We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4+ T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs  Cellular immune responses against any foreign non-self antigens depend upon recognition of peptides presented on the surface of antigen presenting cells (APCs), such as dendritic cells (DCs) + T cell specific. However, it is not known whether these are immunodominant or subdominant within any given CD8+ T cell response.Other approaches for identification of T cell epitopes use predictive computational models of MHC binding affinity to obtain candidate peptides Mass spectrometry (MS) provides an effective means to sample and quantitate MHC II-associated self- peptides from DCs for many MHC II proteins Here, we solved this problem by a four step-approach . First, + T cell epitopes presented on class II MHC complexes are of particular interest because of their role in the development of protective antibody responses induced by vaccination in vitro. We also demonstrate that presentation of MHC II-associated HIV gag peptides by mature DCs takes place intracellularly.To evaluate this strategy we chose HIV gag p24 peptide sequences because the gag protein plays an important role in inducing protective T cell immunity to HIV in vitro with synthetic peptides or with a protein To detect, identify and measure exogenous MHC II-bound peptides presented by DCs among the abundant and complex mixture of endogenous peptides, Flt3L-mobilized DCs are pulsed To overcome this limitation we developed a four step strategy : (I) theUsing this strategy we can rapidly find mass evidence for putative exogenous MHC II peptides and then test their identity by targeted MS/MS. An additional advantage of this strategy is that it identifies the most abundant charge state for subsequent targeted analysis, further reducing the list of candidate masses dramatically.To test the strategy, DCs were loaded with four HIV gag p24 15-mer peptides at concentrations of 1 µM each. These were previously identified by ELISPOT assay as mimetopes after anti-DEC gag p24 antibody vaccine immunization in two distinct mouse haplotypes  [3]. EpiLRAQASQ  and DRFYKT*LRAQASQ  respectively  as an inhibitor of endo-lysosome compartment acidification. In the presence of 20 mM NH4Cl, we did not detect any signal at the expected m/z values  of anti-DEC-205 HIV gag p24 protein and then we eluted MHC II peptides as described above. In this case, EpiSifter generated a list of all 8189 theoretically possible peptides, 7–27 amino acids in length  and DRFYKTLRAEQASQ (D6.1) identified above, a region in the mass chromatogram where the first three isotopic peaks of the peptides were present with similar predicted intensities, in two different charge states, +3 and +4,  and DRFYKT*LRAQASQ (*13C6N1L) . We calculated as 2.1 and 1.98 ppm the mass deviation between the measured m/z values  and the theoretical values, 604.6519 and 606.9905, of the VDRFYKTLRAEQASQ peptide pair. In other words, the exact mass measurement of the VDRFYKTLRAEQASQ peptide pair and co-elution during LC separation led to the unambiguous peptide assignment.We performed MS/MS analysis on the experiment shown in 3C6N1L)  into the3C6N1L)  and DRFY3C6N1L)  isotope 3C6N1L)  and DRFY3C6N1L)  at m/z 63C6N1L)  indicateLRAQASQ (*13C6N1L) peptides were 571.6297 and 573.9688 respectively with only 1.75 ppm and 1.91 ppm mass deviation  HIV gag p24 peptide identified in the first part of our work .We extracted quantitative data for the HIV gag p24 VDRFYKTLRAEQASQ (D6) and DRFYKLTRAEQASQ (D6.1) peptides derived from anti-DEC-205 HIV gag p24 protein in the experiment described in + T cells proliferated actively after restimulation with VDRFYKTLRAQASQ and DRFYKTLRAQASQ peptides as well as in presence of the 56-peptide mixture spanning the entire HIV gag p24 sequence  T cell proliferation assays. Synthetic VDRFYKTLRAQASQ and DRFYKTLRAQASQ HIV gag p24 peptides were added to bulk splenocytes isolated from HIV gag p24 immunized mice sequence . No reac+ T cells, comparably to the response with the 56-peptide mix HIV gag p24  and DRFYKTLRAEQASQ (D6.1) peptides  and DRFYKTLRAQASQ (D6.1) peptides to T cells, peptide pulsed DCs were cocultured with a fixed number of T cells isolated from HIV gag p24 immunized mice 6 and 1.7×106, respectively, recovered from initial 2×106 DC/well). Since treatment with CQ distinctly inhibited T cell-activation by DCs in response to HIV gag p24 peptides, we inferred that intracellular processing of exogenously added VDRFYKTLRAQASQ was required,  and 46% for DRFYKTLRAQASQ peptide . In line with other reports 4Cl was reversible after removal of NH4Cl by extensive washing of DCs with PBS . For comparison, a 30 fold higher concentration of Her2 derived peptide, PDSLRDLSVF, amino acid 420–429, from the Her2/Neu breast cancer antigen Finally, we assessed the functional avidity of T cell responses following VDRFYKTLRAEQASQ and DRFYKTLRAEQASQ peptides presentation by DCs  peptide . The EC5in vitro with HIV gag p24 peptides and we identified two MHC II-bound HIV gag p24 sequences, VDRFYKTLRAQASQ and DRFYKTLRAQASQ, present at concentrations within the range of previously identified MHC II self-peptides on DCs We have described a strategy, which combines the use of a computational tool, EpiSifter, with LC-MS analysis, to identify antigen-specific MHC II peptides eluted from DCs. To test the strategy, we first used DCs that were pulsed There have been only few reports of exogenous MHC-bound peptides identified by LC-MS/MS analysis, and only following exhaustive manual interrogation of thousands of MS/MS spectra or by prior peptide fractionation of complex mixture followed by screening of antigenic peptide fractions in T cell assays + T cells, which have acquired high avidity for these MHC II-bound peptides as a result of the immunization with anti-DEC 205-HIV gag p24 protein  and DRFYKTLRAQASQ (D6.1) peptides derived from the HIV gag p24 protein. Future experiments will examine whether additional peptides derived from the exogenously added DEC-205 HIV gag p24 protein are presented on mouse spleen DCs following the delivery of the HIV gag p24 protein through the DEC-205 