Anti-LCMT1 antibody at ~38Kd . HoweverS-Adenosyl-L-methionine (SAM) is a universal methyl donor in methylation reactions. Following transfer of its methyl group SAM is converted to S-adenosylhomocysteine (SAH). Cellular SAH is broken down to homocysteine and adenosine by the enzyme SAH hydrolase. Homocysteine and adenosine are further metabolized or transported out of the cell. Elevated intracellular SAH is a feedback inhibitor of methyl transferases preventing methylation reactions. Incubation of cells including spermatozoa with adenosine and homocysteine results in increased intracellular SAH levels. L-homocysteine by itself also increases the intracellular SAH levels but to a lesser extent than when adenosine is also present .8 /ml) were incubated at 37°C with 1 mM L-homocysteine with 1mM adenosine, 1 mM L-homocysteine alone, or 1 mM adenosine alone for 10 min in a suspension buffer containing glucose and BSA. Western blot analysis of extracts of sperm following this incubation is shown in Caudal spermatozoa  inhibits PP1 (IC50 of 1 nM) but not PP2A [4 released/minute/2x105 sperm, respectively. As seen in We measured catalytic activity of PP2A in sperm extracts using phosphorylase and PP2A . The linand PP2A ,24. Simiand PP2A . Focusinnot PP2A . PhosphaNext we examined if demethylation of PP2A or PP2A inhibition could initiate sperm motility in distal caput sperm. Distal caput sperm were treated with 1 mM L-homocysteine and 1 mM adenosine to demethylate PP2A or 5 nM okadaic acid to inhibit PP2A. These treatments could not initiate motility in immotile caput epididymal sperm even when L-homocysteine and adenosine concentrations were increased to 10 mM and up to four hours of incubation. However, as previously reported, higher concentration of the okadaic acid (1 uM), a concentration that inhibits both PP1 and PP2A was able to initiate motility in immotile distal caput sperm .°. Motility was quantified with a Computer Assisted Sperm motility Analyzer (CASA) system. For both the L-homocysteine and adenosine- treated and okadaic acid-treated sperm, all motility parameters were increased compared to control  phosphorylation is associated with sperm maturation and motility . It appeWe previously identified that PP2A is one of the two serine/threonine phosphatases in sperm. The other is PP1γ2, which is present both in the soluble and insoluble fractions of sperm sonicates . Here wein vivo and in vitro  and PP2A specific methyl esterase 1 (PME1). The fact that PP2A methylation changes tro Figs  also impin vivo, it is puzzling that the activity of PP2A is lowered, rather than increased, in caudal epididymal sperm following incubation of sperm with adenosine and homocysteine in vitro which results in demethylation of PP2A. It is possible that SAH is both an inhibitor of methyl transferase and of PP2A activity. It is interesting in this regard that pharmacological inhibition with okadaic acid also causes demethylation accompanied by increased tyrosine phosphorylation of PP2A  Sperm extracts from caudal regions of epididymi were prepared by sonication. The soluble fraction of the extracts was serially diluted and analyzed for phosphatase activity with phosphorylase a as the substrate. A 4ul sperm extract aliquot initially containing 4X106 sperm were serially diluted to 0.25X106 sperm. The combined activity of PP1 and PP2A denoted in mmol/min decreases in a linear fashion with decreasing sperm numbers in the extract.  A caudal sperm concentration of 2x106/ml was further used to measure phosphatase activity at various time points with intervals of approximately 3mins. The total phosphatase activity shown in counts per minute (CPM) increases linearly with time. The 10 minute time point was used in all our experiments.(TIF)Click here for additional data file.S2 File without any treatment (Control),  treated with 1mM L-homocysteine and adenosine (L-Hcy + Ado) and  5 nM okadaic acid (OA) after 10 minutes of incubation at 37°C. Sperm treated with 1mM L-homocysteine and adenosine or 5nM okadaic acid show increased percentage motility, velocity and lateral head amplitude.CASA recordings of Bovine sperm in HEPES buffer supplemented with glucose and BSA (WMV)Click here for additional data file.


Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways. Production of ROS by NOX family has long been considered a unique property of phagocytic cells, which utilize this enzyme as a part of host defense immune system. Currently, the regulated ROS production in nonphagocytic cells by NOX was linked to regulation of different processes including proliferation, migration, differentiation, immunomodulation, and oxygen sensing, and therefore, its expression and activity are tissue specific and are tightly controlled [Reactive oxygen species (ROS) play multiple roles in the biology of the cell . NADPH ontrolled , 5. On tntrolled . in vitro and in vivo studies and although their effect was attributed primarily to NOX inhibition, specificity of APO and DPI remains questioned [Despite studies suggesting the importance of NOXs in general, the involvement of individual NOX family members in specific function is still not completely understood. One of the reasons is a lack of highly specific inhibitors that would reliably block particular NOX. The most frequently used inhibitors employed in experiments are vanillin derivative 4-hydroxy-3-methoxyacetophenone  and diphenyleneiodonium chloride (DPI). Both of these drugs were applied in numerousestioned .The proposed molecular mechanism of APO-mediated NOX inhibition is not fully understood but it involves impairment of NOX complex assembly and activation . APO was2∙− mediated apoptosis [DPI was reported not only to affect NOX, but also to interfere with other flavoenzymes, including nitric oxide synthase and xanthine oxidase , 15. Inhpoptosis , inhibitpoptosis . DPI is poptosis . The aboIntracellular formation of ROS leading to overall redox status modulation is important for regulation of pluripotent cells differentiation . In mousβ-catenin signaling pathway plays a vital role in the regulation of mES fate [Several signaling pathways are crucial for the regulation of self-renewal and differentiation of mES. Primarily, the mES pluripotency is controlled by Stat3 together with PI3K/Akt signaling pathways –27. ImpomES fate . SignalimES fate , 32, 33 Our study demonstrates unexpected prooxidant activity of APO and DPI in undifferentiated mES together with impairment of cell proliferation. Further, we show that APO inhibits PI3K signaling accompanied by decrease in protein level of critical ES pluripotency regulator Nanog, whereas DPI enhances both PI3K and Erk activity. Interestingly, APO strengthens Wnt activity, pointing out another unknown mechanism of APO-mediated changes in signaling cascades regulating mES. In contrast to PI3K and Erk, we did not observe any effect of APO or DPI on Stat3 phosphorylation, which is considered to play the major role in mES maintenance.