Genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear.Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines  as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants.Transgenic wild tobacco plants (De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits.The unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines.  Transgenic “ICE” plants expressed an antimicrobial peptide (knottin) from the common ice plant Mesembryanthemum crystallinum  and transgenic “FAB” plants expressed an antimicrobial peptide (fabatin) from the broad bean Vicia faba (synthetic gene similar to the fabatin-1 precursor [GenBank:EU920043]). The sequences of the PNA and FAB constructs were manually adapted to the codon usage table of N. tabacum (http://gcua.schoedl.de/).For heterologous expression of antimicrobial peptides in structed . Plants un.org/)  and wereer (35S) . TransgeN. attenuata Torr. ex S. Watson seeds were originally collected in 1988 from a natural population at the DI Ranch in Southwestern Utah. Wild-type seeds from the 30th inbreed generation were used for the construction of transgenic plants and as WT controls in all experiments. Plant transformation was performed by Agrobacterium tumefaciens-mediated gene transfer as previously described  dCTP (Perkin-Elmer) using the Rediprime II DNA Labeling System  according to the manufacturer′s instructions.A total amount of 6 μg gDNA was digested overnight at 37°C with 140 U CCGC-3′)  and radiN. attenuata tissue. Approximately 150–300 mg ground and frozen tissue was dissolved in 900 μL cell lysis buffer  and shortly mixed. Per sample 300 μL protein precipitation buffer  was added and the tubes inverted ten times. Samples were incubated on ice for 5 min and centrifuged at room temperature in a table top centrifuge (5 min at 16.100 g). The supernatant was collected and extracted with 500 μL chloroform:isoamylalcohol mix (24:1 v/v). After centrifugation (3 min at 16.100 g) the upper aqueous phase was collected and nucleic acids precipitated with 1 volume isopropanol for 15 min at room temperature. Nucleic acids were pelleted in a table top centrifuge (3 min at 16.100 g), washed twice with 400 μL 70% (v/v) ethanol and air dried for 5 min. The final pellet was dissolved in 50 μL nuclease free water (Ambion / Life technologies). The nucleic acid was DNAse-treated using the TURBO DNA-free kit (Ambion / Life Technologies) according to the manufacturer’s instructions. Quality and amount of the remaining RNA was determined using a 1% (w/v) agarose gel and a Nanodrop spectrophotometer (Thermo Scientific). The absence of genomic DNA was tested with 20 ng RNA in a 35 cycle PCR programm  with the same primers as for qRT-PCR. 4 μg of total RNA was reverse transcribed with oligo(dT)18 primers (Fermentas) and the SuperScript II reverse transcriptase enzyme (Invitrogen / Life Technologies). Quantitative Real Time-PCR (qRT-PCR) was performed with 1:10 diluted cDNA (20 ng) on a Mx3005P QPCR System (Stratagene) with either a SYBR Green based PCR Master Mix (Applied Biosystems / Life Technologies) or a qPCR Core kit for SYBR Green (Eurogentec). For amplification the following primers were used: ICE-94F (5′-AATGGAAAAGGATGTCGAGAGG-3′), ICE-167R (5′-CATCCAACCTGACGGTAACAGAA-3′), PNA-86F (5′-GGAGACAAGCTAGTGGGAGGC-3′), PNA-154R (5′-TGGAGCCACAGTAGCCCC-3′), FAB-111F (5′-CAGGTTTAATGGACCATGCTTG-3′), FAB-184R (5′-CACCACCTTTGTAACCTTCTCCC-3′). The used program was 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 1 min and 1 cycle of 95°C for 15 s, 60°C for 30 s, 95°C for 15 s as dissociation curve. For relative gene expression analysis the comparative Δ cycle threshold method (ΔCT ) was used. Gene expression was shown as log2 (ΔCT) relative to N. attenuata actin as the reference gene (Actin-F1 5′-GGTCGTACCACCGGTATTGTG-3′ and Actin-R1 5′-GTCAAGACGGAGAATGGCATG-3′) [Tissue was harvested from rosette-stage leaves and ground in liquid nitrogen to a fine powder. RNA isolation was performed with a salt precipitation method modified from the US patent of Gentra Systems, Inc. publication No. 5973137  and adapCATG-3′) .http://www.urogene.org/methprimer/) and Kismeth [http://cymate.org/) [http://methdb.igh.cnrs.fr/methtools/) [http://weblogo.threeplusone.com/) [DNA methylation analysis was performed by the bisulfite sequencing method . The bis Kismeth  and manu Kismeth  and [126te.org/)  and Methhtools/) . Nucleotne.com/) . For the2 stage seedlings (still resistant) were grown for 10 days on GB5 media supplemented with hygromycin B (35 mg/L). The hypocotyls were cut in small pieces as done for the normal plant transformation procedure but without dipping the scalpel in Agrobacterium suspension. The explant cultures were grown into a callus and regenerated as previously described [3) from the regenerants were germinated on hygromycin containing media and seedlings with 0% sensitivity were brought to the glasshouse for RNA isolation and further propagation to test the subsequent generation (T4) for resistance.Homozygous seedlings of the lines PNA 1.2, ICE 4.4 and ir-ACX1 (A-07-466-1 and A-07-468-3) were chosen for secondary callus regeneration. Tescribed . Fully rManduca sexta. Leaf tissue was collected 60 min after the treatment and was frozen immediately in liquid nitrogen for subsequent analysis. Jasmonic acid was extracted and analyzed as described in [Leaves at nodes +1  from rosribed in .The authors declare that they have no competing interests.AW planned and performed the experiments, analyzed the data and wrote the manuscript. MK performed all experiments with ir-ACX1 lines. ITB participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.Fold difference of transgene expression in consecutive generations.Click here for fileNOS promoter methylation between sensitive and resistant seedlings of line ICE 10.1.A, Methylation status of the NOS promoter among isogenic seedlings from line ICE 10.1. Different methylation sites  were indicated by different colors. Analysis was performed by CyMATE [B. Phenotypes of 10-day-old seedlings used for DNA isolation and bisulfite conversion. Isogenic seedlings of line ICE 10.1.2 were divided into sensitive and resistant seedlings and analyzed separately. Mean methylation rate from five clones is shown for the individual methylation sites . .y CyMATE . B. PhenClick here for fileDetail of 35S promoter methylation analysis of individual clones. Tissue harvested 15, 30, 45 and 60 days post germination of lines ICE 4.4 (T2) and ICE 4.4.1 (T3); ICE 1.1 (T2), ICE 1.1.1 (T3) and ICE 1.1.1.1 (T4); PNA 1.2 (T2) and PNA 1.2.1 (T3); PNA 10.1 (T2) and PNA 10.1.1 (T3); PNA 8.6 (T2) and PNA 8.6.1 (T3).Click here for fileSequence preference in CHH methylation sites. The nucleotide composition of 8-mer sequences around the CHH sites (methylated cytosine in the fifths position) divided in groups with low methylation (0–9%), medium methylation (10–49%) and high methylation (50–100%) frequencies. The pooled frequency data of lines ICE 4.4.1, PNA 1.2.1 and PNA 10.1.1 derived from one time point (60 dpg T3). The logo graphically illustrates the sequence enrichment at particular positions around the methylation site. Maximum sequence conservation is 2 bit, no nucleotide preference is 0 bit. Figures were made with WebLogo 3 [ebLogo 3 .Click here for fileInheritance of the silenced allele after reciprocal crossing with wild-type. The hybrid offspring (hemizygous to the transgene) should be theoretically fully resistant to hygromycin B. The silenced state of the transgene was equally distributed to subsequent generations. A, Percentage of sensitive seedlings after crossing . B, Phenotypes of seedlings on hygromycin B containing GB5 media.Click here for fileTransgene silencing in line ir-ACX1.A, Jasmonic acid accumulation 1 h after wound and oral secretion treatment in rosette leaves of ir-ACX1 and wild-type plants. The T3 generation of ir-ACX1 lost their capacity to suppress jasmonic acid accumulation [B, The T3 seedlings from line ir-ACX1 (A-07-468) developed sensitivity to hygromycin B. C, Transgene activity indicated by jasmonic acid accumulation determined in wound and oral secretion treated leaves of secondary regenerated ir-ACX1 lines. A reduced accumulation of jasmonic acid after wounding compared to wild-type (WT) indicated a functional IR-construct. D, Hygromycin sensitivity of T4 seedlings (direct descendants of the plants used for wound treatment) indicates an ongoing silencing process .mulation . B, The Click here for filePhenotypes after secondary regeneration. A, Photographs of T3 seedlings collected from secondary regenerants of line ICE 4.4 and PNA 1.2. Cell culture-induced variations resulted in variegated pattern of sensitivity on hygromycin B containing GB5 media. B, Photographs of T4 generation seedlings collected from fully resistant secondary regenerated plants. As positive and negative controls conventional propagated T4 seedlings are shown.Click here for file


Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the αvβ3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin αvβ3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer.Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin α Binding between cells and the extracellular matrix (ECM) is critical to complex processes such as cell adhesion and migration. This binding is mediated by interactions between cell surface integrin receptors and ECM ligands. Integrins are heterodimeric α/β transmembrane receptors which bind to ECM ligands such as fibronectin Integrins undergo large-scale conformational changes in order to attain a high-affinity configuration during the process of integrin activation. These receptors are currently understood to exist in equilibrium among three main conformational states . In the 2+ promotes activation while Ca2+ stabilizes the low-affinity conformation Integrin activation can be regulated by signals from the extracellular (“outside-in”) and intracellular (“inside-out”) environments. During outside-in activation, the headpiece-tailpiece interface is destabilized by headpiece opening. This is followed by extension and separation of the leg domains, resulting in adoption of the extended-open conformation +/H+ ion exchanger NHE1, which extrudes an intracellular H+ ion in exchange for an extracellular Na+ ion. Interestingly, it has been shown that NHE1 localizes to adhesion sites It is well known that extracellular pH can become acidic in several biological contexts. For example, while normal physiological pH is 7.4, the average extracellular pH in the tumor environment is generally in the range of 6.2–6.9 Acidic extracellular pH can affect several cell processes, including adhesion and migration. For example, adhesion between neutrophils and endothelial cells was found to strengthen when the extracellular environment was acidified vβ3 and a GRGDSP peptide increased at acidic pH, but did not assess the possible reasons that this occurs vβ3. We also conduct cell-level morphology and migration experiments to assess the consequent effects on cell behavior.While some of these observations have been attributed to changes in intracellular protein expression or protease secretion, the molecular mechanisms facilitating these changes are unknown. In particular, the role of integrin receptors in mediating the observed cellular responses to acidic pH has not been explored in depth. To our knowledge, to date only two studies have addressed this possibility. Stock et al. speculated that an extracellular acid-induced strengthening of the integrin-ligand bond could explain their observed effects of pH on melanoma cell adhesion and migration, but did not directly investigate this hypothesis vβ3 in complex with an RGD peptide vβ3 crystal structure, the headpiece is closed; this configuration provides an opportunity to study the effect of extracellular pH on headpiece opening in silico, independently of other effects that may be coupled in vitro and in vivo.To gain atomistic understanding of how acidic extracellular pH affects integrin conformation, we employed molecular dynamics (MD) simulations. The crystal structure used for these simulations was the extracellular domain of integrin αa values for all titratable residues in the αvβ3 headpiece. MCCE predicts pKa values for residues within the protein context, which can differ appreciably from pKa of these residues in isolation a>8.4. For an effective acidic pH, we protonated residues with pKa>6.2. From the definition of pKa, these residues would be protonated at a pH below the threshold pKa of 6.2 for a majority of integrins (>50%), thus computationally approximating an acidic pH on that order; we refer to this state hereafter as “acidic pH.” MCCE results indicated seven amino acids with pKa values that were elevated from their expected solution values, and were thus protonated in our simulations (We used Multi-Conformation Continuum Electrostatics (MCCE) to predict pKulations , Table 1vβ3 in complex with the fibronectin III10 domain d exhibits a single peak centered at d = 4.2 nm. At acidic pH, the histogram exhibits two peaks, centered at d = 4.1 nm and 4.55 nm  of each pH system, and quantified the amount of headpiece opening that occurred. Puklin-Faucher et al. previously observed partial headpiece opening within 6–8 ns of MD simulations of α 4.55 nm . The emeASPβ127, which is protonated in our acidic pH system, is located in the α1-β1 loop at the top of the α1 helix in the I-like domain, and can coordinate the divalent cation at the ADMIDAS site. As movements within the α1 helix have been implicated in headpiece opening At normal physiological pH, ASPβ127 is negatively charged and coordinates the ADMIDAS ion . When ASvβ3 headpiece, attributable in part to protonation of ASPβ127 in the I-like domain. Headpiece opening in the bent-closed conformation destabilizes the bent state and is expected to lead to integrin extension Our simulations demonstrate that acidic pH promotes opening of the αvβ3vβ3 on live αvβ3 CHO-B2 cell surfaces after incubation in buffer pH over the range 6.0–8.0. Cell shrinkage and detachment indicated a decrease in cell viability at pH≤5.5; therefore these lower pH values were not assessed via flow cytometry. Control experiments with various divalent cations confirmed WOW-1 specificity as a marker of activated αvβ3 -mediated molecular force spectroscopy; this molecular force spectroscopy is defined here as measurement of the distribution of unbinding forces FR between ligand-receptor complexes upon application of load at a given applied loading rate. Previous studies have utilized AFM force spectroscopy to investigate α4β1-VCAM-1 and α5β1-fibronectin interactions on U937 and K562 cells, respectively. In those reports, the applied force required to rupture the induced ligand-receptor complex FR and binding frequency fb increased after integrin activation vβ3-RGD on live αvβ3 CHO-B2 cells, as a function of extracellular pH.To further quantify the conformational state of αvβ3 CHO-B2 cells, far from each cell nucleus . Additional negative controls were performed with cantilevers functionalized with linker molecules, but without GRGDSPC. In identical experiments conducted with these cantilevers, only 3.68±1.75% (mean ± SEM) of the rupture events fell within quadrant II, thereby confirming that events with FR>50 pN and F′>2000 pN/s were specific interactions that occurred infrequently in negative control experiments. Only these specific interactions were included in further analysis.Analysis of rupture forces and effective loading rates before and after blocking demonstrated that unbinding events with vβ3 with antibody LIBS-6 FR among these three conditions  vβ3 we measured after exposure to pH 6.5 and pH 7.0 were not higher than that at pH 7.4, indicating that some level of reversal in activation state does occur after cells are returned to pH 7.4. Accordingly, it is plausible that acid-induced integrin activation also initially occurred in the pH 6.5 and 7.0 conditions, though to not as great a magnitude as for pH 6.0, so that detection in this assay was obfuscated during the antibody binding step.Our flow cytometry results indicate an increased number of activated αo pH 6.0 . It is ivβ3 integrins on cell surfaces to the RGD ligand increases at acidic pH, indicating an increase in the number of activated integrins ; therefore, we chose pH 6.5 for these long-timescale cell-level experiments.vβ3 with Mn2+ ions resulted in pH-independent low cell circularity , antibiotics-antimyotics, non-essential amino acids, and zeocin were purchased from Invitrogen. Fibronectin from human plasma was purchased from Sigma. Fetal bovine serum was purchased from Hyclone.Antibody LM609 was purchased from Millipore and Alexa 488 goat anti-mouse IgG (H+L) F(ab′)vβ3 CHO-B2 cells and CHO-B2 pCDNA cells were provided by Dr. Linda Griffith (Massachusetts Institute of Technology), as subcultures of cell lines developed by Dr. Jean Schwarzbauer (Princeton University) and Dr. Siobhan Corbett (University of Medicine and Dentistry of New Jersey), respectively. Cell culture media consisted of high-glucose bicarbonate-buffered DMEM containing L-glutamine and sodium pyruvate, supplemented with 10% fetal bovine serum, 1% antibiotics-antimyotics, and 1% non-essential amino acids. Media also included 500 µg/mL zeocin or 250 µg/mL G418 for αvβ3 CHO-B2 or CHO-B2 pCDNA cells, respectively. Cells were maintained in an incubator at 37°C with 5% CO2.αvβ3 in complex with an RGD ligand  and a portion of the hybrid domain  of each MD trajectory.To quantify the headpiece opening that occurred during MD simulations, we calculated the y-component of the distance vβ3 CHO-B2 cells, which have been engineered to express the integrin β3 subunit; the parental cell line CHO-B2 does not bind RGD vβ3, and LM609 was used to label all conformations of αvβ3. Alexa 488 goat anti-mouse IgG (H+L) F(ab′)2 fragment was used as the secondary antibody.Flow cytometry experiments were performed with α2PO4, 3.8 mM HEPES, 5.5 mM glucose, 1 mg/ml BSA, and 0.75 mM divalent cations . Buffer was adjusted to desired pH using HCl and NaOH. Experiments were conducted at pH 6.0, 6.5, 7.0, 7.4, and 8.0. Cells were prepared for flow cytometry analysis as follows: cells were washed with  PBS, and detached with trypsin/EDTA. Trypsin was diluted, and cells were centrifuged at 800 rpm for 5 minutes. Cells were resuspended in buffer at desired pH, and 10 µl of each cell type was removed for counting in a hemocytometer. Cells were then centrifuged and resuspended at desired pH at a concentration of 2.3566×106 cells/ml and incubated at room temperature for 20 minutes. All following steps were performed with pH 7.4 buffer. Cells were centrifuged and resuspended at 13.158×106 cells/ml or 5×106 cells/ml for WOW-1 Fab and LM609 experiments, respectively. 