receptor.in vitro its presentation required intracellular processing  F1 mice were used in this study. Animals were maintained under specific pathogen-free conditions and used at 6–8 wk of age in accordance with the IACUC guidelines.All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at The Rockefeller University, New York . Balb/c×C57Bl/6  F1 mice 6 purified DC per well were pulsed with 1 µM of synthetic 15-mer peptides or 1 µg/ml (5 pM) of anti-DEC-205 HIV gag p24 protein for 5–6 h. Twenty five µg/mL polyinosinic:polycytidylic acid (poly IC) was also added as a maturation stimulus. In some experiments, 20 mM of NH4Cl or 100 µM CQ was used to preincubate DCs. Treated and untreated DCs were loaded with peptides.CD11c8 cell equivalents) was injected for LC-MS analysis.MHC II molecules were purified by immunoaffinity from cell lysate We used a Dionex U3000 capillary/nano-HPLC system  interfaced with the Thermo Fisher LTQ-Orbitrap mass spectrometer, operated in a data-dependent acquisition mode cycling through a single MS full-scan  followed by 6 MS/MS scans in the ion trap www.perl.org) and R (www.r-project.org). First, sequences of the proteins of interest are cut to generate all possible MHC II peptides, and corresponding m/z’s are calculated for charge states +1 to +4, considering oxidized and unoxidized methionine. Second, the mass chromatograms for the possible peptides, modifications, charge states, and isotopic peaks, and interfering peaks are extracted from the data within the mass accuracy of the mass spectrometer (10 ppm). Third, the mass chromatograms are plotted together with the expected intensity ratio’s using R.EpiSifter was implemented using Perl (MHC II peptide mixture (10 µL) in 0.1% TFA/water was mixed with 10 µL of a solution containing known amounts (1 ng) of the two synthetic isotope labeled peptides dissolved in 50% acetonitrile/0.1% TFA/water. The entire peptide mixture was subjected to LC-MS analysis 6 splenic CD11c+ DCs were pulsed with poly IC and different HIV gag p24 peptide concentrations. NH4Cl-, CQ-treated or untreated DCs were loaded with peptides, washed and added to CFSE labeled HIV gag p24 primed T cells. After 4 days of culture, cell proliferation and cytokine production were determined by intracellular cytokine staining and FACS analysis.Antigen-specific bulk splenocytes were isolated from F1 mice primed intraperitoneally with 5 µg anti-DEC-205 HIV gag p24 protein and 50 µg of polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly ICLC), as adjuvant, and boosted under the same condition 4–6 weeks later Figure S1Query of DC MHC II- bound HIV gag p24 peptides after DCs were pulsed in vitro with a peptide mixture. Panels A, B and C show the full scan generated by EpiSifter for the 3 HIV gag mimetopes (imetopes . Note thimetopes .(PPTX)Click here for additional data file.Figure S2Identification of MHC II-bound HIV gag p24 peptides. Comparison of MS/MS spectra of eluted HIV gag p24 peptides VDRFYKTLRAEQASQ Click here for additional data file.Figure S34Cl inhibits MHC II- binding of HIV gag p24 peptides VDRFYKTLRAQASQ and DRFYKTLRAQASQ.NH  The same experiment as in 4Cl. No peptide ions (m/z = 906.4736 and m/z = 856.9394) corresponding to the peptides above could be detected.(PPTX)Click here for additional data file.Figure S4Quantitation of MHC II-associated VDRFYKTLRAEQASQ and DRFYKTLRAEQASQ HIV gag p24 peptides. Heavy isotopes peaks are indicated with a (*). MS profile of VDRFYKTLRAEQASQ (A) and DRFYKTLRAEQASQ (B) isotope peptide pair identified as MHC II-bound peptides. The detected m/z values are indicated in bold letters.(PPTX)Click here for additional data file.Figure S5Quantitation analysis of a selected endogenous MHC II peptide. Heavy isotopes peaks are indicated with a (*). MS profile of the ApoE isotope peptide pair  identified in the MHC II peptide mixture eluted from the experiment shown in (PPTX)Click here for additional data file.Figure S6Immuno-reactivity of HIV gag p24 mimetopes measured by FACS analysis. CFSE-labeled bulk splenocytes from HIV gag p24 immunized mice were stimulated with negative control HIV gag p17 peptide mix, positive control HIV gag p24 peptide mix and with the four HIV gag p24 mimetopes listed in (PPTX)Click here for additional data file.Figure S7Presentation of HIV gag p24 VDRFYKTLRAEQASQ peptide in DC:T cell cocultures. Untreated (medium) and NH4Cl-treated DCs, prepared from the experiment described in + IFNγ+ T cells pooled from two independent experiments.(PPTX)Click here for additional data file.Figure S8Functional avidity of MHC II–bound HIV gag p24 peptides in DC:T cell cocultures. For each peptide concentration (−logM), the frequencies of CD4+ IFNγ+ T cells measured in (−6 M). The indicated EC50 represents the peptide concentration of VDRFYKTLRAEQASQ and DRFYKTLRAEQASQ peptides, respectively, required to attain 50% of maximal IFNγ production by HIV gag p24 specific CD4+ T cells.sured in  were exp(PPTX)Click here for additional data file.Table S1List of all theoretically predicted peptides, m/z ratios and relative intensities for the isotope distributions, calculated by EpiSifter from the HIV gag p24 mimetope sequences shown in .(XLS)Click here for additional data file.Table S2List of identified DC MHC II-bound peptides. Peptide sequences were analyzed by tandem MS and identified by searching the IPI database for mouse protein sequences plus the HIV gag p24 protein sequence using the X! Tandem algorithm Click here for additional data file.Table S3List of isotope labeled peptides synthesized for quantitation analysis and their MS parameters.(a)Mass corresponds to the monoisotopic form of the selected peptide. (b)Predicted mass shift due to the incorporation of the isotopically labeled amino acid.(DOCX)Click here for additional data file.Table S4List of all theoretically predicted peptides, m/z ratios and relative intensities for the isotope distributions, calculated by EpiSifter from the HIV gag p24 protein sequence (aa 133–363 derived from HIV isolate BH10).(XLSX)Click here for additional data file.Materials and Methods S1Detailed materials and methods.(DOCX)Click here for additional data file.