β-mercaptoethanol , supplemented with 5 ng/mL of leukemia inhibitory factor  referred to here as the complete medium.A feeder-free adapted mES line R1 was propagated as described previously  in an un2O2), N-acetylcysteine (NAC), and LY294002 (LY) were provided from Sigma-Aldrich. Stock solutions of APO (0.4 M), DPI (10 mM), and LY (10 mM) were prepared by dissolving the compounds in dimethyl sulfoxide. Aliquots were stored at −20°C. NAC was prepared as a 0.5 M stock solution in serum-free DMEM medium, pH was adjusted to 7.4, and filter-sterilized aliquots were stored at −20°C. Drugs were added directly to the incubation medium or freshly prediluted in sterile phosphate-buffered saline (PBS) to desired concentration.APO, DPI, hydrogen peroxide . Protein concentration was determined using DC protein assay  kit according to manufacturer's instructions.The cell proliferation was determined by estimation of overall cellular protein mass in whole cell lysates that reflects the cell number as demonstrated previously . ES cellμg of total RNA was used for cDNA synthesis with DyNAmo cDNA Synthesis Kit  according to the manufacturer's instructions. qPCR was performed in LightCycler 480 instrument using LightCycler Probes Master with Universal Probe library probes  according to manufacturer's instructions. Ribosomal protein L13A (RPL13A) was used as reference gene; data are presented as 2−(Cq(target)−Cq(reference)). The primers and probes used were as follows:Total RNA was extracted using the UltraClean Tissue & Cells RNA Isolation Kit  for ESC and RNAzol RT  for mouse tissues according to manufacturer's instructions. 1 NOX1: 5′tggattttctaaactaccgtctcttc, 5′caaagtttaatgctgcatgacc3′, #20; NOX2: 5′tgccaacttcctcagctaca3′, 5′gtgcacagcaaagtgattgg3′, #20; NOX3: 5′tgaacaagggaaggctcatt3′, 5′catcccagtgtaaagctatgtga3′, #20; NOX4: 5′catttgaggagtcactgaactatga3′, 5′tgtatggtttccagtcatccag3′, #5; DUOX1: 5′acagatggggcagcaaag3′, 5′gctggtagacaccatctgcat3′, #20; DUOX2: 5′cagacagctttttgctcaggt3′, 5′cacttgctgggatgagtcc3′, #64; RPL13A: 5′catgaggtcgggtggaagta3′, 5′gcctgtttccgtaacctcaa3′, #25.μM, Sigma-Aldrich). Live imaging of prepared samples was performed at 37°C and 5% CO2 atmosphere using the high-content screening microscope ImageXpressMicroXL . Seven images per well were acquired during 120 minutes with scanning interval of 10 minutes. By Gaussian thresholding of the HE fluorescence images, mask area of the viewfield covered with metabolizing cells was detected using the Otsu method of fitting individual objects. The 10640 images for every measured plate were analyzed with CellProfiler [The cells were seeded to 96-well plate designed for live imaging fluorescence microscopy . After 24 hours, cells were pretreated with tested drugs for 15 minutes and loaded with hydroethidine  which is formed in the reaction of O2∙− with HE [μM was added. The medium after centrifugation was stored for optional HPLC analysis. To extract the HE products, ice cold methanol was added to the cells [μL sample was injected into the HPLC system (Agilent series 1100) equipped with fluorescence and UV detectors (Agilent series 1260) to separate the 2-OH-E(+) product. Fluorescence was detected at 510 nm (excitation) and 595 nm (emission). The mobile phase consisted of H2O/CH3CN. Kromasil C18 (4.6 mm × 250 mm) column was used as the stationary phase. Elution conditions for the analysis of HE and its products were used from Nature Protocols [The HPLC detection of O with HE , 39. Beshe cells  for 15 mrotocols .β-actin , p-Akt (S473), Akt, p-Stat3 (Y705), Stat3, Erk1/2, p-Erk1/2 (T202/Y204), and p-GSK3β (S9) . Following immunodetection, each membrane was stained by amido black to confirm the transfer of the protein samples. The total level of β-actin was detected as loading control.Western blot analysis, cell sample harvesting, and preparation were performed by a standard procedure as presented previously . We usedμL per 1 μg of DNA. Super8X TOPflash construct, Renilla luciferase construct, and expression vector for mutant nondegradable β-catenin, and S33-β-catenin  were used in concentration of 0.5 μg per well in a 24-well plate 24 hours after seeding. 6 hours after transfection medium was changed and cells were treated with NOX inhibitors or LY for 24 hours. For cell stimulation Wnt3a or control conditioned medium [ Renilla luciferase expression.Cells were transiently transfected using polyethyleneimine in a stoichiometry of 4 d medium  was addet-test or by one-way analysis of variance ANOVA and Bonferroni's Multiple Comparison posttest. The values of P < 0.05 were considered statistically significant .Data are expressed as mean + standard error in the mean (SEM). Statistical analysis was assessed by To confirm the assumption of the negligible presence of NOX and DUOX homologues in the undifferentiated mES, the gene expression was compared to the selected mouse tissues. Particular organs were chosen based on the described presence of NOX homologues by various authors , 6. In aBoth APO and DPI affected growth of mES in dose-dependent manner in the concentration range 0.1, 0.25, 0.5, 1.0, and 2.0 mM for APO  and 10, 2∙−, the 2-OH-E(+), and also nonspecific product, ethidium (E+), were determined by HPLC .Finally, we examined the impact of APO and DPI on Akt and Erk activity after 24 hours in absence or presence of NAC . Contrarβ-catenin which specifically induces the transcriptional activity of TCF/LEF [β-catenin, both known agonists, to promote its activation. These interventions induced transcription activity of reporter gene 45 and 25 times, respectively  to determine the level of canonical Wnt activation mediated by  TCF/LEF . Firstlyectively .