0.5×106 cells were incubated with 12 µl WOW-1 Fab or 2 µg LM609 for 30 minutes at room temperature or on ice, respectively. During antibody incubations, cells were agitated every 10 minutes. After incubation, 200 µl buffer was added and cells were centrifuged. Cells were then resuspended in 100 µl buffer, and 100 µl of secondary antibody solution (1∶300 dilution in PBS) was added. Cells were incubated in secondary antibody for 30 minutes on ice, and agitated every 10 minutes. After incubation, 200 µl of buffer was added and cells were centrifuged. Cells were resuspended in 0.5 ml buffer and analyzed on a BD FACSCalibur flow cytometer. All experiments were performed with triplicate samples, and at least two independent experiments were performed for each pH . For each experiment, the average αvβ3 CHO-B2 geometric mean fluorescence intensity (MFI) was normalized to the average CHO-B2 pCDNA geometric MFI. This quantity is referred to as the normalized geometric MFI.The buffer used for all flow cytometry experiments consisted of 137 mM NaCl, 2.7 mM KCl, 3.3 mM NaHk∼25 pN/nm. Cantilever spring constant varied by approximately +/− 10% from this value. Cantilevers were cleaned in piranha solution  and then rinsed thoroughly with 18 MΩ Millipore water. Rinses in following steps were performed in PBS+1 mM EDTA. To conjugate a polyethylene glycol (PEG) linker to the cantilever surface, a 1 mM solution of SH-PEG-NH2 (3.4 kDa) in PBS-EDTA was allowed to react with cantilevers for one hour at room temperature. Cantilevers were rinsed and then allowed to react with a solution of Sulfo-LC-SPDP cross-linker for 30 minutes at room temperature. Solution was prepared by dissolving Sulfo-LC-SPDP in ultrapure water at 20 mM, and diluting this solution 1∶40 in PBS-EDTA. Cantilevers were rinsed and then incubated in a 1 mg/ml solution of GRGDSPC peptide overnight at room temperature. Cantilevers were rinsed thoroughly once again to remove excess peptide before use. Functionalization was confirmed using fluorescent GRGDSPC peptides.All AFM force spectroscopy measurements were performed on an MFP-3D  system using Olympus TR400PB gold-coated silicon nitride cantilevers, with spring constant 2PO4, 3.8 mM HEPES, and 1 mM MgCl2. Buffer was adjusted to pH 7.4 or pH 6.0 using HCl and NaOH. Experiments were conducted on αvβ3 CHO-B2 cells adhered to 60 mm-diameter tissue-culture treated polystyrene Petri dishes . Cells were incubated at room temperature for 20–30 minutes before measurements were taken. In experiments with LIBS-6 and LM609, antibody was added at 25 µg/ml or 20 µg/ml respectively, and allowed to react for 30 minutes at room temperature. For each cell, 200 force cycles were conducted in a 10×20 grid on a 2×2 µm area. For each force cycle, the AFM cantilever was positioned above a cell away from the nucleus. Cantilevers were moved toward the cell at a velocity v = 5 µm/s until a trigger force of 150 pN was reached. Cantilevers were then held on the cell surface for 0.1 s before retraction at 5 µm/s. All unbinding measurements were conducted at the same applied loading rate. Cantilevers were held above the cell surface for 1 s to allow cells to recover between measurements. Unbinding events were detected as jumps in the retraction portion of the force-displacement data.Experiments were performed at room temperature in buffer containing 137 mM NaCl, 2.7 mM Kcl, 3.3 mM NaHF-d) data were analyzed with a customized Matlab script. For every F-d response exhibiting a visible unbinding event (jump in force during retraction), rupture force FR was calculated as the difference between the average force following rupture and the minimum point at rupture. Effective loading rate F′ was calculated as the product of the slope immediately before rupture (keff) and the cantilever velocity v >) were calculated using the method of non-overlapping intervals S. Mean-squared displacements as a function of time were fit to a persistent random walk model: <2d(t)> = 2S2P[t−P(1−et/P−)] 2<0.5 were not included in the calculation of mean cell speed. At least 40 cells with R2>0.5 were analyzed for each condition. Cells plated on 10, 20, and 30 µg/ml fibronectin were also analyzed to measure circularity  at the 8-hour timepoint. Experiments at different fibronectin concentrations showed consistent results; presented data correspond to the fibronectin concentration of 10 µg/ml.Unless otherwise noted, media for cell experiments consisted of high-glucose bicarbonate-buffered DMEM containing L-glutamine and sodium pyruvate, supplemented with 1% antibiotics-antimyotics, 1% non-essential amino acids, and 500 µg/mL zeocin. For migration experiments, 50 mm-diameter Petri dishes with tight-fitting lids  were coated with 0.1–30 µg/ml fibronectin in PBS for 1 hour at room temperature. Dishes were then rinsed twice with PBS. αvβ3 CHO-B2 cells were plated on dishes in serum-free media at a density of approximately 6000 cells/cm2 and allowed to adhere for 3 hours. After 3 hours, media was changed to pH 6.0 and refreshed to pH 6.0 every hour; between media changes, pH increased to ∼7.2. Cells were imaged in phase contrast every 5 minutes for 8 hours in an incubator at 37°C with 5% CO2. Projected cell area was measured using ImageJ. An independent experiment with 10 µg/ml fibronectin coating showed similar cell area oscillations (data not shown).For pH oscillation experiment, 60 mm-diameter glass-bottom Petri dishes (MatTek) were coated with 30 µg/ml fibronectin as described above. αFor comparison of two conditions, p values were calculated with an unpaired t-test. For comparison of three or more conditions, p values were calculated with a Bonferroni post-test following one-way ANOVA.Figure S13 subunit, and αvβ3 CHO-B2 cells exposed to Mn2+, which activates integrin αvβ3.Representative flow cytometry fluorescence intensity histograms illustrating WOW-1 Fab binding for CHO-B2 pCDNA cells, which do not express the integrin β(TIF)Click here for additional data file.Figure S2vβ3 CHO-B2 cells plated from the suspended state into media at pH 6.0 or pH 7.4.Mean spread area of α No. 1 glass coverslips or glass-bottom P60 dishes (MatTek) were coated with 15 µg/ml fibronectin in PBS for 1 hour at room temperature. Coverslips or dishes were then rinsed with PBS. αvβ3 CHO-B2 cells were plated on coverslips or dishes in serum-free media with an initial pH of 6.0 or 7.4 and allowed to adhere for 30 minutes in an incubator at 37°C with 5% CO2 before imaging in phase contrast. Media initially set to pH 6.0 remained below pH 7.0 for the duration of the incubation. Cell area was measured using ImageJ. Two independent experiments were performed: in each experiment, approximately 40 cells were measured for each pH. Independent experiments showed consistent results, and data presented are from a single experiment. Results demonstrate that the mean cell area was significantly higher at pH 6.0 than at pH 7.4 (p = 0.0249). Error bars represent SEM.(TIF)Click here for additional data file.Figure S3Kymography experiments demonstrate that membrane protrusion lifetime increases and protrusion velocity decreases at acidic extracellular pH. (A) Example kymograph illustrating αvβ3 CHO-B2 membrane dynamics. (B) Mean protrusion lifetime for cells in pH 6.5 or pH 7.4. (C) Mean protrusion velocity for cells in pH 6.5 or pH 7.4. Experiments were conducted as follows: glass-bottom P60 dishes (MatTek) were coated with 30 µg/ml fibronectin in PBS for 1 hour at room temperature. Dishes were then rinsed with PBS. αvβ3 CHO-B2 cells were plated on coverslips or dishes in serum-free media and allowed to adhere for 3 hours before media was changed to bicarbonate-free serum-free media at pH 6.5 or 7.4. Cells were imaged in phase contrast at 40× magnification. Images were collected every 5 seconds for a duration of 25 minutes. Each kymograph was produced by drawing a one-pixel-wide line perpendicular to the cell membrane at an active lamellipod. The images along this line at all timepoints were then sequentially compiled into a single image, illustrating the membrane dynamics at that specific location on the cell. For each visible protrusion event on a kymograph, a straight line was drawn from the beginning of the event to its peak, or to the beginning of a plateau. Events with a height of less than 4 pixels were neglected. The slope of this line represents the protrusion velocity. Protrusion lifetime was quantified as the x-axis projection of this line, with the addition of plateau duration, if applicable. Two independent experiments were conducted: in each experiment, approximately 10–20 cells were imaged and 300–400 protrusion events were analyzed for each pH condition. Independent experiments showed consistent results, and data presented are from a single experiment. Results demonstrate that protrusion velocity is significantly decreased and protrusion lifetime is significantly increased at acidic pH (asterisks represent p<0.001). Error bars represent SEM.(TIF)Click here for additional data file.Figure S4vβ3 CHO-B2 cells or CHO-B2 pCDNA cells.Mean cell circularity 8 hours after changing media pH for adhered α Petri dishes of 50 mm diameter with tight-fitting lids  were coated with 10 µg/ml fibronectin in PBS for 1 hour at room temperature. Dishes were then rinsed twice with PBS. αvβ3 CHO-B2 or CHO-B2 pCDNA cells were plated on dishes in serum-free media at a density of approximately 6000 cells/cm2 and allowed to adhere for 2 hours before media was changed to bicarbonate-free serum-free media at pH 6.5 or 7.4. MnCl2 (1 mM) or soluble GRGDSPC peptide (200 µg/ml) was also added within this media exchange for some sample conditions, as indicated. Eight hours after the media change, optical images of cells were acquired and analyzed to measure circularity . At least 50 cells were analyzed for each condition. Results demonstrate that circularity is significantly decreased for αvβ3 CHO-B2 cells in pH 6.5 . There was no significant difference in circularity for αvβ3 CHO-B2 cells in the presence of RGD at pH 6.5 vs. pH 7.4 (Columns 3 and 4), CHO-B2 pCDNA cells at pH 6.5 vs. pH 7.4 (Columns 5 and 6), or αvβ3 CHO-B2 cells in the presence of Mn2+ at pH 6.5 vs. pH 7.4 (Columns 7 and 8).(TIF)Click here for additional data file.Figure S5Mean migration speed as a function of fibronectin coating concentration on glass-bottom dishes for cells in media at pH 7.4. Maximum migration speed occurred at 30 µg/ml fibronectin, compared to 1 µg/ml fibronectin when cells are plated on fibronectin-coated polystyrene ((TIF)Click here for additional data file.