K50A).The eukaryotic translation initiation factor 5A1 (eIF5A1) is a highly conserved protein involved in many cellular processes including cell division, translation, apoptosis, and inflammation. Induction of apoptosis is the only function of eIF5A1 that is known to be independent of post-translational hypusine modification. In the present study, we investigated the involvement of mitogen- and stress-activated protein kinases during apoptosis of A549 lung cancer cells infected with adenovirus expressing eIF5A1 or a mutant of eIF5A1 that cannot be hypusinated (eIF5A1K50A, the mechanism by which unhypusinated eIF5A1 induces apoptosis was investigated by Western blotting, flow cytometry, and use of MAPK and p53 inhibitors.Using adenoviral-mediated transfection of human A549 lung cancer cells to over-express eIF5A1 and eIF5A1K50A, along with phosphorylation and stabilization of the p53 tumor suppressor protein. Synthetic inhibitors of p38 and JNK kinase activity, but not inhibitors of ERK1/2 or p53 activity, significantly inhibited apoptosis induced by Ad-eIF5A1. Importantly, normal lung cells were more resistant to apoptosis induced by eIF5A1 and eIF5A1K50A than A549 lung cancer cells.Phosphorylation of ERK, p38 MAPK, and JNK was observed in response to adenovirus-mediated over-expression of eIF5A1 or eIF5A1Collectively these data indicate that p38 and JNK MAP kinase signaling are important for eIF5A1-induced cell death and that induction of apoptosis was not dependent on p53 activity. Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein that is post-translationally modified on a conserved lysine residue by two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH), which transfer a butylamine group from spermidine to a conserved lysine residue to produce the amino acid, hypusine. Two isoforms of eIF5A sharing 84% homology exist in humans but appear to have distinct biological functions . EIF5A1 Although the physiological role of eIF5A1 has not been fully elucidated, it has been found to function both as a translation elongation factor during protein synthesis  and as a2-terminal kinase (JNK), has been implicated in the activity of numerous chemotherapy and genotoxic drugs. MAPK can regulate apoptosis through specific phosphorylation of downstream mediators of apoptosis, including the tumor suppressor p53, thus linking cellular stress signaling and regulation of p53 activity. Phosphorylation of p53 can regulate p53 activity by altering protein stability, interaction with co-activators, and transcription of target genes .The development of cancer gene therapies requires agents that target pathways that maximize anti-cancer activity. EIF5A1 has been identified as a viable cancer target that can be adapted for use in gene therapy approaches since its over-expression has been demonstrated to induce apoptosis in a wide variety of cancer types ,13-16. AK50A was found to induce apoptosis in A549 lung cancer cells. The similarity in cellular response to eIF5A1 and eIF5A1K50A over-expression can be attributed to the rate-limiting activity of DHS and DOHH  followed by brief sonication. Protein concentration was quantified using the Bicinchoninic Acid Kit (Sigma-Aldrich). One to ten micrograms of protein was separated by SDS-PAGE and western blot analysis was performed by incubating with primary antibodies for either one hour  or overnight at 4°C . After incubation with HRP-conjugated secondary antibodies, the antibody-protein complexes were visualized using enhanced chemiluminescence . Densitometry analysis was performed using TotalLab TL100 vs2006 software. In order to distinguish between the different post-translational modification states of eIF5A, two-dimensional gel electrophoresis followed by western blot analysis using eIF5A antibody was performed as described . BrieflyTotal RNA was isolated from cells infected with adenoviral constructs using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). Reverse transcription was performed on 1.2 micrograms of total RNA using AMV reverse transcriptase (Roche Applied Science) according to the manufacturer’s instructions. PCR reactions contained 500 nM of each primer, 1× of iQ SYBR Green Supermix (Bio-Rad), and 1 μL of cDNA. Real time PCR was performed in a MiniOpticon Real Time PCR Detection System (Bio-Rad) for 40 cycles using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene. The tumor suppressor p53 was amplified using the following primers : forwardApoptosis was quantified by labeling cells with Annexin V-FITC and propidium iodide using the FITC Annexin V Apoptosis Detection Kit II, according to the manufacturer’s instructions, followed by analysis on a BD FACSVantage SE system (BD Bioscience) with an argon laser source. A minimum of five thousand cells was counted and the data was analyzed using WinMDI 2.8 software.t-test was used for statistical analysis. Significance was determined by a confidence level above 95% (P < 0.05).Student’s 2-terminal kinase; LacZ: β-galactosidase; MAPK: Mitogen-activated protein kinases; MEK: MAPK kinase; MOI: Multiplicities of infection; NF-κB: Nuclear Factor Kappa B; PI: Propidium iodide; RT-qPCR: Quantitative reverse transcription polymerase chain reaction; siRNA: Small interfering RNA; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; SAPK: Stress activated protein kinase; TNFR1: Tumor necrosis factor receptor 1; UV: Ultraviolet light.AP-1: Activating protein-1; Bcl-2: B cell lymphoma-2; DHS: Deoxyhypusine synthase; DOHH: Deoxyhypusine hydroxylase; DMSO: Dimethylsulfoxide; eIF5A1: Eukaryotic translation initiation factor 5A1; ERK: Extracellular signal-regulated kinase; FBS: Fetal bovine serum; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; GC7: N1-guanyl-1,7-diaminoheptane; HRP: Horseradish Peroxidase; JNK: c-Jun NHK50A as anticancer agents.This work was supported by a research contract from Senesco Technologies Inc. J.E.T. is Chief Scientific Officer for Senesco Technologies Inc. and holds Senesco shares. C.A.T. holds stock options in Senesco Technologies. Senesco Technologies Inc. holds patents pertaining to the use of eIF5A1 and eIF5A1CT designed the study, performed western blot analysis, RT-qPCR, and apoptosis analysis on experiments using A549 cells, and drafted the manuscript. QZ performed the 2-D gel electrophoresis and western blot analysis for experiments using WI-38 cells. ZL amplified the adenoviruses and performed apoptosis analysis on WI-38 cells. CT and JT wrote the final manuscript. All authors read and approved the final manuscript.A549 lung carcinoma cells were infected with adenovirus expressing either LacZ (L) or eIF5A1 (5A). A) Forty-eight hours later the cell lysate was harvested and analyzed by western blot analysis for expression of phosphorylated c-Jun (ser63), p53, or eIF5A.Click here for file