β-catenin mediated transcription activity of TCF/LEF (β-catenin (PI3K inhibitor LY294002 (LY) and both NOXs inhibitors APO and DPI did not change the spontaneous  TCF/LEF . In cont TCF/LEF . On the  TCF/LEF . However-catenin .β-catenin signaling, the GSK3β S9 phosphorylation, allowing accumulation of β-catenin, was evaluated [β S9 phosphorylation in this setup as shown by the western blot analysis . However, in the intracellular milieu, the presence of redox metal ions or hemeproteins with peroxidase activity or other one-electron oxidants can oxidize HE to several nonspecific products, including the ethidium E(+) and dimeric products [2∙− production after APO and DPI treatment. The only observed effect was significant DPI-mediated elevation of E(+). APO did not induce generation of HE oxidation products {both 2-OH-E(+) and E(+)}, which is partially in contrast with the results from live imaging fluorescent microscopy where all nonspecific HE oxidation fluorescent products are summarized [2∙−, are responsible for increase in HE fluorescence.Further, we assessed how NOX inhibitors affected ROS production in our model. Due to their natural short half-life and high reactivity, precise detection of ROS represents a tremendous challenge, especially in nonphagocytic cells. We were aware of limits in ROS measurement with respect to specificity and generation of possible artefacts; therefore, we used two different assays, live imaging fluorescent microscopy and HPLC, both utilizing ROS-sensitive probe HE. The reaction between Oproducts , 59, 60. in vivo experiments [Although NOX inhibitors should generally relieve oxidative stress, several lines of evidence for APO and DPI exist demonstrating the opposite. It was reported that DPI inhibits pentose phosphate pathway (PPP) responsible for the synthesis of NADPH, a redox cofactor important for many antioxidant enzymes, thus making the cells more prone to oxidative stress . Suggesteriments , further2O2-induced phosphorylation. Similarly, we can assume that DPI-mediated ROS production is responsible for the observed effects on Akt and Erk activation which is in contrast to the response to APO. Properties of general antioxidant NAC in sense of attenuation of kinase phosphorylation were more profound during treatment in complete medium. Notably, we did not observe changes of Stat3 phosphorylation in presence of any drugs tested in our study. The most striking effect was detected downstream of PI3K on the level of S473 Akt phosphorylation when APO treatment was employed. In serum starved cells, in FBS or LIF activated cells, and also in the presence of complete medium, APO decreased Akt phosphorylation when short term impact was studied. It was earlier reported that vanillin and some of its derivatives, including APO, readily inhibited PI3K in lung adenocarcinoma cell line [Critical features of mES, pluripotency, self-renewal, and unlimited proliferation, are predominantly exerted through actions of cytokine LIF, which is routinely added to the culture medium. Binding of LIF to its receptor triggers the activity of three major intracellular signaling cascades: JAK/Stat3, PI3K/Akt, and MAPK/Erk. These pathways converge to regulate the gene expression pattern typical for mES . To clarell line . These a via PI3K inhibition. To further elucidate impact of APO-induced PI3K inhibition, we examined the level of homeodomain transcription factor Nanog, which was suggested as a downstream target of PI3K and is also recognized as an important intrinsic regulator of stem cell pluripotency [Remarkably, critical role of PI3K was also described for the process of NOX activation ; thus itipotency . After 2β-catenin on multiple sites that enhances its subsequent degradation [β and S21 in GSK3α) [β-catenin signaling when insulin and constitutively active Akt were administrated [β-catenin degradation, represents just a minor fraction of GSK3 cellular content and this complex is not accessible to Akt phosphorylation, thus preventing PI3K/Akt/Wnt cross-talk [β S9 phosphorylation, further questioning the simplified model of PI3K/Wnt cross-talk mediated solely by level of GSK3 inhibition. However, after Wnt3a induction, both LY and APO augmented Wnt transcription activity. Notably, PI3K inhibition was recently shown to increase the amount of active nuclear β-catenin and to promote induction in TOPflash assay in epithelial cell culture system [β-catenin signaling. Although Erk kinase was not dramatically affected by APO treatment in our system, we are not excluding the possibility of a different MAPK involvement in observed phenomena, as numerous convergence points were described between PI3K and MAPKs [Next, we examined the impact of NOX inhibitors on canonical Wnt signaling, as it also plays a distinctive role in regulation of embryonic stem cells , 71 and radation . GSK3 isn GSK3α) . Therefoistrated . Moreoveoss-talk . In our e system . In agree system , 77, autnd MAPKs .Altogether, our study suggests prooxidant activity of APO and DPI in mES. Moreover, treatment with those drugs results in different modulation of intracellular pathways critical for regulation of proliferation and differentiation. APO markedly downregulates activity of Akt and its downstream Nanog and augments Wnt signaling. DPI promotes Akt and Erk activation. Taking into account described negligible NOXs levels in mES we suggest that actions of drugs observed in our experiments are rather independent of their typical function as NOX inhibitors. Therefore, caution should be taken to potential applications of these NOX inhibitors and interpretation of obtained results, especially in studies focused on the stem cell biology and intracellular and redox signaling.