The aim of this study was to evaluate treadmill VO2peak, CVD risk factors and quality of life (QOL) in patients with schizophrenia .Peak oxygen uptake . Risk factors were compared above and below the VO2peak thresholds; 44.2 and 35.1 ml·kg-1·min-1 in men and women, respectively.33 patients, 22 men (33.7 ± 10.4 years) and 11 women (35.9 ± 11.5 years), were included. Patients VO2peak was 37.1 ± 9.2 ml·kg-1·min-1 in men with schizophrenia; 74 ± 19% of normative healthy men (p < 0.001). VO2peak was 35.6 ± 10.7 ml·kg-1·min-1 in women with schizophrenia; 89 ± 25% of normative healthy women (n.s.). Based on odds ratio patients were 28.3 (95% CI = 1.6-505.6) times more likely to have one or more CVD risk factors if they were below the VO2peak thresholds. VO2peak correlated with the SF-36 physical functioning (r = 0.58), general health (r = 0.53), vitality (r = 0.47), social function (r = 0.41) and physical component score (r = 0.51).VO2peak than the general population. Patients with the lowest VO2peak have higher odds of having one or more risk factors for cardiovascular disease. VO2peak should be regarded as least as important as the conventional risk factors for CVD and evaluation of VO2peak should be incorporated in clinical practice.Men with schizophrenia have lower VO Patients suffering from schizophrenia have a mortality risk that is two to three times that of the general population and the leading cause of death is cardiovascular disease (CVD) ,2. Altho2peak) is a strong predictor of CVD and all-cause mortality [2peak have indicated reduced risk of CVD, coronary heart disease and all cause mortality [2peak is often a stronger predictor of mortality than conventional risk factors for CVD [2peak. This might be especially important considering that higher levels of VO2peak seems to attenuate or eliminate the increased health risk associated with obesity [2peak below 44.2 ml·kg-1·min-1 were eight times more likely to have a cluster of CVD risk factors, compared to men above 50.5 ml·kg-1·min-1 [Cardiorespiratory fitness, measured as peak oxygen uptake  [2peak and perceived QOL are not evaluated.Results from the Aerobics Center Longitudional Study further suggest that people with low VOll being . High lefe (QOL) . Body mafe (QOL)  but the 2peak have rarely been presented in patients with schizophrenia. The classical study by Carlson et al. [2peak after eight weeks of high aerobic intensity training in patients with schizophrenia [-1·min-1). This VO2peak value are much lower than the VO2 required for walking in patients with schizophrenia [Objective measures of VOn et al.  were theophrenia . Recentlophrenia  publisheophrenia , and at ophrenia .2peak during walking or running in men and women with schizophrenia compared to VO2peak in healthy individuals from the Nord-Trøndelag Health Study (HUNT). We hypothesized that patients with schizophrenia had reduced VO2peak compared to normative healthy individuals. The secondary aim was to evaluate relationships between VO2peak, risk factors for cardiovascular disease, and quality of life.The primary aim of this study was to evaluate objectively measured VOWe included 33 patients, 11 women and 22 men, with ICD-10 schizophrenia, schizotypal or delusional disorders (F20 to F29) in the study. Patients were in- and out-patients at a University hospital and had agreed to take part in exercise interventions studies. All patients were under antipsychotic medical treatment. 24 patients were smokers. Exclusion criteria were known coronary artery disease, known chronic obstructive pulmonary disease, and not being able to perform physical treadmill testing and exercise. Patients were examined by a physician at inclusion to the study and the exclusion criterions were confirmed by medical records.2peak and peak heart rate (HRpeak), using the Cortex Metamax II portable metabolic test system  and the Polar S610i heart rate monitor , respectively. The protocol has previously been described in patients with schizophrenia as well as in healthy individuals [An individualized protocol was applied to measure VOividuals ,16.peak. The test started from warm-up speed (with minimum 5% inclination) after which the speed or the inclination was increased every minute  to a level that brought the patient to exhaustion. The highest oxygen uptake and heart rate (HR) recorded during the last minute of the test were determined as VO2peak and HRpeak, respectively. VO2peak where also presented as ml·kg-0.75·min-1 to normalise for the differences in bodyweight between the patients [The patients were carefully familiarized with the test procedures and the treadmill when entering the laboratory. Warm-up was ten minute walking or running on the treadmill at an intensity corresponding to 60-70% HRpatients .2peak with age and sex specific strata from the Nord-Trøndelag Health Study (the HUNT Study) [2peak in 4 631 healthy individuals (20 to 90 years) using mixing chamber gas-analyzer ergospirometry  and an individualised protocol that has close resemblance to the protocol used in the present study. 14.1% of the participants reported to be inactive, defined as no activity or exercising less than once per week. For each patient with schizophrenia, we estimated a normative VO2peak, namely the mean value defined in the HUNT Fitness study strata for the corresponding sex and age. We titled the VO2peak estimated from sex and age strata independent of physical activity level, as HUNT general. The VO2peak from age and sex strata for healthy inactive men and women were titled HUNT inactive. The percent of HUNT general and HUNT inactive VO2peak was calculated as: (achieved VO2peak ÷ age predicted VO2peak) · 100.We compared the patients VOT Study) . The HUN-1·min-1 and 35.1 ml·kg-1·min-1, respectively, were associated with higher cardiovascular risk factor profile [2peak values were used as threshold values when evaluating conventional CVD risk factors.In the HUNT Fitness study men and women below 44.2 ml·kg profile . The sam-1; elevated total cholesterol, > 6.1 mmol·L-1 in patients < 30 years old, > 6.9 mmol·L-1 in patients 30-49 years old and > 7.8 mmol·L-1 in patients ≥ 50 years old; elevated LDL-cholesterol, 4.3 > mmol·L-1 in patients < 30 years old, 4.7 > mmol·L-1 in patients 30-49 years old and > 5.3 mmol·L-1 in patients ≥ 50 years old; reduced HDL-cholesterol, < 1.0 mmol·L-1; elevated triglyceride, > 2.6 mmol·L-1; obesity, BMI ≥ 30.0 kg·m-1 [Morning fasting blood levels were taken. Serum glucose was analysed using Reflotron Plus system . HDL (high-density-lipoprotein) cholesterol, total cholesterol and triglyceride concentrations in serum were measured using a Modular P chemistry analyzer . LDL cholesterol was calculated using the Friedewald equation . BP  was used to assess the physical health and mental health aspects of health related quality of life . SF-36 cThe Positive and Negative Syndrome Scale (PANSS) was used to evaluate the severity of symptoms of schizophrenia . PANSS c2peak thresholds as well as between measured VO2peak and HUNT general and HUNT inactive VO2peak. We used the Pearson chi-square test to detect whether there was a significant association between patients above/below the VO2peak threshold and prevalence of risk factors. We calculated the odds ratio for having one or more risk factors in the patients below threshold. The analysis was adjusted for age and sex. In multiadjusted analysis we also adjusted for the potential cofounding effect of smoking.We used the independent samples T-test to compare differences between men and women, between patients below and above the VO2peak (ml·kg-0.75·min-1) and each domain of the SF-36. The significance level (α) was set at p <0.05 (2-tailed). Data are described as mean and standard deviation (SD), unless otherwise noted. SPSS statistical package, version 18.0 (SPSS Inc.), was applied to analyse results.We used Pearson r to analyse correlations between VOThe study was approved by the regional committees for medical and health research ethics, middle Norway and conducted according to the Helsinki declaration. Written informed consent was obtained from all the included patients after the procedures were fully explained.Age was 33.7 ± 10.4 years and 35.9 ± 11.5 years in men and women, respectively. The total PANSS, total positive PANSS and total negative PANSS score was 65 ± 17, 15 ± 6 and 17 ± 8 in men, and 68 ± 23, 16 ± 6 and 18 ± 8 in women, respectively.2peak for the men and women with schizophrenia are presented in Table 2peak values are plotted against age as well as normative VO2peak strata from the HUNT Fitness study in Figure 2peak in the men with schizophrenia was 84 ± 21% of age predicted HUNT inactive (p < 0.001) and 74 ± 19% of HUNT general (p < 0.001). The VO2peak in the women with schizophrenia was not different from HUNT inactive (101 ± 28%) and HUNT general . Age predicted VO2peak was 44.5 ± 2.9 in HUNT inactive men, 50.3 ± 4.1 ml·kg-1·min-1 in HUNT general men, 35.2 ± 1.8 in HUNT inactive women and 40.0 ± 3.2 ml·kg-1·min-1 in HUNT general women.The VO2 = 7.6, df = 1, p = 0.006). Based on the odds ratio adjusted for age and sex patients were 24.2 (95% CI = 1.5-505.6) times more likely to have one or more risk factors if they were below the VO2peak threshold. When we also adjusted for smoking the odds ratio was 28.3 (95% CI = 1.6-505.6). Among the patients below the VO2peak thresholds 10 patients had hypertension, 11 elevated glucose, 12 reduced HDL-cholesterol, 11 elevated triglyceride and 14 had obesity. Above the thresholds 2 patients had hypertension, 2 elevated glucose and 1 was obese. There were 8 smokers above the thresholds and 16 below. Differences in mean levels are presented in Table Risk factor assessment was lost in one male patient. Risk factors were present in 24 of 32 patients and of these five were above and 19 were below the thresholds. Among the eight patients without risk factors, six were above and two were below the thresholds . These low VO2peak values is to some degree explained by the high body weight (mean BMI of 36.7 ± 7.5 m·kg2). However, there are some indications of an underrating of these patients' VO2peak. First, the patients only reached a low peak heart rate (142 ± 21 beats·min-1). Secondly, both Carlsson et al. [2max compared with a maximal treadmill test, even when HRpeak is not significantly different [The present results highlight reduced VOumptions . Strassnn et al. . Thirdlyifferent ,25.2peak in the women was similar to the men with schizophrenia, even though the age was similar (36 years in women versus 34 years in men). Women normally have about 10 ml·kg-1·min-1 lower VO2peak compared to men at the same age [2peak.In contrast to Strassnig et al. , the pre2peak from the HUNT Fitness study, confirm our hypothesis that VO2peak is reduced in men with schizophrenia. The VO2peak in the women with schizophrenia was almost identical (101%) to inactive healthy HUNT women. Even lower VO2peak in men with schizophrenia compared to normative inactive men might suggest that more than just inactivity contribute the reduced VO2peak. The VO2peak in the men with schizophrenia is similar to normative healthy men aged 60-69 years [2peak in the men with schizophrenia is comparable to healthy men that are about 30 years older. Patients with schizophrenia actually have 15-25 years shorter life expectancy than the general population [2peak presented in the HUNT Fitness study is somewhat higher than previous described populations with regard to objectively measured VO2peak [The comparison with normalised VOdentical 1% to inapulation ,27. It i VO2peak -31.2peak are consistently being associated with increased risk of cardiovascular and all-cause mortality. Kodama et al. [-1·min-1 (1 MET) increases were associated with 13% and 15% reductions in all-cause mortality and CVD/coronary heart disease, respectively. Aspenes et al. [-1·min-1 lower VO2peak correspond to 56% higher odds of having a cluster of cardiovascular risk factors.People with reduced VOa et al.  found ths et al.  found th2peak thresholds suggested by Aspenes et al. [2peak thresholds. When comparing mean levels above and below thresholds, all risk factors, except glucose, was better in the patients above the thresholds. These findings suggest a strong connection between the patients VO2peak and the conventional risk factors for CVD, as confirmed in other populations [The comparison of patients with schizophrenia below and above the VOs et al.  confirm ulations ,32.2peak have been described as a strong independent predictor of metabolic syndrome [Our data are not quite consistent with findings from US suggesting that especially women with schizophrenia are at high risk of developing metabolic syndrome . This issyndrome .2peak should be incorporated into routine clinical practice for risk prediction. The prognostic value of VO2peak is beyond that predicted from other conventional risk factors [2peak seem to confer a significant protective effect [2peak is a modifiable risk factor, and eight weeks aerobic high intensity interval training has provided significant improvements of VO2peak both in healthy populations [2peak than increasing physical activity level alone [These results emphasize that evaluation of VO factors ,34. Evenulations  and in pulations . Furtherel alone ,36.2peak suggest major gender differences in self-perception. Only the correlation with between SF-36 physical functioning and VO2peak was significant in men, whereas six correlations with the SF-36 were significant in women. In all subjects together the VO2peak correlated with the patient's perception of physical function, general health, vitality, social function, and physical component score. With some exceptions, these findings are consistent with correlations between SF-36 variables and BMI in patients with schizophrenia [2peak mainly is perceived as a physical health problem, not mental. Contrary, both the mental and physical health components of QOL are found related to estimated VO2peak in healthy men [2peak seemed to experience lower vitality and social functioning. Sedentary people are associated with greater risk of low vitality [Our findings of lower SF-36 social function, role emotion and mental component score among women than among men might reflect a sex difference in the general population. Lower scores for women than for men have been identified in normative adults . The genophrenia . In lineophrenia  we foundlthy men . An intevitality . QOL arevitality .There are some limitations of the study. First, the sample size is low. Secondly, the patients were included in the study based on request to take part in exercise intervention studies. However, all eligible patients at the department were asked to participate in these studies. Thirdly, severe ill patients with schizophrenia, with poor insight to their illness, might have difficulties to evaluate their perception of QOL.2peak than men in the general population. Patients with a VO2peak below 44.2 ml·kg-1·min-1 (men) and 35.1 ml·kg-1·min-1 (women) have higher odds of having one or more risk factors for cardiovascular disease. Low VO2peak compromise patients' perceived physical health. VO2peak should be regarded as least as important as the conventional risk factors for CVD and evaluation of VO2peak should be incorporated in clinical practice. Finally, these finding represent an urging need for developing effective physical training interventions for patients with schizophrenia.Men with schizophrenia have lower VOThe authors declare that they have no competing interests.2peak testing and other data acquisition. GM and JH undertook the statistical analysis and JH wrote the first draft of the paper. All authors have contributed to and have approved the final manuscript.GM, JH, JH and JH designed the study. JH and GEN recruited patients, performed VOThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/11/188/prepub


Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD+ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and Ki values between ∼1–15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.Mitochondrial  Compounds for screening were arbitrarily selected from a diverse, non-combinatorial library obtained from ChemBridge (∼10 mM in DMSO). New stocks of primary hits and their structural analogs were also sourced from ChemBridge (and dissolved to 80 mM in DMSO). The biological activity of iGP-1 (ChemBridge ID 5224148) was validated using compound obtained from Vitas-M (ID STK017597) and found to be essentially identical. All other reagents were from Sigma-Aldrich. sn-Glycerol 3-phosphate was added as disodium rac-α/β-glycerol phosphate  and will be referred to as glycerol phosphate. Where indicated, total and free calcium concentrations in assay buffers were calculated using the Extended MaxChelator program available through Chris Patton on the Stanford University Web site at http://maxchelator.stanford.edu.CaClSkeletal muscle mitochondria were isolated from hindlimbs of 5–8 week old female Wistar rats (Harlan Laboratories) as previously described 2O2 after dismutation by endogenous or exogenous superoxide dismutase. Rates of total superoxide/H2O2 production were measured fluorimetrically using Amplex UltraRed in the presence of exogenous superoxide dismutase and horseradish peroxidase in a Varian Cary Eclipse fluorimeter as previously described F) plus matrix NAD-linked dehydrogenases (collectively "site IF/DH") with 5 mM glutamate, 5 mM malate, and 4 µM rotenone Q) with either 0.5 or 5 mM succinate F) with either 15 µM palmitoylcarnitine, 2.5 µM antimycin A, and 2 µM myxothiazol or 0.5 mM succinate, 4 µM rotenone, 2.5 µM antimycin A, and 2 µM myxothiazol Qo) with either 0.5 or 5 mM succinate, 4 µM rotenone and 2.5 µM antimycin A 2O2 calibration curves were performed in the presence of tested compounds.Superoxide was measured as H−1) were incubated with 5 µM TMRM in the presence of inhibitors or vehicle. ΔΨm was generated by sequential addition of substrates of complex I , complex II (4 µM rotenone then 0.5 or 5 mM succinate), and mGPDH (2 µM atpenin A5 then 1.7 or 16.7 mM glycerol phosphate). The effect of putative small-molecule inhibitors was normalized as the % change in fluorescence between vehicle treatment and complete depolarization by the uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone  added at the end of each run. When indicated, the K+/H+ exchanger nigericin (80 ng ⋅ ml−1) was added to collapse the pH gradient across the mitochondrial inner membrane (ΔpH) and maximize ΔΨm.Mitochondrial membrane potential (ΔΨm) was estimated fluorimetrically as described previously 2O2 and ΔΨm measurements was 120 mM KCl, 5 mM HEPES, 1 mM EGTA, 0.3% (w/v) BSA without or with 250 nM free calcium at pH 7.0. All assays were performed with stirring at 37°C.The assay buffer for both HExcitation and emission spectra of iGP-1 fluorescence were acquired with the Varian fluorimeter in standard buffer with pH adjusted to between 1.5 and 11 as indicated.2O2 production as described in the previous section with minor changes. Targeted sites included site IF/DH with 5 mM glutamate, 5 mM malate, and 4 µM rotenone, site IQ with 5 mM succinate, site IIF with 15 µM palmitoylcarnitine, 2.5 µM antimycin A, and 2 µM myxothiazol, mGPDH with 16.7 mM glycerol phosphate, 4 µM rotenone, 2.5 µM antimycin A, 2 µM myxothiazol, 1 mM malonate, and no added calcium, and site IIIQo with 5 mM succinate, 4 µM rotenone and 2.5 µM antimycin A. Compounds and assay components were distributed by a Biomek FX liquid handling workstation (Beckman) into 96-well black plates (Nunc) to a final volume of 200 µL, and incubated in the dark at room temperature for 30–40 min after the addition of substrate. Endpoint fluorescence was measured on a Perkin Elmer Victor V3 fluorescent plate reader. The fluorescence intensity of each well was normalized as % change from the average of eight DMSO controls on the same plate. Duplicate wells of each of four modulators of mitochondrial metabolism known to inhibit specific sites of superoxide/H2O2 production were included on each plate as positive controls and the resulting fluorescence levels set as −100% change from DMSO controls. Known inhibitors included 20 mM aspartate (for site IF/DH), 1 µM FCCP (for site IQ), 10 mM malonate (for site IIF), and 2 µM myxothiazol (for site IIIQo). The background fluorescence of the FCCP wells in the site IQ assay was used to normalize mGPDH plates run in parallel. To minimize position-dependent effects across each assay plate, Tukey’s two-way median polish was applied to fluorescence values after initial normalization 3,200 compounds were tested initially at 2.5 µM  in duplicate on different plates against a panel of five distinct assays of site-specific superoxide/H2O2 production by mGPDH were identified as compounds whose average effect was to decrease H2O2 production by at least 10% without altering H2O2 production in other assays by more than ±10%. Select hits in the mGPDH assay were re-sourced from ChemBridge and retested at 0.08–80 µM  in duplicate on the same microplate as described above against the eight conditions listed under “Measurement of mitochondrial H2O2 production and membrane potential.”Novel inhibitors of superoxide/H2O2 production as described above. Additionally, four microplate assays for ΔΨm were run in parallel to the H2O2 assays. These assays were as described under “Measurement of mitochondrial H2O2 production and membrane potential” except that each substrate condition was assayed in a separate microplate. Compounds were tested against the following substrate conditions: 5 mM glutamate and 5 mM malate both without and with 80 ng ⋅ mL−1 nigericin, 5 mM succinate and 4 µM rotenone with 80 ng ⋅ mL−1 nigericin, and 16.7 mM glycerol phosphate and 4 µM rotenone with 80 ng ⋅ mL−1 nigericin. Plates were incubated in the dark at room temperature for 30–40 min (H2O2) or 10–15 min (ΔΨm) after the addition of substrate, and endpoint fluorescence was measured on the Victor plate reader. Endpoints were chosen for the H2O2 assays to provide good signal-to-noise ratios and linear rates of change without the indicator dye limiting the reaction. Endpoints for the ΔΨm assays were determined in pilot experiments as the times for TMRM accumulation to reach a steady state. At room temperature, this was typically ∼ 5 min and remained stable for at least 30 min. Endpoint fluorescence values were normalized as described in the previous section with wells containing FCCP serving as positive controls for the ΔΨm assays.Structure/activity analysis was performed on 18 structural analogs of two structurally-related hits from the primary screen (iGP-1 and iGP-2). These 20 compounds were tested in duplicate at 0.08–80 µM  in microplate format for effects on site-specific Hex = 365 nm, λem = 450 nm). Activity of cGPDH in frozen-thawed synaptosomes was measured at 37°C in a BMG Labtech PHERAstar Plus microplate reader as the linear rate of change in NADH absorbance (λ = 340 nm) specific to the addition of 2.5 mM DHAP and insensitive to 4 µM rotenone. Rates were converted to nmol NADH using an extinction coefficient