Recently, excessive SOCE was proposed to play a central role in the pathogenesis of malignant hyperthermia (MH), a pharmacogenic disorder of skeletal muscle. We tested this hypothesis by characterizing SOCE current (ISkCRAC) magnitude, voltage dependence, and rate of activation in myotubes derived from two mouse models of anesthetic- and heat-induced sudden death: 1) type 1 ryanodine receptor (RyR1) knock-in mice (Y524S/+) and 2) calsequestrin 1 and 2 double knock-out (dCasq-null) mice. ISkCRAC voltage dependence and magnitude at -80 mV were not significantly different in myotubes derived from wild type (WT), Y524S/+ and dCasq-null mice. However, the rate of ISkCRAC activation upon repetitive depolarization was significantly faster at room temperature in myotubes from Y524S/+ and dCasq-null mice. In addition, the maximum rate of ISkCRAC activation in dCasq-null myotubes was also faster than WT at more physiological temperatures (35-37°C). Azumolene (50 µM), a more water-soluble analog of dantrolene that is used to reverse MH crises, failed to alter ISkCRAC density or rate of activation. Together, these results indicate that while an increased rate of ISkCRAC activation is a common characteristic of myotubes derived from Y524S/+ and dCasq-null mice and that the protective effects of azumolene are not due to a direct inhibition of SOCE channels. Store-operated calcium entry (SOCE) channels play an important role in Ca Malignant hyperthermia (MH) is a pharmacogenetic disorder triggered by exposure to halogenated anesthetics  and depolarizing skeletal muscle relaxants (e.g. succinylcholine) . MH epis2+ release from the sarcoplasmic reticulum (SR) that ultimately leads to a dramatic and sustained rise in intracellular Ca2+ Max), calculated from the peak of the first derivative of the normalized ISkCRAC activation time course, was significantly increased in myotubes from Y524S/+ mice  . Similarfication  and was fication  and 1 µMmyotubes . Specifi WT mice . In addiS/+ mice .SkCRAC activation would be observed in Casq-null myotubes since Casq deficiency should result in faster store depletion due to the combined effects of reduced SR Ca2+ storage and loss of Casq-dependent RyR1 inhibition ryanodine binding to SR vesicles [Dantrolene, a skeletal muscle relaxant ,41, is cmyotubes , as wellvesicles , two sepvesicles ,48. An ivesicles , likely vesicles , was dem2+ imaging and Mn2+ quench assays, these studies demonstrated that pretreatment with 20 µM azumolene for 1-2 min inhibited SOCE activated by subsequent addition of caffeine and ryanodine to promote RyR-dependent SR Ca2+ store depletion in mouse myotubes [2+ release, while SR Ca2+ reuptake was inhibited by high concentrations of intracellular EGTA. While this approach activates SOCE linked to RyR1 channel activity [SkCRAC magnitude nor rate of activation were significantly altered by pretreatment of WT and Y524S/+ myotubes with 50 µM azumolene. Similarly, application of 50 µM azumolene did not significantly alter ISkCRAC current density even when applied after maximal ISkCRAC activation (data not shown). These data indicate that azumolene is not a direct SOCE channel inhibitor. The therapeutic action of dantrolene and azumolene in MH was recently suggested to result from an inhibition of SOCE coupled to RyR1 activity -23. Usinmyotubes . Interesactivity , we foun2+ store depletion and uncontrolled Ca2+ entry through SOCE channels is a key component of an MH reaction as suggested above, how could dantrolene (or azumolene) be highly effective in preventing and reversing MH crises if it does not act by blocking SOCE channels? As one possibility, since Orai1-dependent SOCE lies downstream of store depletion, anesthetic- or heat-induced RyR1-dependent Ca2+ leak would first lead to store depletion and only then be followed by SOCE activation, Ca2+ entry, and myoplasmic Ca2+ overload. According to this mechanism, MH events could be attenuated by agents that either reduce RyR1 Ca2+ leak and store depletion (e.g. dantrolene/azumolene) or directly inhibit Orai1-dependent Ca2+ influx when stores are depleted (e.g. SOCE channel inhibitors). Moreover, an agent that inhibits RyR1-mediated store depletion (e.g. dantrolene) would be expected to indirectly reduce SOCE activation even if the drug does not directly block the channel once it is activated. While our results demonstrate that azumolene does not directly inhibit Orai1 Ca2+ current, the drug could still indirectly limit ISkCRAC activation by protecting stores from becoming depleted. In this context, the reduction in SOCE (i.e. Mn2+ quench) when azumolene was applied prior to caffeine/ryanodine exposure reported previously [2+ release, and thus, limiting the relative degree of SR Ca2+ store depletion and SOCE activation achieved. If SR Caeviously -23 couldSkCRAC) in myotubes derived from two separate mouse models for heat- and anesthetic-induced MH. We demonstrated a similar significant increase in the rate of ISkCRAC activation during repetitive stimulation in myotubes derived from both models. While dantrolene and azumolene may minimize ISkCRAC activation by reducing RyR1 Ca2+ release, and thus store depletion, the drugs do not to directly inhibit SOCE channels. Nevertheless, it will be important for future work to determine the potential therapeutic value of inhibiting SOCE channels in skeletal muscle in the prevention and management of MH environmental/exertional heat illness.We used a previously described voltage clamp approach  to direcFile S1Table S1. Times for 10%, 50%, and 90% activation of ISkCRAC at room temperature in C57Bl6, Y524S/+, and dCasq-null myotubes.Table S2. Times for 10%, 50%, and 90% activation of ISkCRAC at physiological temperature in C57Bl6, Y524S/+, and dCasq-null myotubes.Table S3. Effect of azumolene  on times for 10%, 50%, and 90% activation of ISkCRAC at in C57Bl6 and Y524S/+ myotubes.Figure S1.SkCRAC current density and pharmacology.I (A) Average (±SE) ISkCRAC current density recorded from WT C57Bl6 , Y524S/+, and dCasq-null myotubes at -80 mV in control  and after addition of 1 µM Gd3+ (open bars). Data for wild-type are taken from Yarotskyy and Dirksen (2012). Numbers of paired experiments are shown in bars. *p < 0.05. (B) Average (±SE) ISkCRAC current density recorded from WT C57Bl6, Y524S/+ , and dCasq-null myotubes at -80 mV in control  and after addition of either 100 µM (C57Bl6 and Y524S/+) or 10 µM (dCasq-null) 2-APB (open bars). Numbers of paired experiments are shown in bars. *p < 0.05.Figure S2.SkCRAC magnitude and activation rate in myotubes from WT C57Bl6, Y524S/+, and dCasq-null mice.The effect of PT on I (A) Average (±SE) time required for 10% (T10%), 50% (T50%), and 90% (T90%) activation of ISkCRAC in WT C57Bl6, Y524S/+, and dCasq-null myotubes. (B) Average (±SE) maximum rate of ISkCRAC activation in WT C57Bl6, Y524S/+, and dCasq-null myotubes. *p < 0.05. (C) Average (±SE) peak ISkCRAC current density recorded at -80 mV in myotubes from WT, Y524S/+, and dCasq-null mice.(DOCX)Click here for additional data file.


Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting  due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice ( The secondary wall is a plant-specific, stiff and thick cell wall composed of lignin, cellulose and hemicelluloses in a complicated bridged structure. The secondary wall is synthesized just outside the plasma membrane of vascular vessels and fiber cells in xylem, which require mechanical rigidity and stability to retain water in the vessel, endure the internal pressure of water transport and support the plant’s aerial structures. Some species also produce secondary walls in the internal cell layer of anthers and siliques; the different shrinkage rates of cell layers with or without secondary walls produces tension that promotes dehiscence of anthers and siliques during dehydration . In contSome secondary cell wall components can negatively affect downstream uses of plant biomass. For example, monocots are traditionally important forage crops; however, high lignin content results in poor digestibility. Also, several monocot plants, such as miscanthus and switchgrass, have recently emerged as potential feedstock crops for cellulosic biofuels ; howeverArabidopsis thaliana. These studies revealed that a specific set of NAC transcription factors acts as master regulators of vascular vessel differentiation and secondary wall formation in fiber cells (VASCULAR ASSOCIATED NAC DOMAIN PROTEIN (VND) genes inhibited the differentiation of vascular vessels (NAC SECONDARY WALL THICKENINGS PROMOTING FACTOR 1 (NST1) and NST2 showed an indehiscent anther phenotype due to failure of secondary wall formation in the anther endothecium (NST1 and NST3/SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1) caused a complete absence of secondary wall in fiber cells of stem and hypocotyl  also function in secondary wall formation  or VND, NST/SND AND SMB RELATED PROTEINs (VNSs) because their expression patterns are not readily classified into either VND or NST types as in Arabidopsis , were recently shown to restore the drooping phenotype of Arabidopsis nst1 nst3 double mutants and induce ectopic secondary formation when overexpressed in Arabidopsis  of Arabidopsis and rice, using NST1 as query. Full-length amino-acid sequence alignment was prepared using MAFFT , OsNST2C (5′-TACGTTATTCATGGTCGTCAAGTCTGCGTG-3′) and OsNST2S (5′-TCAACGGTCACCGTCGAGCAGTTGCTCGGG-3′). For OsSWN2S, OsNST1N (5′-GATGAGCATCTCGGTGAACGGGCAGTCGTG-3′), OsNST1C (5′-TGGCCCAGTCCGTGCGGTGGTGGT-3′) and OsNST1S (5′-TCATGGCCCAGTCCGTGCGGTGGTGGT-3′). Each CDS was ligated into the SmaI sites of the pActSRDXG, p35SG and pNST3_EntG vectors. ProZmUBQ1:OsSWN1/2S-SRDX was prepared similarly, based on the pUBQ1SXG vector, which was modified from the pActSRDXG vector. For the cloning of OsSWN promoters, the following primer sets were used. For OsSWN1, OsNST2pFA3 (5′-AAATTTGGCGCGCTTGCCGACGCTCGGAGCGTGCGAGCTG-3′) and OsNST2proR2Bam (5′-AAATTTGGATCCACCGACTGCCCGTTCACCGATATGC-3′). For OsSWN2, OsNST1pFA (5′-AAATTTGGCGCGCCACATATGTTGGATGTATTCTCCGAAA-3′) and OsNST1pRB (5′-AAATTTGGATCCATTGATGATCTTCTTCTTCTTCTCCTT-3′). The amplified fragments were cloned into the AscI/BamHI site of the pSRDX_NOSG vector, which does not have a plant promoter, and into the pGUS_Ent vector for promoter-GUS analysis. The OsSWN CDSs were cloned into the SmaI site of promoter-inserted pSRDX_NOSG vectors. The contents of these vectors were transferred into the binary vector pBCKH  and AscNSTx3Not_R (5′-GGCCGCAGTTTCTTCATTCACAAGGTATACAGTTTCTTCATTCACAAGGTATACAGTTTCTTCATTCACAAGGTATACGG-3′), AscmSNBEx3Not_F (5′-CGCGCCGTATACAAGGTGAATGAAGAAACTGTATACAAGGTGAATGAAGAAACTGTATACAAGGTGAATGAAGAAACTGC-3′) and AscmSNB-Ex3Not_R (5′-GGCCGCAGTTTCTTCATTCACCTTGTATACAGTTTCTTCATTCACCTTGTATACAGTTTCTTCATTCACCTTGTATACGG-3′) containing three tandem repeats of the binding sequence for NST transcription factors  was artificially synthesized and cloned into the SmaI site of the p35SG vector.escribed , 2007. Tor pBCKH  to prepars SNBE;  or mutatrs SNBE;  terminatArabidopsis thaliana ecotype Columbia-0 was grown on soil at 23°C, under a 16 h day/8 h night cycle. For the genetic transformation, the floral dip method was employed . All observations by light and fluorescence microscopy were made with the Axioskop2 plus system (Zeiss Inc.). For observations of lignin autofluorescence, we used a filter with the following specifications: excitation filter: 365 nm short pass; dichroic mirror: 395 nm; emission filter: 400 nm long pass. To observe ectopic secondary wall thickening, we cleared tissues by incubating them overnight in 70% lactic acid at 50°C. To stain lignin specifically, sections were stained with 2% (w/v) phloroglucinol in 95% ethanol for 2–5 min, washed in 10 N HCl for 1 min, and mounted in 5 N HCl.For GUS staining, plant tissues were incubated in 100 mM sodium phosphate buffer, pH 7.0, containing 0.1% Triton X-100, 1 mM 5-bromo-4-chloro-3-indolyl-β-Arabidopsis rosette leaves was performed as described  and collected by centrifugation at 100 × g for 5 min. The pellet was washed twice with W5 buffer containing 150 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES, followed by centrifugation at 100 × g for 5 min. The protoplast suspension was kept at 4°C for 30 min. After removing the supernatant, the protoplast cells were resuspended in MMg solution containing 0.4 M mannitol, 15 mM MgCl2, and 4 mM MES and then adjusted to 1.5–2.0 × 105 cells/ml. The protoplast suspension, plasmids and transduction solution containing 40% polyethylene glycol, 0.2 M mannitol, and 0.1 M CaCl2 were thoroughly mixed with a vortex mixer at 900 rpm for 15 s and then kept at room temperature for 10 min. After introducing the plasmids, the protoplasts were washed three times with W5 buffer and then incubated at 22°C for 18 h in dark. Firefly luciferase driven by a minimal promoter with three tandem repeats of the NST binding sequence or its mutated version was employed as a reporter. The details of its construction are described in the Section “Plasmid Construction.” Along with the reporter construct, 35S:OsSWN1, 35S:OsSWN2S or 35S:OsSWN2L was co-introduced as the effector. As the internal reference, a modified Renilla luciferase  gene driven by the 35S promoter  in 50 mM potassium-phosphate buffer (pH 7.0) containing 5% ethanol for 18 h after pre-incubation in protease solution for 2 h at 37°C. The protease-treated explants were washed with distilled water and stored in methanol. Force-extension relationships were measured with a tensile-testing instrument . After measurement, the explant was dried and