O-GlcNAc modification regulates functions of several important components of insulin signal pathway. In this study, we identified that PTP1B is modified by O-GlcNAcylation at three O-GlcNAc sites . Palmitate acid (PA) impaired the insulin signaling, indicated by decreased phosphorylation of both serine/threonine-protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) following insulin administration, and upregulated PTP1B O-GlcNAcylation in HepG2 cells. Compared with the wild-type, intervention PTP1B O-GlcNAcylation by site-directed gene mutation inhibited PTP1B phosphatase activity, resulted in a higher level of phosphorylated Akt and GSK3β, recovered insulin sensitivity, and improved lipid deposition in HepG2 cells. Taken together, our research showed that O-GlcNAcylation of PTP1B can influence insulin signal transduction by modulating its own phosphatase activity, which participates in the process of hepatic insulin resistance.Protein tyrosine phosphatase 1B (PTP1B), which can directly dephosphorylate both the insulin receptor and insulin receptor substrate 1 (IRS-1), thereby terminating insulin signaling, reportedly plays an important role in insulin resistance. Accumulating evidence has demonstrated that  Insulin resistance, a state in which peripheral tissues demonstrate a diminished response to the glucose-lowering properties of insulin, plays a central role in the development of type 2 diabetes mellitus (T2DM). After insulin stimulation, insulin receptor (IR) is autophosphorylated. Activated IR phosphorylates the downstream docking protein insulin receptor substrate 1 (IRS-1), which, subsequently, through the activation of the phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B (PKB) pathway, leads to the translocation of glucose transporter type 4 (GLUT4) vesicles to the cell surface and triggers cellular glucose uptake [The involvement of protein tyrosine phosphatase 1B (PTP1B) in insulin signaling has been suggested in numerous reports ,4,5,6. PO-linked-N-acetylglucosamine (O-GlcNAc). O-GlcNAc rapidly cycles on serine and threonine residues in a fashion analogous to phosphorylation. Unlike phosphorylation, the addition of O-GlcNAc is performed by a single catalytic subunit , and the removal is achieved by O-GlcNAc selective N-acetyl-β-d-glucosaminidase . Emerging evidence suggests O-GlcNAc’s putative roles in the pathogenesis of insulin resistance. Increased intracellular protein O-GlcNAc modification has been observed in diabetes and hyperglycemic states, like FoxO1, ChREBP, CRTC2 et al. [O-GlcNAcylation of proteins involved in proximal and distal steps in the PI-3 kinase signaling pathway [Glucose flux through the hexosamine biosynthetic pathway (HBP) leads to the post-translational modification of cytoplasmic and nuclear proteins by 2 et al. ,15,16,17 pathway ,19. In a pathway ,21,22.O-GlcNAc modified remains unclear. In this study, we observed that PTP1B has three O-GlcNAc sites: Ser104, Ser201, Ser386; O-GlcNAcylation of PTP1B is increased in insulin resistance; and intervention PTP1B O-GlcNAcylation can recover insulin sensitivity and improve lipid deposition in HepG2 cells. This study helps us to further understand the contribution of post-translational modification of PTP1B to the etiology of insulin resistance.Diverse post-translational modifications have been reported to regulate PTP1B functions, including oxidation, nitrosylation, sulfyhydration, and phosphorylation ; howeverO-GlcNAc functioned as a nutrient sensor, we tested whether PTP1B was O-GlcNAc modified in HepG2 cells. O-GlcNAcylated using the anti-O-GlcNAc antibodies, RL-2 and CTD110.6 , another approach to increase protein O-GlcNAc concentrations, we also observed increased O-GlcNAc levels of PTP1B, which further confirmed that PTP1B was an O-GlcNAc modified protein  level is commonly associated with obesity and T2DM. Palmitate acid (PA) is one form of saturated FFAs, which are frequently used to induce insulin resistance . So we fO-GlcNAc level was altered in this cell model of insulin resistance. We treated HepG2 cells as previously described and found that O-GlcNAcylation of total protein was increased by palmitate . Then we detected PTP1B O-GlcNAcylation level when O-GlcNAc sites were mutated. All three mutants showed lower amounts of O-GlcNAcylation when compared with the wild-type, further confirming they are O-GlcNAc sites (Next we conducted site-directed mutants containing Ala substitution at Ser104 (S104A) and Ser201 (S201A) to block Ac sites C.O-GlcNAcylation regulated PTP1B phosphatase activity. We found site-directed mutant PTP1B phosphatase activity was impaired when compared with the wild-type, and the level of its phosphatase activity was concomitant with its O-GlcNAcylation level. Mutation at Ser201 was more significant. Constitutive phosphorylation mutant S201D partially reversed phosphatase activity when compared with 201A  overlap with its phosphorylation sites. Because how phosphorylation regulates PTP1B phosphatase activity is unclear and simple site-directed mutations will be unable to distinguish between the biological functions mediated by either O-GlcNAcylation or phosphorylation, it is complex to investigate the function of PTP1B O-GlcNAcylation, despite the fact that the interplay between O-GlcNAcylation and phosphorylation has been reported in other proteins [O-GlcNAc sites might be important for regulating PTP1B phosphatase activity and functions. So we constructed site-directed mutants to further investigate its role in insulin resistance: Ser104 and Ser201 were mutated to alanine to block O-GlcNAcylation, while Ser201 was also mutated to aspartic acid to mimic constitutive phosphorylation. All the mutants represented a decreased state of PTP1B O-GlcNAcylation when compared with the wild-type, displaying markedly reduced phosphatase activity, and in turn resulting in the significant recovery of normal insulin signal transduction. Importantly, the effect of S201A was more significant, and the level of its phosphatase activity was concomitant with its O-GlcNAcylation level. This is possibly because Ser201 residue is closer to the essential catalytic cysteine 215, which is located at the base of phosphatase’s active site cleft. Constitutive phosphorylation mutant S201D partially reversed its phosphatase activity when compared with S201A, suggesting that the crosstalk between O-GlcNAcylation and phosphorylation is more complex than the “yin-yang hypothesis,” and this interplay is not a simple binary model with reciprocal on or off status. However, we were unable to test this possibility due to the lack of available phospho-site-specific anti-PTP1B antibodies. We know that PTP1B is phosphorylated on tyrosine and