of 6.22 mM−1 ⋅ cm−1 and a calculated pathlength of 0.6 cm.The effects of small-molecule inhibitors on cGPDH and mGPDH enzymatic activity were tested according to established procedures. Activity of purified rabbit muscle cGPDH was measured as described by the manufacturer as the rate of oxidation of NADH in the presence of DHAP on the Varian fluorimeter  −1. Hill Slope and IC50 were determined using unconstrained, sigmoidal dose-response curve fits of % remaining activity against concentration of iGP. Kic and Kiu were determined using plots of rate−1vs iGP and GP ⋅ rate−1 vs iGP, respectively x-coordinate at which the best-fit lines intersect. These x-coordinates correspond to -Kic and -Kiu in their respective plots −1 ⋅ cm−1 and a calculated pathlength of 0.6 cm.Activity of mGPDH in frozen-thawed skeletal muscle mitochondria was measured as 2,6-dichlorophenolindophenol (DCPIP)-linked reduction by glycerol phosphate as described previously in the presence of 1 µM free calcium −1 oligomycin), and to reveal the non-mitochondrial rate (2 µM myxothiazol and 2.5 µM antimycin A). Each concentration of compound was tested in at least two wells on each plate. At least three biological replicates of each titration in each substrate condition were performed.The effects of small-molecule inhibitors on respiration were tested in plate-attached skeletal muscle mitochondria using a Seahorse XF24 Analyzer according to published protocols , 0.5 mM aminooxyacetate, or iGP-1 with aminooxyacetate, and either 10 mM pyruvate or 15 mM glucose. Plates were equilibrated for 20 min at 37°C then loaded into the instrument followed by measurement of basal respiration and injection of 5 µM FCCP  with 4 µg ⋅ mL−1 oligomycin . In some experiments, 10 mM oxamate was added with the FCCP/oligomycin to minimize regeneration of cytosolic NAD+ by lactate dehydrogenase. Inhibition of the malate-aspartate shuttle was defined as the difference between maximal rates ± aminooxyacetate, expressed as % of control. Inhibition of the glycerol phosphate shuttle was the difference between maximal rates with aminooxyacetate ± iGP-1, as % of control.Synaptosomal respiration was tested in the Seahorse XF24 using published protocols 2PO4, 20 mM TES, 5 mM NaHCO3, 1.2 mM Na2SO4, 1.5 mM CaCl2, 0.5 mM MgCl2, 2 mM Glucose, and 0.2% BSA, pH 7.4) containing DMSO, 250 nM bafilomycin A1 or 100 µM iGP-1. Cells were imaged on a Nikon Eclipse Ti-PFS inverted microscope equipped with a Cascade 512B camera , a Plan Fluor 100X/1.3 oil objective, an LB-LS17 Xe-arc light source (attenuated), 10–3 excitation and emission filter wheels , and an MS-2000 linear encoded motorized stage . The filter sets, given as excitation, dichroic mirror, emission in nm/bandwidth, were, for iGP-1 (1 s exposure), 340/26, 409, 460/80 and for LysoTracker Red (100 ms exposure), 543/22, 562, 588/21 . Image acquisition was controlled by NIS Elements 4.0 . Images of cell-free wells containing buffer with DMSO or iGP-1 were used for background subtraction in Image Analyst MKII .The ability of iGP-1 to cross cellular membranes was evaluated in STHdhQ7 cells cultured at 33°C as described p values <0.05 were considered significant.Data are presented as mean ± S.E. or mean ±95% C.I. where indicated. Statistical differences between conditions were analyzed by one-way ANOVA with Newman-Keuls post-test as specified in the figure legends. 2O2 production and levels of ΔΨm in mitochondria provided with different substrates . Out of 3200 compounds arbitrarily selected from a structurally diverse chemical library and tested at 2.5 µM, 87 (2.7%) decreased the signal in the mGPDH assay by at least 10% compared to DMSO controls  selective mGPDH inhibitors . In contrast, iGP-2  against a panel of eight assays of HH, iGP-1 , remainet, iGP-2  was slig2O2 production by mGPDH. These 20 compounds were retested for effects on eight assays of site-specific H2O2 production and four assays of ΔΨm utilizing different mitochondrial substrates . However, the much larger increase in ΔΨm observed in the absence of nigericin was found to represent a true change in ΔΨm, most likely a collapse in the ΔpH component of the protonmotive force. This decrease in ΔpH may explain the greater effect of these structural analogs on H2O2 production by site IQ since this site is known to be uniquely sensitive to ΔpH versus mGPDH ROS production. Changing the relative positioning of these groups from ortho- to para- lowered the potency by more than 5-fold (iGP-8). Importantly, altering the carboxyl end of the succinamic acid group decreased potency and selectivity for mGPDH, if any inhibition remained at all (iGP-9– iGP-16). Similarly, the benzimidazole ring was required for inhibition of mGPDH (iGP-17– iGP-20). Ultimately, while our structure/activity analysis yielded no compound with improvements to both potency and selectivity against mGPDH, it provided insight into the structural elements essential to mGPDH inhibition and useful clues as to which chemical features should likely be targeted in future optimization studies.Motivated by the observation that subtle changes in structure resulted in changes to both the potency and selectivity of our novel mGPDH inhibitors, we tested an additional 18 compounds structurally related to the top hits in our primary screen to identify structural features that determined the relative potency and selectivity for inhibition of Hbstrates . To idenbstrates . First, 2O2 production by mGPDH but also its enzymatic activity  succinate but not by high (5 mM) succinate    and alsotaining) . To addrtaining) . Further+. However, because of a lack of cell-permeant selective inhibitors of mGPDH and compensation by other NAD+ regenerating mechanisms, there is a lack of evidence of functional mGPDH activity in intact systems. We tested the role of mGPDH-dependent metabolism using iGP-1 in synaptosomes because mGPDH is active in neurons −/− mice, and synaptosomes are a tractable intact system for studying neuronal energetics under tightly controlled cell-, substrate-, and demand-specific conditions.mGPDH is a critical component of the glycerol phosphate shuttle for regenerating cytosolic NAD−1 ⋅ mg protein−1; mean ± SE, n = 3). To demonstrate the likelihood of a functional shuttle system, we measured cGPDH activity in the same preparations. There was a significant rate of DHAP-specific, rotenone-insensitive NADH oxidation that we attribute to cGPDH .We verified that the high mGPDH activity reported in brain was present in synaptosomes. In frozen-thawed cortical presynaptic terminals, mGPDH activity was high   in synapneration . 100 µM neration . With glneration , confirm+ regeneration has a significant permissive role in synaptosomal metabolism of glucose but not pyruvate. There was also faster extrasynaptosomal acidification with glucose as substrate, suggesting efflux of lactate (with H+) to regenerate cytosolic NAD+ at lactate dehydrogenase  and H2O2 production . Inhibition of mGPDH by both iGP-1 and iGP-5 displayed a selective, one-site binding profile  and uncompetitively with the enzyme-substrate complex (defined by the uncompetitive inhibitor dissociation constant Kiu). Values for these dissociation constants were in the 10 µM range for iGP-1 and the 1 µM range for iGP-5 . Both inhibitors have Hill slopes near unity, suggesting they interact with mGPDH at a single, allosteric binding site. Although the analysis of inhibition kinetics was performed in the presence of activating calcium, our evidence from assays of mGPDH-specific H2O2 production (data not shown) and ΔΨm driven by glycerol phosphate  and good selectivity. Apart from a subtle effect on succinate oxidation at high concentrations, iGP-1 does not alter mitochondrial oxidation of numerous substrates including a second dicarboxylate, malate. Therefore, it is unlikely that the subtle effect on succinate oxidation is due to inhibition of the dicarboxylate transporter by the succinamide of iGPs. Indeed, analogs iGP-17– iGP-19 retain the succinamide without or with the attached phenyl ring yet do not alter succinate oxidation as assessed by H2O2 production by succinate alone Click here for additional data file.


NHE1 activity remained stimulated during 1–5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1–24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H+ homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI.Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na Each hippocampal astrocyte is in contact with several hundred dendrites from multiple neurons and envelope hundreds of thousands of synapses +/H+ exchanger isoform 1 (NHE1) is a membrane protein that functions to regulate intracellular pH (pHi) by extrusion of one H+ in exchange for one Na+in vitro ischemia . We also detected concurrent elevation of NHE1 activity, increased intracellular sodium concentration ([Na+]i) and intracellular calcium concentration ([Ca2+]i), and release of glutamate and cytokines from hippocampal astrocytes following OGD/REOX. Interestingly, inhibition of NHE1 activity significantly reduced all of these events. Therefore, we concluded that over-stimulation of NHE1 promotes gliotransmitter and cytokine release from reactive astrocytes, which can subsequently contribute to hippocampal neuronal damage under hypoxic and ischemic conditions.NaHanks balanced salt solution (HBSS) and L-glutamine were from Mediatech Cellgro . BCECF-AM, fura2-AM, Sodium Green-AM, and the Amplex® Red Glutamic Acid/Glutamate Oxidase kit were from Invitrogen . Gramicidin, ionomycin, nigericin, calcein-AM, propidium iodide (PI) and L-leucine methyl ester were purchased from Sigma . HOE 642 was a kind gift from Aventis Pharma . DL-threo-b-benzyloxyaspartate (TBOA) was from Tocris . Fetal bovine serum (FBS) was obtained from Valley Biomedical . Rabbit anti-NHE1 polyclonal antibody was purchased from Abcam . ELISA kits for the cytokines IL-1, IL-6, and TNF-α were from R & D . Mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody was from Cell Signaling . Monoclonal antibody cocktail against GFAP derived from the Bigner-Eng clones MAb1B4, MAb2E1 and MAb4A11 was from Convance . Mouse anti-α tubulin monoclonal antibody was from Promega . m.4 cells/6 well, 1×103 cells/24 well) were either plated on glass coverslips (22×22 mm) coated with collagen type 1 placed in petri dishes, six well plastic culture plates, 24-well culture plates or 35 mm glass bottomed culture dishes (15 mm). Cultures were maintained in a 5% CO2 atmosphere at 37°C. To obtain morphologically differentiated astrocytes, confluent cultures  were treated with EMEM containing 0.25 mM dibutyryl cyclic AMP (dBcAMP) to induce differentiation.This study was carried out in strict accordance with the recommendation in the Guide for the Care and Use of laboratory Animals of the National Institute of Health. The protocol was approved by the University of Wisconsin-Madison, Institutional Animal Care and Use Committee (Protocol G000614). Harvesting of tissue for culture was done under isoflurane anesthesia and all efforts were made to minimize pain. Dissociated hippocampal astrocyte cultures were prepared from the hippocampus of 3–4 day old mice as described before In some studies, three-day old astrocyte cultures were supplemented with 5 mM L-leucine methyl ester (LME) for 10 days to remove possible microglial contamination in the astrocyte cultures. LME is a microglial cytotoxic agent that has been used extensively as a method to eliminate proliferating microglia 3, 120 NaCl, 5.36 KCl, 0.33 Na2HPO4, 0.44 KH2PO4, 1.27 CaCl2, and 0.81 MgSO4. The