weighed for calculation of its mass per length. Young’s modulus was calculated by employing Cleland’s formula  = 0.578 × delta absorbance - 0.0072.To analyze water transport in Short pieces of rice leaf blades were fixed in 30 mM HEPES containing 2% paraformaldehyde and 2% glutaraldehyde then fixed in HEPES containing 2% osmium tetroxide. Fixed tissues were embedded in Q651 resin . Sections of 80–90 nm thick were post-stained with uranyl acetate and lead citrate and observed with a JEM1200EX transmission electron microscope  at an accelerating voltage of 80 kV. Thickness of secondary walls in several layers of cortical fiber cells beneath the epidermis was measured for more than 10 cells in each of 7–9 pictures.Aspergillus niger in 0.1 M sodium maleate buffer at pH 6.0) at 37°C for 16 h with shaking. After amylase treatment, the sample was washed with ethanol and water, then dried at 65–70°C for 3 days and designated as the amylase-treated sample. The saccharification rate of the amylase-treated sample was measured by enzymatic reaction with Celluclast 1.5 l and Novozyme 188  as previously described (Culms of rice were collected after the harvest of mature seeds and dried at 70°C for 1 week. The amylase-treated sample was prepared as follows. The samples were ground and the resulting powder (100 mg) was treated with amylase solution (3 U/ml of α-Amylase from porcine pancreas and 30 U/ml of amyloglucosidase from escribed .g for 15 min with a swing rotor at room temperature. The supernatant (0.4 ml) was transferred to a 1.5 ml tube that contained 0.3 mL of 0.3 M NaOH and 0.5 mL of acetic acid. After mixing, 0.1 ml of 0.5 M hydroxylamine was added to each tube and the absorbance at 280 nm was measured. Lignin treated with alkali carboxylate  was used as a standard for lignin content. All measurements were run in triplicate for each sample.We quantified lignin content by the acetyl bromide method, following the protocol of 100 - 100(A′ - A)/S(G/100). S, A, G, A′ represent weight of the sample, weight of container + filter paper, dry matter percentage and weight of container + filter paper + residue, respectively.Digestibility as forage was analyzed by a modified method as described previously . Approxi2SO4 and 2% (w/v) CTAB was added to about 0.5 g of sample and placed on the heating unit for 60 min, after which the solution was filtered through a fritted glass. After washing with distilled water and acetone, the fritted glass crucible with residue was dried at 105°C overnight. After cooling in a desiccator and weighing, the crucible with residue was placed at 500°C for 3–4 h in a muffle furnace. After cooling, the ash was weighed. ADF was calculated by the following equation: 100(A - B)/S(G/100). S, A, B and G represent weight of the sample, weight of the crucible + residue, weight of the crucible + ash and dry matter percentage, respectively.Acid detergent fiber (ADF) was analyzed according to g for 5 min using a swing rotor and then dried at 80°C with N2 aeration. The dried sugars were dissolved in distilled water for the analysis of sugar composition. The TFA insoluble residues were rinsed with distilled water and ethanol and then completely dried at 80°C for 24 h. The dried residue was treated with 200 μl of 72% (w/w) sulfuric acid and incubated for 1 h at room temperature with agitation. Then the mixture was diluted into 4 ml of distilled water and autoclaved at 121°C for 1 h. Sulfuric acid hydrolysate (TFA insoluble fraction) was neutralized with calcium carbonate and then used for the sugar composition analysis. Mono-saccharide composition of each hydrolysate was analyzed by Shimadzu Prominence HPLC system  with fluorescence detector  as described by Matrix polysaccharides of cell wall in culm sample (3–5 mg) treated with α-amylase and amyloglucosidase were hydrolyzed in 1 ml of 2 M trifluoroacetic acid (TFA) at 121°C for 60 min . After cArabidopsisNSTs, which act as master regulators of secondary wall formation. We found two close orthologous genes , OsSWN1 (Os06g0131700) and OsSWN2 (Os08g0115800), which were previously reported by GUS) as a reporter, generating transgenic rice expressing ProOsSWN1:GUS and ProOsSWN2:GUS. The approximately 3 kbp promoter regions of both OsSWN1 and OsSWN2 were active in leaf veins but examination of leaf cross sections revealed slight differences in their expression patterns . The OsSWN2 promoter was particularly active in bundle sheath, including vascular vessels in the xylem  and the OsSWN1 promoter was preferentially active in several layers of sclerenchymatous cells beneath the epidermis in addition to the bundle sheath . This pattern of OsSWN1 promoter activity was consistent with the previously reported in situ RNA hybridization data  is shorter than the predicted protein from the JN634071 cDNA (OsSWN2L) and seems to lack the transcriptional activation domain. OsSWN2L was not described in the most recent study .Because the bidopsis , 2007. Tnt study  but was nst1-1 nst3-1 mutant, we hypothesized that OsSWN1 may act as a strong activator. Also, based on its length, OsSWN2L may act as an activator, but OsSWN2S likely lacks the ability to activate transcription. To test the activation activities of OsSWN1 and OsSWN2S/L, we performed transient effector-reporter experiments using Arabidopsis protoplasts derived from rosette leaves. For the reporter, we used a construct with three repeats of the SNBE sequence, which was previously reported to be a consensus binding site for NST transcription factors . As expected, the OsSWN1 effector activated the expression of the reporter gene by as much as 20-fold, and the OsSWN2L effector activated the reporter more than 30-fold . By contrast, OsSWN2S showed very weak activation compared with the control effector . However, when OsSWN2S was fused with the VP16 activation domain derived from herpes simplex virus (OsSWN2S-VP16), the effector activated the reporter gene more than 20-fold . In the negative control, OsSWN1, OsSWN2S-VP16 and OsSWN2L showed very little activation of reporter expression driven by the repeated mSNBE . These results indicate that OsSWN1, OsSWN2S and OsSWN2L bind to the SNBE sequence specifically but OsSWN2S does not have adequate activation activity. We further tested whether OsSWN1 can activate native promoters of Arabidopsis genes that are known to function downstream of NST transcription factors. As shown in Figure 3D, OsSWN1 activated promoters of MYB46, CELLULOSE SYNTHASE A 7 (CESA7), FRAGILE FIBER 8 (FRA8) and Caffeoyl-CoA 3-O-methyltransferase1 (CCoAOMT1), suggesting that