serine residues, which can either enhance or attenuate its enzymatic activity [Next we determined PTP1B with three proteins ,29,30. Cactivity ,27, but O-GlcNAcylation represents a novel regulation of PTP1B activity and functions. Inhibition of PTP1B O-GlcNAcylation can recover insulin sensitivity, facilitate hepatic glucose uptake, and improve lipid deposition. Numerous studies have reported the beneficial effects of PTP1B inhibitors in enhancing insulin sensitivity. However, structural homologies in the catalytic domain of PTP1B with other PTPs like leukocyte common antigen-related, CD45, SHP-2, and T-cell-PTP present a challenging task of high selectivity [O-GlcNAc sites of PTP1B are expected. It may provide a potential therapeutic target for the treatment and prevention of diabetes.In summary, our results emphasize the importance of PTP1B in the control of hepatic insulin signaling and reveal that O-GlcNAc antibodies (CTD110.6 and RL-2), O-(2-acetamido-2-deoxy-d-glucopyranosylidene), amino-N-phenylcarbamate (PUGNAc), anti-PTP1B, anti-myc, anti-GAPDH antibodies, palmitate acid, and Oil Red O were from Sigma . Lipofectamine 2000 was obtained from Invitrogen .Akt, phospho-Akt (Ser473), GSK3α/β, phospho-GSK3α/β (Ser21/9) antibodies were obtained from Cell Signaling . 2 (5%). Then the cells were treated with palmitate acid  for 24 h. Palmitate acid was prepared by conjugating it with bovine serum albumin and diluting it to appropriate concentrations as previously reported [Human HepG2 cells were grown in 5 mmol/L (low glucose) or 25 mmol/L (high glucose) Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum at 37 °C with humidified air and COreported . For somAnimal studies were conducted in accordance with the National Institutes of Health Animal Care and Use Guidelines. Male six-week-old C57BL/6J mice were housed with 12 h light/dark cycle with free access to water and given a normal diet (ND) or high fat diet (HFD) containing 50% carbohydrate, 20% protein, and 30% fat for 12 weeks. A glucose tolerance test was conducted two weeks before sacrifice. Mice were fasted overnight and then dosed orally with glucose (2 g/kg). The tail vein blood glucose level was measured at 0, 20, 40, 60, and 120 min after administration.Cells were lysed in M-PER mammalian protein extraction reagent containing protease inhibitor mixture and phosphatase inhibitor mixture (Sigma), and fractional protein was extracted according to the manufacturer’s instructions. The protein level was quantified using the Bradford procedure. Two hundred micrograms of whole cell extracts were separated by SDS-PAGE and transferred to an enhanced nitrocellulose membrane. The blots were blocked with 5% skim milk for 1 h, and incubated overnight at 4 °C with the primary antibody at the dilutions recommended by the supplier. The membrane was then washed with TBST , and the secondary antibodies conjugated to horseradish peroxidase were incubated for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence system . ImageJ was used to analyze the densities of the bands.N-acetylglucosaminidase and lysed in 1.5% SDS containing Complete™ protease inhibitor cocktail  and 5 µM PUGNAc. The lysate (7.5 mg) was diluted 1.5-fold, quenched with one volume of NETFS buffer containing 6% (v/v) NP-40 and then was further diluted to 2 mg/mL with NETFS buffer. The sample was passed over 400 μL of anti-PTP1B M2 affinity gel three times, washed with 10 mL of NETFS containing 1% (v/v) NP-40, eluted in 400 μL of 4% SDS, 100 mM Tris separated pH 7.9, and concentrated to a volume of 20 μL. After SDS-PAGE (4-12% Bis-Tris gels), the PTP1B band was excised and manually digested in gel with trypsin or Glu-C at 37 °C. LC-MS analysis was carried out on LTQ mass spectrometer . Peptide mixtures were on a C18 column. Mass spectrometric data were searched by SEQUST against PTP1B protein sequence. The relevant searching parameters of mass range, intensity threshold, minimum ion count, precursor ion mass tolerance, and fragment ion mass tolerance were set as 500–5000, 500, 15, 3.0, and 1.0, respectively. Trypsin or Glu-C was designated as the protease, and up to two missed cleavages were allowed. Carbamidomethylation of Cys was a fixed modification, and phosphorylation (80.0 Da) and O-GlcNAcylation (203.2 Da) of Ser/Thr, and oxidation of Met (16 Da) were allowed as variable modifications.Human HepG2 cells were treated with PUGNAc to inhibitβ-Human wild-type PTP1B plasmid was obtained from Genechem , inserted into pcDNA3.1/myc-His (−) vector. Site-directed mutants  were performed by PCR using mutagenic oligonucleotides using human wild-type PTP1B plasmid as template. The primers used in the mutations were the following: S104A, forward, 5-TGGGAGCAGAAAGCAAGGGGTGTCGT-3, and reverse, 5-ACGACACCCCTTGCTTTCTGCTCCCA-3, S201A, forward, 5-AAAGTCCGAGAGGCAGGGTCACTCAG-3, and reverse, 5-CTGAGTGACCCTGCCTCTCGGACTTT-3, S201D, forward, 5-AAAGTCCGAGAGGATGGGTCACTCAG-3, and reverse, 5-CTGAG TGACCCATCCTCTCGGACTTT-3. Mutagenized plasmids were checked by sequencing, and a region containing the mutation was systematically subcloned into the original pcDNA3.1/myc-His (−) vector using endogenous restriction sites to prevent mutations that may have occurred elsewhere on the plasmid.Wild-type PTP1B, different site-directed mutations plasmids were constructed as previously described. Using lipofectamine 2000, 5 mg of plasmid was transfected for 36 h in HepG2 cells according to the manufacturer.O-GlcNAc antibodies rotating at 4 °C, followed by incubation with 30 μL of protein A/G PLUS-agarose  for 3 h at 4 °C. Immunoprecipitates were extensively washed, resuspended in 2× sample buffer, boiled for 15 min, and analyzed by immunoblotting. Another method to detect terminal GlcNAc-specific lectin is to add succinylated wheat germ agglutinin (WGA) agarose beads to cell lysate and analyze by western blot with PTP1B antibody.Cells were transfected with myc-His-tagged full-length wild-type PTP1B or different point mutations. After 36 h, cells were collected in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 with protease inhibitors and then lysed by vortexing for 2 h. Equal amounts of lysate were incubated overnight with 5 μL of anti-In vitro PTP1B phosphatase assays were carried out according to the manufacturer’s instruction .To detect PTP1B phosphatase activity, HepG2 cells were transfected with wild-type PTP1B and its mutants for 36 h, followed by insulin stimulation for 20 min. Cells were lysed and 50 μL of different cell extracts were used as samples. HepG2 cells were transfected with wild-type PTP1B and its mutants for 36 h. Insulin was added during the last 20 min of incubation. A quantity of 50 μL of medium was sampled for measurement