cells were incubated in the OGD solution for 2 h in a hypoxic incubator  containing 94% N2, 1% O2, and 5% CO2. Normoxic control cells were incubated for 2 h in 5% CO2 and atmospheric air in a buffer identical to the OGD solution except for the addition of 5.5 mM glucose. For REOX, the OGD solution was replaced with EMEM containing 10% FBS and incubation at 37°C in 5% CO2 and atmospheric air.Hippocampal astrocyte cultures were rinsed with an isotonic OGD solution (pH 7.4) containing (in mM): 0 glucose, 21 NaHCOi measurement and prepulse treatment were performed as described previously 3–free HEPES-MEM solution : 140 NaCl, 5.36 KCl, 0.81 MgSO4, 1.27 CaCl2, 0.44 KH2PO4, 0.33 Na2HPO4, 5.55 glucose and 20 HEPES. The chamber was mounted on the stage of the Nikon TI Eclipse inverted epifluorescence microscope and the cells visualized with a 40X objective. The cells were excited every 10–30 s at 440 and 490 nm, and the emission fluorescence at 535 nm recorded. Images were collected using a Princeton Instruments MicroMax CCD camera and analyzed with MetaFluor  image-processing software. The ratio of the background-corrected fluorescence emissions (F490/F440) was calibrated using the high K+/nigericin technique pH4+/NH3 solution as described previously 4+/NH3 solutions were prepared by replacing 30 mM NaCl in the HCO3− -free, HEPES-buffered solution with an equimolar concentration of NH4Cl. pHi recovery rate during the first min after NH4+/NH3 prepulse was determined from the slope of a fitted linear regression at ∼ pH 6.5 For the pre-pulse treatment, cells were subjected to an acid load by a transient application (2.5 min) of a 30 mM NH3-MEM at 37°C. Using a TI Eclipse inverted epifluorescence microscope (40X objective), cells were excited every 10 sec at 340 and 380 nm, and the emission fluorescence at 510 nm was recorded. Images were collected and analyzed with the MetaFluor image-processing software. At the end of each experiment, the cells were exposed to 1 mM MnCl2 and 5 µM Br-A23187 in Ca2+-free HEPES-MEM. The Ca2+-insensitive fluorescence was subtracted from each wavelength before calculations 2-corrected 340/380 emission ratios were converted to [Ca2+]i as described previously Hippocampal astrocytes grown on coverslips were incubated with 5 µM fura-2AM for 2 h at 37°C. The cells were rinsed with PBS and quickly (<2 min) placed on an open-bath imaging chamber and superfused (1 mL/min) with HCO+]i was determined using the fluorprobe sodium green-AM. Hippocampal astrocytes grown on glass-bottomed culture dishes  were loaded with 15 µM sodium green-AM at 37°C for 2 h under normoxia, OGD, or REOX conditions. For OGD treatment, cells were incubated at 37°C with 200 µl OGD buffer for 2 h in a hypoxic incubator containing 94% N2, 1% O2, and 5% CO2. Normoxic control cells were incubated for 2 h in 5% CO2 and atmospheric air in a buffer (200 µl) identical to the OGD solution except for the addition of 5.5 mM glucose. Cells were then washed with HEPES-MEM and placed in a stage top incubator  at 37°C on the stage of Nikon A1 confocal microscope equipped with a perfect focus system and images collected every 20 sec  using a 60X objective. After a one min baseline collection, cells were subjected to an in-situ calibration using the ionophores gramicidin and monensin. Absolute [Na+]i was determined as described before i in hippocampal astrocytes during OGD/REOX. The resting level of [Na+]i in hippocampal astrocytes was 10.0±2.6 mM and did not change significantly when NHE1 was inhibited (12.1±0.1 mM). Following 2 h OGD, [Na+]i was not different from normoxia (A). Upon REOX, [Na+]i increased to 22.1±1.8 mM at 1 h REOX and 34.5±1.1 mM by 5 h of REOX . At 24 h of REOX, [Na+]i decreased by 45% but remained significantly elevated compared to normoxic control (18.9±1.4 mM). In contrast, inhibition of NHE1 activity with 1 µM HOE 642 reduced [Na+]i by ∼ 75% (13.3±1.8 vs. 22.1±1.8 mM) at 1 h REOX and ∼ 50% (22.5±2 mM vs. 34.1±1 mM) at 5 h REOX (A). After 24 h REOX, [Na+]i in the HOE-treated cells returned to normoxic levels (12.1±1.8 mM). Taken together, these data indicate that activation of NHE1 activity led to sustained Na+i overload in reactive astrocytes after OGD/REOX.We investigated whether overstimulation of NHE1 results in Na+i overload could induce an elevation in [Ca2+]i via reversal of Na+/Ca2+ exchanger (NCXrev) in hippocampal astrocytes following OGD/REOX. Basal [Ca2+]i in hippocampal astrocytes was 44.2±12.1 nM and was not affected by 1 µM of HOE 642 (65.2±21.2 nM). [Ca2+]i remained unchanged following 2 h OGD and at1 h REOX (B). However, at 3 h REOX, [Ca2+]i in astrocytes increased significantly (416.7±43.9 nM), and remained elevated at 5 h of REOX . Interestingly, in hippocampal astrocytes treated with 1 µM HOE 642 the increase in [Ca2+]i was much less at either 3 h REOX (155.2±28.6 nM) or at 5 h REOX . This implies that the rise in [Ca2+]i may result from increased NCXrev subsequent to Na+i overload . To test this possibility, we examined whether SEA 0400 (1 µM), a potent inhibitor of NCXrev, could block the increases in Ca2+ after OGD/REOX in hippocampal astrocytes. As shown in B, inhibition of NCXrev with SEA 0400 abolished the OGD/REOX-mediated rise in [Ca2+]i. Interestingly, SEA 0400 effects are similar to the inhibition of NHE1 by HOE 642. These data illustrate that NHE1 activation in hippocampal astrocytes can couple with reverse mode operation of NCXrev and result in Ca2+ elevation subsequent to intracellular Na+ overload.We further investigated whether Na+ and H+ homeostasis. To test this, we measured glutamate release from astrocytes after OGD/REOX. No detectable glutamate release was found in medium of the normoxic hippocampal astrocyte cultures (A). Following 2 h of OGD or 1 h REOX, low levels of glutamate (0.04±0.04 pmol/µg protein) were released from hippocampal astrocytes. These data suggest that hippocampal astrocytes release minimal amount of glutamate after OGD or early REOX. In contrast, following 5 h REOX, glutamate release from hippocampal astrocytes increased dramatically to 9.6±1.7 pmol/µg protein (A). At 24 h REOX, although the glutamate levels in the media were reduced, they remained elevated compared to normoxic levels . Inhibition of NHE1 activity in hippocampal astrocytes with 1 µM HOE 642 had no effect on the release of glutamate at 2 h OGD or 1 h REOX . However, blocking NHE1 activity decreased the glutamate release by ∼ 50% at 5 h and ∼ 35% at 24 h REOX (A). It is unclear how blocking NHE1 activity can reduce glutamate release from astrocytes following OGD/REOX.We speculate that overstimulation of NHE1 activity may affect glutamate release and/or uptake via disruption of Na+-dependent excitatory amino acid transporter (EAATs) subsequent to increases in Na+i, we investigated if the competitive non-transportable EAAT blocker TBOA can block the glutamate release. 100 µM TBOA had no effects on the glutamate release at 2 h OGD or 1 h REOX . The lack of effect on glutamate release further implies that little glutamate is released during this initial OGD/REOX period. However, TBOA nearly abolished the glutamate efflux (∼95%) at 5 h REOX and reduced the glutamate release by 80% at 24 h REOX. These data further suggest that glutamate release during REOX was primarily through reversal of EAATs. To further confirm glutamate release through reversal of the transporter, we calculated the reversal potential for the transporters using the following equation.rev is the reversal potential. The values for [Na]o, [Na]i, [H]o, [H]i and [K]o are known, the [Glu]o were estimated to be 0.01 µM at baseline, 0.1 µM at 5 h REOX and 0.05 µM at 24 h REOX. Assuming a value for [Glu]i of 5 mM and [K]i of 70 mM, then Vrev = −53.6 mV. Cultured cortical astrocytes have a large range of resting plasma membrane potentials, but average ∼ −60 mV when measured either by whole cell or perforated electrophysiological recordings o was very low, the glutamate transporters were near their reversal potentials and little or no flux through the transporter would be expected. In contrast, because of the stoichiometry of three Na+/glutamate anion transporter, the reversal potential was sensitive to changes of [Na]i (A inset). At 5 h REOX with a [Na]i of 34.5 mM and [K]i of 45.5 mM, the reversal potential dropped to −78.2 mV and efflux of glutamate through the transporters would be favored. At 24 h REOX, when [Na]i decreased to 19 mM the reversal potential fell to −59.6 mV, making glutamate release less likely.To test whether glutamate release was mediated via reversal of NaB–D). However, after 5 h REOX, we detected a transient increase in the release of IL-1β, IL-6 and TNF-α by 6-fold, 33-fold and 33-fold, respectively. Similar to the glutamate release, the release of the proinflammatory cytokines from hippocampal astrocytes returned to control levels by 24 h REOX (B–C). Interestingly, in the presence of the NHE1 inhibitor HOE 642 (1 α µM), the release of cytokines at 5 h REOX was reduced between 26% and 45% (p<0.05). These data imply that inhibition of NHE1 activity also decreases cytokine release from reactive astrocytes.Next, we accessed the release of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α from hippocampal astrocytes following OGD/REOX. Neither OGD nor 1 h REOX resulted in significant release of the cytokines . The cell death rate was 0.83±0.14% (B). When astrocytes were subjected to OGD, followed with either 5 h REOX  or 24 REOX , the cell death rates remained at ∼1% (B). Inhibition of NHE1 with HOE 642 did not change the cell death in astrocytes . These data suggest that reactive astrocytes were resistant to ischemia-mediated cell death.We further investigated the impact of the above cellular changes on astrocyte viability. Under normoxic conditions, astrocytes in the culture retained the calcein dye with minimum PI staining , suggesting that inhibition of NHE1 and subsequent reduction of Na+ overload would help to prevent EEATs-mediated glutamate release from astrocytes following ischemia. These findings are consistent with other previous reports. Investigators have reported neuroprotection with the use of glutamate transporter inhibitors during bilateral carotid occlusion/hypotension in rats in vitro by raising extracellular K+In this study, we detected concurrent intracellular Na+/K+-ATPase activity because it could be blocked by the application of ouabain +/K+-ATPase activity appears to still be functional. Therefore, increased Na+ influx in hippocampal astrocytes after OGD/REOX, rather than altered Na+ pump function or membrane depolarization, seem to drive reversal of EAATs in hippocampal astrocytes.Xie et al measured changes in astrocytic plasma membrane potential when hippocampal slices were exposed to OGD + and Ca2+ levels in hippocampal astrocytes after OGD/REOX may be the trigger behind astrocytic activation and subsequent cytokine release. Collectively, these results suggest that astrocytes-induced cytokines release following in vitro HI may be regulated by NHE1 and changes of Na+ and Ca2+.It is well recognized that the activation of astrocytes is accompanied by increased production of potentially neurotoxic factors, including cytokines 2+ as a signal pathway for astrocyte mediated cytokines release 2+/PKCs/MAP kinases/NF-κB/STAT1727 pathways and the cytokines produced subsequently re-activate astrocytes via Jak/STAT1701, which then release more cytokines thereby contributing to the exacerbation of experimental allergic encephalomyelitis in mice. The authors reported that activation of astrocytes is usually manifested as a rise of intracellular Ca2+ level due to release of Ca2+ from internal stores as well as Ca2+ uptake from the extracellular space. This suggests that NHE1 may have an important role in astrocyte mediated inflammatory responses following HI. Recently, we have shown that NHE1 is required for microglial activation and proinflammatory responses after ischemia. In cultured microglia, activation by several stimuli depends on NHE1 mediated H+ homeostasis and inhibition of NHE1 with HOE 642 also reduced the production of superoxide anions as well as proinflammatory cytokines IL-1β, IL-6, and TNF-α induced by LPS or in vitro ischemia in vivo ischemia studies where activation of microglia leading to proinflammatory cytokine production and phagocytosis is significantly reduced with NHE1 inhibition or genetic knockdown In a recent interesting study, Kim et al. (2011) demonstrated a role for intracellular Cain vitro ischemia (+i] and [Ca2+i] increased, leading to cytokine and glutamate release from hippocampal astrocytes but did not cause cell death. Interestingly, although inhibition of NHE1 activity did not change reactive astrocyte death, it significantly reduced the release of glutamate and cytokines. Therefore, we concluded that over-stimulation of NHE1 promotes gliotransmitter and cytokine release from reactive astrocytes, which can subsequently contribute to hippocampal neuronal damage under hypoxic and ischemic conditions.In summary, NHE1 protein expression was up-regulated in reactive hippocampal astrocytes after ischemia Figure 7