OsSWN1 induces secondary wall formation by a mechanism similar to that of Arabidopsis NST transcription factors.We next directly tested whether OsSWN1 and 2S/L can activate transcription. Based on its ability to induce ectopic secondary wall formation and to rescue the  factors  and we uOsSWN genes in rice and examine their potential utility for cell wall engineering in rice, we prepared transgenic rice lines that express chimeric repressors (OsSWNs-SRDX), which consist of each OsSWN fused with the modified plant specific repression domain [ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif] derived from SUPERMAN , and found that ProOsSWN2:OsSWN2S-SRDX caused severe retardation of shoot growth and rolling of vegetative leaves, resulting in needle-like leaf shape, 4–6 weeks after culture under normal water conditions. In addition, the rolled leaves of ProOsSWN2:OsSWN2S-SRDX lines turned brown at the maximum tillering stage. We describe this phenotype of ProOsSWN2:OsSWN2S-SRDX as “para-wilting”; the para-wilting phenotype was observed in 499 out of 568 ProOsSWN2:OsSWN2S-SRDX transgenic plants but never observed in 779 control transgenic plants expressing the ProOsSWN2:GUS construct and 396 ProOsSWN2:OsSWN1-SRDX transgenic plants .To analyze the role of onstruct , 2007. TOsSWN2 promoter is active in the vascular bundle, we examined water transport in the ProOsOSWN2:OsSWN2S-SRDX plants using a tracer, red Fantasy dye solution. Even though no morphological abnormalities were observed in ProOsSWN2:OsSWN2S-SRDX rice leaves at 1–2 weeks after cultivation in the greenhouse, the excised shoots from transgenic plants did not absorb water as fast as the those from control transgenic plants . Quantification of dye uptake supported this observation . These data strongly suggest that water transport was severely impaired in these plants. This might be partly related to incomplete development of vascular vessels, as shown previously in Arabidopsis plants that expressed 35S:VND7-SRDX .Because the ND7-SRDX , since dOsSWN1 promoter, which is preferentially active in several layers of sclerenchymatous cells beneath the leaf blade epidermis and shows little activity in vascular vessels. Because OsSWN2S-SRDX effectively induced abnormalities in the experiment described above, we focused on OsSWN2-SRDX in this experiment. 192 out of 471 ProOsSWN1:OsSWN2S-SRDX rice plants exhibited an apparent drooping phenotype , a slight decrease in height (length of leaves). Detailed examination of leaf cross sections of the ProOsSWN1:OsSWN2S-SRDX rice plants revealed a reduction of secondary wall thickenings in the sclerenchymatous cells that form cortical layers beneath the epidermis and in bundle sheath fibers surrounding vascular vessels in xylem . Moreover, transmission electron microscopy showed that the secondary walls were thinner in the transgenic rice plants . Staining of the cross sections with phloroglucinol, which specifically stains lignin polymers, revealed reduced accumulation of lignin in the ProOsSWN1:OsSWN2S-SRDX rice plants . We quantified the lignin content of culms by the acetyl-bromide method and found that it was reduced more than 20% in the transgenic plants compared to the control plants . We further examined the neutral sugar compositions of the culms using liquid chromatography and found that TFA-soluble xylose was significantly reduced, but TFA-soluble arabinose presumably derived from arabinoxylan was not changed , suggesting that xylan content was reduced in the cell walls of the ProOsSWN1:OsSWN2S-SRDX rice plants. Because some cultivars of rice and related monocot species are consumed as forage, we examined the ADF value, which shows the digestibility of the plant, with higher numbers indicating a higher lignin and crystalline-cellulose content, and therefore lower digestibility. Indeed, the ADF value was also significantly reduced in the ProOsSWN1:OsSWN2S-SRDX rice plants . Also, the enzymatic saccharification rate of the cellulosic material was higher in the ProOsSWN1:OsSWN2S-SRDX rice plants than in the control transgenic plants . The digestibility as forage was also significantly enhanced in the ProOsSWN1:OsSWN2S-SRDX transgenic rice . These data suggest possible mechanisms for future genetic improvement of monocotyledonous forage and bioenergy species using this gene construct.Next, we expressed the chimeric repressors under control of the Arabidopsis NST transcription factors that regulate secondary wall formation. We found that OsSWN1 and OsSWN2 are expressed in cells where secondary cell walls are formed and can alter secondary cell wall formation in rice. Ectopic expression of OsSWN1 induced secondary wall formation in Arabidopsis and OsSWN2S chimeric repressors driven by the OsSWN1 promoter reduced secondary wall formation in rice. These results might reflect their abilities to activate transcription and we found that OsSWN1 can strongly activate transcription. However, OsSWN2S does not activate transcription, even though it can bind to the same cis-element as OsSWN1. It is likely that OsSWN1-SRDX did not function as a repressor in our assays because OsSWN1 itself can strongly activate transcription; this may counteract the negative activity of SRDX. The extent of ectopic secondary wall formation was greatest in the Arabidopsis plants overexpressing OsSWN1, compared to the other transgenic lines tested. In addition, we used the SRDX sequence, which is mainly used in Arabidopsis, and which may not function well in rice. However, OsSWN2S-SRDX efficiently suppressed secondary wall formation, probably because OsSWN2S has no endogenous transcriptional activation activity in our assays to counteract SRDX function. By contrast, OsSWN2L, the recently identified longer version of OsSWN2, has comparable transcriptional activation activity to OsSWN1. These results suggest that either: (i) OsSWN2L functions in planta but OsSWN2S does not function, or (ii) OsSWN2L promotes, and OsSWN2S inhibits secondary wall deposition in planta to fine-tune the formation of secondary wall, similar to a mechanism previously suggested in poplar . We expect further studies of the secondary wall in monocots will improve our understanding of plant cell wall formation, structure, and function and allow key improvements for forage and biofuel crops.