of glucose concentration using a Glucose Colorimetric/Fluorometric Assay Kit . The primary medium was used as control. Glucose concentration was normalized with cellular protein concentration.After transfection with myc-His-tagged wild-type PTP1B or different point mutations for 36 h, cells were washed three times with PBS and then fixed in 4% paraformaldehyde for 30 min and washed with 60% isopropyl alcohol. Then the cells were stained for 60 min in 0.2% Oil Red O dissolved in 60% isopropyl alcohol. Excess stain was removed by washing with 70% ethanol. The stained cells were finally washed three times with distilled water. Cells were imaged using a microscope.post hoc comparisons. The critical α level (p) was set at 0.05; p < 0.05 was considered statistically significant. GraphPad Prism 5 statistical software was used for analyses.Data are expressed as the mean ± SEM of results of three or more independent experiments. Statistical analyses were performed using one-way ANOVA, followed by individual O-GlcNAc modified protein and O-GlcNAcylation of PTP1B is increased in insulin resistance. O-GlcNAc site-directed mutants can partially inhibit PTP1B phosphatase activity, thereby reversing insulin signal transduction, facilitating glucose uptake, and improving lipid metabolism.In conclusion, our findings show that PTP1B is an


Ice and marine sediment records demonstrate that atmospheric dust supply to the oceans increased by up to an order of magnitude during glacial intervals. However, poor constraints on soluble atmospheric Fe fluxes to the oceans limit assessment of the role of Fe in glacial–interglacial change. Here, using novel techniques, we present estimates of water- and seawater-soluble Fe solubility in Last Glacial Maximum (LGM) atmospheric dust from the European Project for Ice Coring in Antarctica (EPICA) Dome C and Berkner Island ice cores. Fe solubility was very variable (1–42%) during the interval, and frequently higher than typically assumed by models. Soluble aerosol Fe fluxes to Dome C at the LGM (0.01–0.84 mg m−2 per year) suggest that soluble Fe deposition to the Southern Ocean would have been ≥10 × modern deposition, rivalling upwelling supply.Relief of iron (Fe) limitation in the Southern Ocean during ice ages, with potentially increased carbon storage in the ocean, has been invoked as one driver of glacial–interglacial atmospheric CO Elevated deposition of bioavailable atmospheric iron may have enhanced carbon storage in the glacial Southern Ocean. Conway et al. apply a novel rapid-filtration technique to iron trapped in Antarctic ice cores and show that glacial soluble iron deposition was an order of magnitude greater than the modern. While these calculated soluble Fe fluxes are not fully representative of bioavailable Fe fluxes to the Southern Ocean, they enable an improved estimate of the atmospheric Fe that would have been deposited and dissolved in seawater at the LGM compared with previous studies. Our measured aerosol Fe solubilities and soluble aerosol Fe fluxes are higher than typically used in models of ocean biogeochemistry, and thus we suggest that models should consider higher and more spatially and temporally variable soluble atmospheric Fe fluxes to the Southern Ocean during glacial intervals.Here we present high-resolution fluxes of total and soluble atmospheric Fe, as well as calculated aerosol Fe solubility, from the Last Glacial Maximum ) of the EPICA Dome C ice core15192022Aerosol Fe solubility and calculated total and soluble Fe fluxes from EDC and Berkner ice cores are shown compared with other ice-core parameters in n=23) and 17% (n=11) at pH ∼5.3 and in seawater, respectively, and a median of 6% at pH ∼5.3  combines to mean that soluble Fe fluxes show great variability (3 per year), with mean values of 0.09±0.17 mg m−2 per year (1 s.d.) for pH ∼5.3 soluble Fe and 0.15±0.13 mg m−2 per year (1 s.d.) for seawater-soluble Fe. Despite the lower Fe solubility at Berkner Island compared with Dome C, higher atmospheric Fe fluxes at the LGM (0.95±0.07 mg m−2 per year), probably attributable to greater dust deposition owing to closer proximity to South American dust sources−2 per year) compared with those at Dome C. Total Fe fluxes at Berkner Island were almost an order of magnitude lower during Antarctic Warm Events A1 and A2 than during colder periods  are considerably higher than previous estimates based on acidic (pH ∼1–2) leaching or mineral dust proxies at several Antarctic locations . The difference may partially reflect the high-resolution variability in Fe flux at EDC, but also the fact that, similar to modern dust2219The aerosol Fe flux data presented here can be compared with previous ice-core estimates of Fe fluxes to Antarctica and the nearby Southern Ocean, during the LGM. We find that our total EDC aerosol Fe fluxes  represent a ∼30–50 × increase over EDC (0.3–0.4 μmol m−2 per year), and a ∼10–20 × increase over coastal sites such as Law Dome (ref. −2 per year). The modern gradient in dust deposition between coastal sites and EDC is thought to be largely influenced by wet deposition, which dominates at coastal sites and is a minor factor inland, and thus might be somewhat smoothed out during drier glacial intervals−2 per year represents a >10-fold increase over modern soluble Fe fluxes (even assuming a similar high solubility in modern dust), and is ∼10 × higher than modern calculated Southern Ocean atmospheric Fe fluxes  is also similar to modelled modern upwelling Fe flux values for entrainment  and vertical diffusion (1.2–3.6 μmol m−2 per year), which have been suggested to sustain surface water Fe concentrations in the modern Southern OceanBy comparison with estimates of Holocene Fe fluxes to Antarctica2. While the surface ocean is Fe-limited , surface waters can act as a source for pCO2. Deposition of atmospheric dust only supplies Fe and thus provides the means to increase utilization of upwelled carbon and nutrients in surface waters. A full understanding of the role of atmospheric Fe supply in carbon cycling of the past thus requires not only knowledge of upwelling and dust deposition Fe fluxes but also the resulting change in the carbon to bioavailable Fe concentration ratio in surface waters, as well as how both Fe and carbon are recycled and/or taken to depth. However, models do suggest that an increase in bioavailable atmospheric Fe supply of 5–10 × could double export production92 only has 2 p.p.m.v. sensitivity to dust globally, lower than the up to 40 p.p.m.v. suggested by recent Sub-Antarctic productivity/nutrient utilization recordsOf course, upwelling of deep water supplies both Fe and carbon, and it is the proportion of this carbon that is utilized by primary