The presence of these compounds in seawater suggests relevant effects on phytoplankton. Indeed, we provide evidences of the negative effect of sunblocks on the growth of the commonly found marine diatom Chaetoceros gracilis (mean EC50 = 125±71 mg L-1). Dissolution of sunscreens in seawater also releases inorganic nutrients  that can fuel algal growth. In particular, PO43− is released by these products in notable amounts (up to 17 µmol PO43− g−1). We conservatively estimate an increase of up to 100% background PO43− concentrations (0.12 µmol L-1 over a background level of 0.06 µmol L-1) in nearshore waters during low water renewal conditions in a populated beach in Majorca island. Our results show that sunscreen products are a significant source of organic and inorganic chemicals that reach the sea with potential ecological consequences on the coastal marine ecosystem.A growing awareness of the risks associated with skin exposure to ultraviolet (UV) radiation over the past decades has led to increased use of sunscreen cosmetic products leading the introduction of new chemical compounds in the marine environment. Although coastal tourism and recreation are the largest and most rapidly growing activities in the world, the evaluation of sunscreen as source of chemicals to the coastal marine system has not been addressed. Concentrations of chemical UV filters included in the formulation of sunscreens, such as benzophehone 3 (BZ-3), 4-methylbenzylidene camphor (4-MBC), TiO We conduct field and laboratories studies to evaluate the presence of chemicals products released from sunscreens in coastal seawater and its effect on the marine phytoplankton. Particularly, (1) we present the results for UV chemical filters levels in different fractions of surface marine waters of three Majorca areas; (2) we evaluate the contribution of sunscreen products to the total dissolved P in nearshore waters of a populated beach in Majorca island; and (3) we test the effect of sunscreens on the growth rate of a marine diatom . No specific permit was required for sampling in the Ses Salines Cape. The maritime area of Ses Salines is not private and protected for sampling. The study did not involve endangered or protected species.-1 shoreline), and the third one, considered a control, was an open and scarcely used beach located in a pristine area  was collected using a peristaltic pump and pumped through acid-cleaned Teflon tubing coupled to a C-flex tubing , filtered through an acid-cleaned polypropylene cartridge filter  for the dissolved fraction, and collected in a 0.5 L low-density polyethylene plastic bottle Surface waters (microlayer and subsurface) were collected from a zodiac during August 20-1) of BZ-3 and MBC in ethanol were prepared. Then, a multicomponent aqueous stock solution (25 ng mL-1) of BZ-3 and MBC was prepared from these, and it was used to prepare multicomponent aqueous working solutions (50–250 ng L−1 or 200–1000 ng L-1). On the other hand, a stock solution (20 µg mL-1) of deuterated benzophenone (benzophenone-d10 (BZ-d10)), which was used as surrogate, was prepared. Then, an aqueous working solution (65 ng mL-1) was prepared from this.Organic UV filters (i.e. BZ-3 and 4-MBC) in seawater were preconcentrated by dispersive liquid-liquid microextraction (DLLME). Stock solutions (500 ng mL−1 helium constant flow rate. The oven temperature program was: from 70 °C (1 min) to 170 °C at 10 °C min−1, then to 200 °C at 2 °C min−1 and finally to 280 °C (6 min) at 10 °C min−1. The transfer line and ion source temperatures were set at 280 and 250 °C, respectively. The chromatograms were recorded in selected ion monitoring (SIM) mode at the following mass/charge (m/z) ratios: 151, 227 (quantifier) and 228 for BZ3; 128, 211 and 254 (quantifier) for 4-MBC; and 82, 110 (quantifier) and 192 for BZ-d10.Ten mL of each of the standard working solutions were pH adjusted with glacial acetic acid to 2–4, then 1–1.5 g of sodium chloride were added to reach a final content within 10–15% (m/v), and finally 25 µL of the working surrogate solution were added. Then, they were subjected to DLLME in 15-mL polyethylene centrifuge tubes, by rapidly injecting pre-mixed 940 µL of acetone with 60 µL of chloroform solutions. Once the cloudy solutions were formed, they were centrifuged at 6000 rpm for 5 min. After centrifugation, approximately 25 µL of each of the organic sedimented phases were collected with the aid of a 50 µL syringe and transferred into 100 µL inserts placed inside 1.5 mL injection vials. Then, 2 µL were injected into the GC system coupled to a mass spectrometry (MS) detector operated in positive electron ionisation mode at ionisation energy of 70 eV and with a multiplier voltage set at 1400 V. The inlet temperature was 280 °C and the injection was accomplished in splitless mode (splitless time: 1 min). The separation was run at a 1 mL mini/Asur , each one obtained by using its quantifier ion) versus target analyte concentration.Calibration was performed by plotting AFor the determination of the soluble fraction of organic UV filters, 10 mL of filtered water samples were pH adjusted, and sodium chloride and surrogate solution added as previously described. For the determination of the total content , 10 mL of unfiltered water samples were sonicated for 10 min, then they were filtered and treated as described before for the determination of the soluble fraction. Then, they were subjected to DLLME and injected into GC-MS system as previously described for standards. For each target analyte, Ai/Asur was obtained and interpolated in the corresponding calibration line, and the concentration was finally obtained.43−, NO3−, NO2−, SiO2 and NH4+) were determined with an autoanalyzer (Alliance Futura) using colorimetric techniques 43−, NO3−, NO2− and SiO2, respectively. Zinc in seawater was preconcentrated by the APDC/DDDC organic extraction method Titanium in seawater samples was analyzed with MSFIA-LWCC -1 sunscreen was used. Water renewal at the beach was calculated from a series of 6 hours averages of historical current measurements in the sampling site during the same season, obtained using moored Nortek ADCPs The experiment on the release of nutrients from sunscreen was carried out by dissolving 15 g of the sunscreen number 11  into 5004 initial cellular density populations of Chaetoceros gracilis, obtained from the ICMAN marine culture collection, included in the BIOCISE index. Exposition was carried out at 20±1°C under continuous white light (35.2±1.1 µmol(quantum) m−2 s−1) in a controlled culture chamber (Ibercex) in 50 mL of exposition media disposed in 125 mL borosilicate conical flasks topped with synthetic cotton -Perlon-. After a previous wide-range concentration experiment developed in order to get a narrower concentration interval Standard microalgal toxicity bioassays were carried out on ASTM -1; 4-MBC: 113±7 ng L-1; and Ti: 38±7 µg L-1; Zn: 10.8 µg L-1; -1, respectively) were measured in the total fraction of SML, and in subsurface water , whilst Ti was only detected in the total water fraction of the SML (23.7±1.7 ng L-1). This suggests a high degree of alongshore connectivity due to the persistence of these products that results in their generalized impact around the island.Chemical analysis of the surface nearshore waters of three areas around Majorca Island showed that four of the main chemicals used in commercial sunscreens were detected in the surface waters, with the highest concentrations measured in the unfiltered fraction of the surface microlayer (SML)  which may affect algal growth. A release kinetic experiment consisting of shaking a commercial sunscreen (num. 11 in -1 NaCl) showed rapid dissolution of silicate and nitrogen compounds (in the first 16 hours), and lower rates (if any) thereafter (43- was fairly more progressive and linear (0.01 µmol g-1 h-1). Nutrient release kinetics varies depending on sunscreen composition, but since a majority of the tested products stabilized their release after 72 h we assumed that concentrations at this time to be indicative values for total content. The 13 commercial sunscreens tested provided final concentrations in water of 2±5 µmol g-1 of PO43-, 0.2±0.4 µmol g-1 of NO3-, 0.001±0.002 µmol g-1 of NO2-, 2±2 µmol g-1 of SiO2 and 0.02±0.01 µmol g-1 of NH4+  43- concentrations in Palmira Beach  after 72h incubation, resulting in an average of 125±71 mg L-1. The sunscreen num. 5  presented the lowest effects (EC50 = 218±17 mg L-1) (-1) are higher than environmental concentrations measured in our studied areas. These amounts of sunscreen (at which EC50 occurs) are reflecting the threshold for acute toxicity. However, even at very low sunscreen concentrations certain inhibitory effect is observed (We tested the effect of sunscreens on the growth rate of the marine phytoplankton  mg L-1) . The conobserved . The higMore than half of today’s world population live in coastal areas, and estimates for the future suggest that in three decades from now nearly 75 percent of the world’s population will live along coasts Figure S143− (µM) at Palmira Beach (Majorca) estimated from sinusoidal variations of beachgoers and 6 hours averaged current velocities.Modeled variations of PO Dashed line indicates shelf water background concentration.(TIF)Click here for additional data file.Figure S2Diel variation of beach users at Palmira during a labor day (bars) and sinusoidal adjustment used in model simulations (line).(TIF)Click here for additional data file.Table S1-1 released from commercial sunscreens in seawater after 72 h shaking.Concentration (average ± SDV) of nutrients in nmol g Samples were analyzed by triplicate except for nutrients from sunscreen 4. SPF (Sun Protection Factor).(DOCX)Click here for additional data file.Table S2-1  in commercial sunscreens.Concentration of metals in µg g SPF (Sun Protection Factor). Other elements such as Ag, Cd, Co, Cr, Cu, Mn, Mo, Ni, Si, Sr, Pb, Tl, V, and Zr were not detected.(DOCX)Click here for additional data file.