productivity in surface waters, and thus retained in the ocean, which determines the surface Southern Ocean's role in setting pCO2 release.In conclusion, our EDC soluble Fe fluxes suggest that soluble Fe fluxes to the Southern Ocean during the LGM and glacial times were likely higher than previously considered. Both Fe content (8 wt%) and mean Fe solubility  in EDC dust are greater than typically assumed by models  at the British Antarctic Survey, Cambridge, and all sample processing and analysis were carried out in clean laboratories at the University of Cambridge. Seawater analysis was carried out at Old Dominion University, USA. All clean work was carried out within a Class 1,000 HEPA-filtered clean laboratory and/or under Class 100 laminar flow air.All water used was ultrapure (18.2 MOhm cm) and acids were either quartz-distilled (QD) to high purity from reagent grade or purchased as ultrapure (Fisher Scientific). Strict trace metal protocols were used, including wearing polyethylene (PE) gloves to minimize metal contamination during cleaning, processing and sampling. All plastic equipment was rigorously acid-cleaned following the procedures outlined below, which were based on previously published work3 (24–48 h) and warm 50% (v/v) reagent-grade HCl (24–48 h). Teflon equipment was re-cleaned by immersion in either warm 50% (v/v) HNO3 or HCl (24 h). All Teflon equipment was thoroughly rinsed with ultrapure water following each step and then left to dry on a clean drying rack under laminar flow. For any Teflon equipment that was to be in contact with ice or samples at near-neutral pH, the plastic was filled with ultrapure water to condition the surface. Nalgene low-density PE and polycarbonate (PC) bottles were cleaned following previously published cleaning methods for preparation of equipment for seawater sampling3 and twice with ultrapure water before use (non-seawater samples).New perfluroalkoxy (PFA) or fluorinated ethylene propylene (FEP) Teflon filter rigs, vials, jars, bottles and micro centrifuge tubes were cleaned by sequential immersion in weak (∼5%) Decon detergent (24 h), warm 50% (v/v) reagent-grade HNOFor handling and decontamination of ice samples, Kyocera ceramic knives and polytetrafluroethylene (PTFE) Teflon boards were used. Knives were cleaned by sequential immersion in 6 M reagent-grade HCl (24 h) and 1 M reagent-grade HCl (24 h), followed by extensive rinsing with ultrapure water. PTFE boards were cleaned by immersion in 6 M reagent-grade HCl and then rinsed extensively with ultrapure water. Arm-length PE gloves for handling ice were cleaned by immersion in weak (∼0.01% v/v) QD-HCl (24 h) and rinsed thoroughly with ultrapure water to ensure no transfer of acid to samples.−1 dust; ref. 15Detailed information about the age of ice sampled in this study is shown in −1 levels. Preliminary testing in this study demonstrated that, as expected, ultrapure water ice was severely contaminated following cutting with a bandsaw . The small size of ice samples used in this study prevented the use of techniques that remove several cm of core, and so we developed a technique using Kyocera ceramic blades that sequentially removed contaminated ice-core material. Each piece of ice was placed on a PTFE board and held with a clean PE glove, while the outside few mm were scraped off with a clean ceramic blade. This was repeated three times using separate clean equipment. To achieve the lowest contamination levels, all ice sampling equipment and ice were only handled with clean PE gloves, and all ice decontamination was carried out under Class 100 laminar flow air within the cold room (−20 °C) at the British Antarctic Survey.The outside of ice core material is typically unavoidably contaminated with trace metals from drilling, cutting with bandsaws and handling with gloves225152−1 for Fe and 116 pg g−1 for Al , and testing established that using acid-cleaned equipment had no effect on the natural pH of ice (pH ∼5.3). Our simpler technique did not achieve the full decontamination of more rigorous chiselling methods−1 c.f. 4 pg g−1 Fe). However, our method can be applied to smaller pieces of ice, and the procedure reduces Fe contamination by factor of ∼100 × , resulting in insignificant contamination that represents <0.1% of typical natural Fe concentrations within ice from the high dust portion of the LGM.Decontamination blanks were 47 pg g3 (conc.) per 5 ml. A 5-ml aliquot was evaporated to dryness, refluxed overnight at ∼80 °C with 100 μl QD-HNO3 (conc.) and 200 μl Teflon-distilled conc. HF, evaporated to dryness, redissolved in 100 μl of 6 mol l−1 QD-HCl, refluxed at ∼80 °C overnight and evaporated to dryness. Finally, samples were redissolved in 250–500 μl of 0.1 mol l−1 QD-HNO3 for analysis by inductively coupled plasma optical emission spectrometry (ICP-OES). Procedural blanks (n=26) were 1.9 ng g−1 (Fe), 5.7 ng g−1 (Al) and 2.8 ng g−1 (Ca), all <0.5% of natural concentrations.Following decontamination, ice samples for total digestion were allowed to melt within PFA vials and acidified with 50 μl of QD-HNO3 to acidify samples to pH 0. On completion of melting, dust was leached with three 25 ml aliquots of ultrapure water, similarly acidified. This procedure, analogous to instantaneous leaches of modern aerosols3 precipitation or wall adsorption, which are concerns with flow-through and long leaching procedures  were 17 pg g−1 (Fe), 244 pg g−1 (Ca) and 83 pg g−1 (Na), all corresponding to 0–2% of natural concentrations.Following decontamination, ice samples for soluble analysis were processed by either ‘rapid-filtration'  or ‘seawater-leaching' methods (see later sections). Ice samples for rapid filtration were allowed to melt at room temperature (∼2 h) on a 0.2-μm Whatman polycarbonate membrane inside a 47-mm PFA Savillex filter rig, under vacuum. Rigs were covered to prevent contamination. Liquid that formed was quickly filtered and collected in a PFA reservoir containing an appropriate volume of conc. HNOocedures . Acidifin=10). Long-term reproducibility of the standard was 100±1% for all elements (n=60). Detection limits for Fe were 0.1–0.4 ng g−1. We assign 5% uncertainty on data to account for weighing, pipetting and %RSD of six ICP-OES analyses .Samples from total digestion or rapid filtration were analysed for dissolved Al, Ca, Fe and other cations using a Varian Vista ICP-OES at the University of Cambridge. Each sample was analysed six times and a mean concentration was determined by reference to a quadratic calibration curve based on five standards. Single-session accuracy on a 250-ng per gram internal multi-element reference standard for Al, Ca and Fe was >99.6% with precision of 0.41%  released from bubbles trapped in ice-core material5556572 measurements obtained using other techniques in BernSublimation allows the extraction of gases or dust from ice-core material while preventing melting, which could compromise the sample fidelity, and is achieved by lowering the pressure and temperature of ice below the triple point . Sublimation has been previously used to investigate concentrations and isotope ratios of gases . Rates of sublimation were relatively slow, dependent on ice mass and volume, with a maximum rate of ∼250 g per 24 h. Small samples (5–10 g) sublimed in several hours, but were typically left overnight to ensure all ice was gone.Once all ice had sublimed, the desiccator was sealed, disconnected and returned to the laminar flow bench to slowly allow clean air to enter the chamber; this prevented both dust being blown away by a sudden increase in pressure and the entry of dirty air from the pump. The bottles containing the dry dust samples were then carefully bagged and taken for ‘seawater-leaching' procedures (see next section). Blank contamination for the whole procedure over 24 hours was found to be acceptably low (∼40 pg Fe per ice sample).−1 , consistent with previous determination63−1. Each aliquot of seawater was allowed to leach the dust momentarily, vacuum was applied and then the liquid was pulled through and acidified to pH 1.6 with 120 μl of conc. ultrapure HCl. Samples were left acidified for >2 weeks before being analysed for dissolved Fe by FIA-8HQ, following published techniques−1) and analytical precision is typically 5–10% during routine analysis; we therefore applied 10% uncertainty on seawater Fe concentration. Fe concentrations from four leaches were combined in final calculations.Ice samples for seawater-soluble Fe analysis (‘seawater-leaching') were placed on a membrane on a PFA rig within a 1 l PFA jar within a glass desiccator at −20 °C, and quickly placed cleanly under vacuum (before moisture could form) at ∼18 °C. Samples remained under vacuum until all the ice had sublimed, leaving dry dust on the membrane (see ‘Sublimation' section). The dust on the membrane was then leached with four 60-ml aliquots of low-Fe Ross Sea seawater at 2 °C. The Ross Sea seawater used in leaching was previously collected18O record18O record so that EDML δ18O, Berkner δD and Berkner Fe data are all shown on approximately the EDC3 age scale. Dust and temperature in 152 concentration (pCO2) in both 2 record of Schilt et al.69The ice core at Berkner Island was drilled to bedrock, and completed in 2005 ref. . DeuteriElemental fluxes were calculated:−1) in the ice, AR is the calculated ice-accumulation rate , combined from multiple leaches.where [pH ∼5.3 or seawater-soluble Fe] and [total Fe] are the concentration in ice meltwater  and seasalt (0.038), respectivelywhere ss denotes from seasalt, nss from non-seasalt sources, [dNa] dissolved Na concentration at pH ∼5.3, [dCa] dissolved Ca concentration at pH ∼5.3, How to cite this article: Conway, T. M. et al. Constraints on soluble aerosol iron flux to the Southern Ocean at the Last Glacial Maximum. Nat. Commun. 6:7850 doi: 10.1038/ncomms8850 (2015).Supplementary Figure 1-3, Supplementary Discussion and Supplementary ReferencesIce core dataset


This was achieved using either trains of high frequency action potentials to evoke localised [Ca2+]i increases at active zones, or a calcium ionophore to raise [Ca2+]i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca2+]i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca Central nerve terminals rely on the efficient recycling of a limited supply of synaptic vesicles (SVs) in order to maintain neurotransmission. This is achieved via recruitment of a number of SV endocytosis modes , 2. ClatTriggering of ADBE is dependent on the dephosphorylation of the large GTPase dynamin I at two serine residues (Ser774 and Ser778) [GSK3 has a high basal activity which is regulated via its phosphorylation status . Phospho2+]i) via either action potentials or a calcium ionophore. We found that localised [Ca2+]i increases via voltage gated calcium channels were essential for Akt activation. This activation was dependent on phosphatidylinositol 3-kinase (PI3K) but not calmodulin activity. This reveals a key role for PI3K in the activity-dependent control of GSK3 activity, but leaves the identity of the calcium sensor still undetermined.It is currently unclear how action potential stimulation is transduced into activation of Akt. To address this we monitored the phosphorylation status of both Akt and GSK3 after elevating intracellular free calcium  cultures were prepared from the cerebella from P7 Sprague–Dawley rat pups of either sex . Culture2PO4, 20 mM TES [N-tris(hydroxy-methyl)-methyl-2-aminoethane-sulfonic acid], 5 mM NaHCO3, 5 mM glucose, 1.2 mM Na2SO4, 1.2 mM MgCl2, 1.3 mM CaCl2 at pH 7.4) for 10 min. This medium was then replaced by media supplemented with (2R)-amino-5-phosphonovaleric acid , 6-cyano-7-nitroquinoxaline-2,3-dione  and various antagonists as described in the respective figure legends. For electrical field stimulation experiments coverslips were placed in a Warner imaging chamber (RC-21 BRFS) and either challenged with a train of 800 action potentials delivered at 80 Hz or no stimulus . In experiments using ionomycin, cells were treated in an identical manner, with the exception that the challenge with 2.5 µM ionomycin or a vehicle control (DMSO) was for 1 min. In all experiments antagonists were present during stimulation with the exception of those using either BAPTA-AM or EGTA-AM. Low calcium incubation medium had the following alterations, CaCl2 was reduced to 100 µM, MgCl2 was increased to 10 mM and EGTA was added at 50 µM. After stimulation with either action potentials or ionomycin, CGNs were immediately lysed in 25 µl of SDS sample buffer  and boiled at 95 °C for at least 5 min.Cerebellar granule neurons (CGNs) were repolarised in incubation medium  lysates were subjected to Western blotting as described before . Primary2+]i. To assess the importance of increased [Ca2+]i on both Akt and GSK3 phosphorylation, the calcium ionophore ionomycin was employed to uniformly increase [Ca2+]i independent of depolarisation. Phosphorylation of both Akt and GSK3 was observed when CGNs were challenged with ionomycin in the presence of extracellular calcium  we used the antagonist calmidazolium. Incubation with calmidazolium (10 μM) during stimulation with 800 action potentials had no significant effect on the activity-dependent phosphorylation of either Akt or GSK3  is dependent on localised calcium microdomains formed by voltage-gated calcium channels. The identity of the sensor or release mechanism which transduce increased [Ca