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transcription factor Oct1 regulates multiple cellular processes. It is known to be phosphorylated during the cell cycle and by stress, however the upstream kinases and downstream consequences are not well understood. One of these modified forms, phosphorylated at S335, lacks the ability to bind DNA. Other modification states besides phosphorylation have not been described.pS335 concentrates at centrosomes, mitotic spindle poles, kinetochores and the midbody. Oct1 siRNA knockdown diminishes the signal at these locations. Both Oct1 ablation and overexpression result in abnormal mitoses. S335 is important for the overexpression phenotype, implicating this residue in mitotic regulation. Oct1 depletion causes defects in spindle morphogenesis in Xenopus egg extracts, establishing a mitosis-specific function of Oct1. Oct1 colocalizes with lamin B1 at the spindle poles and midbody. At the midbody, both proteins are mutually required to correctly localize the other. We show that phospho-Oct1 is modified late in mitosis by non-canonical K11-linked polyubiquitin chains. Ubiquitination requires the anaphase-promoting complex, and we further show that the anaphase-promoting complex large subunit APC1 and Oct1pS335 interact.We show that Oct1 is phosphorylated at S335 in the Oct1 DNA binding domain during M-phase by the NIMA-related kinase Nek6. Phospho-Oct1 is also ubiquitinated. Phosphorylation excludes Oct1 from mitotic chromatin. Instead, Oct1These findings reveal mechanistic coupling between Oct1 phosphorylation and ubquitination during mitotic progression, and a role for Oct1 in mitosis. The Oct1 (POU2F1) transcription factor is a potent regulator of metabolism and tumorigenicity One of the aforementioned phosphorylation events occurs at Ser335 within the DNA binding domain. Ser335 mutation to aspartic acid blocks Oct1 binding to all tested DNA recognition sites pS335), we generated a phospho-specific polyclonal antibody. The peptide sequence used to generate the antibody (EALNLS335FKNMC) aligns perfectly to the POU-specific portion of the DNA binding domain of human Oct1, Oct2 and Oct11. This region is less conserved in other human POU domain proteins and does not match any other protein sequences in the non-redundant protein databases (not shown). The predicted molecular weight of unmodified human Oct1 is 80 kDa, but the standard form migrates at ∼90 kDa in denaturing SDS-polyacrylamide gels. Initial characterization of the antibody using HeLa whole cell extracts and Western blotting indicated the presence of an intense band of high apparent molecular weight (>200 kDa) in cells arrested in mitosis by nocodozole  and total Oct1. A band of the same size corresponding to the high molecular weight form was observed in both cases  assays , which resulted in the strong diminution of the high molecular weight form, as well as other Oct1 forms, but had no effect on total Oct1 . No incr) assays .pS335 antibody . Early mitotic cells also showed staining at two puncta suggestive of duplicated centrosomes. Oct1pS335 was enriched at the prophase nuclear envelope (see prophase detail). Work of others has associated Oct1 with the nuclear periphery pS335 was largely excluded from DNA, except at small puncta consistent with kinetochores. We corroborated spindle pole body and centrosome localization in mitotic and interphase cells using γ-tubulin antibodies  identified a consensus Nek6 kinase target site at S335. Nek6 is a NIMA-related kinase required for normal mitosis in HeLa cells pS335 staining and disrupted the organization of mitotic spindles (pS335 detected at the midbody (right panels). We quantified the degree of pan-Oct1 or Oct1pS335 signal intensity in all mitotic cells following control or Nek6 siRNA transfection, observing an overall two-fold decrease in Oct1pS335 but not pan-Oct1 in Nek6 but not control siRNA . The high molecular weight form of Oct1 enriched in mitosis interacted only poorly with lamin B .To determine whether phosphorylated Oct1 and lamin B interact, we performed co-immumoprecipitation experiments using whole cell extracts from untreated or nocodozole-arrested HeLa cells. As expected, immunoprecipitation with lamin B antibodies enriched total Oct1 in cycling HeLa cells  using a spindle assembly assay stimulated by magnetic beads coated with the mitotic kinase Aurora A Prior studies in Xenopus Oct1 could have functional effects in this assay, although there is a distinct possibility of redundancy with the Oct2 protein. To test this prediction, we immunodepleted Oct1 from XEE and reconstituted the assay. Overall Oct1 immunodepletion was estimated at ∼85% based on band intensity, although the antibody showed some variation in the ability to immunodeplete different Oct1 species (The above result suggested that loss of  species . Immunod species .pS335 and Western blotted using Cdk11 and control pan-Oct1 antibodies (We expressed Oct1 fused to GFP in MEFs and used anti-GFP coupled beads to identify associated proteins via mass spectrometry (unpublished data). One of the identified proteins was Cdk11 (not shown), which regulates mitotic spindle stability. Cdk11 depletion results in similar in vitro defects in spindle assembly tibodies . The respS335 localization during metaphase and anaphase. Lamin B1 silencing did not change Oct1 protein levels, but resulted in early mitotic defects such as mitotic spindle disruption and unfocused spindle poles , suggesting that lamin B1 recruits phospho-Oct1. Oct1 knockdown (lower panels) also quantitatively depleted lamin B1+B2 at the midbody. These findings indicate that during cytokinesis lamin B1 and Oct1 are mutually required for midbody localization.We next silenced lamin B1 using siRNAs and monitored Oct1le poles , as repole poles . This repS335 midbody localization and localization to other mitotic structures: midbody association is maintained after most signal is eliminated  cipitate . To demo protein  was effi protein . Furthertibodies , lane 3,tibodies .pS335 is displaced from mitotic chromatin and concentrated at spindle pole bodies and the midbody during mitosis. Interphase cells show Oct1pS335 staining at centrosomes. The signal detected during mitosis is qualitatively different from that observed during interphase, and cannot be explained simply by increased total Oct1 levels. With the exception of the midbody, Oct1pS335 staining is rapidly lost at the anaphase-telophase transition. We identify Nek6 is an upstream Oct1pS335 kinase. Previous studies have shown that Nek6 localizes to the centrosomes in interphase cells, and to the spindle poles and midbody during mitosis. Nek6 loss of function also results in mitotic abnormalities and apoptosis in HeLa cells Here we show that Oct1 phosphorylated at position S335 by Nek6 is ubiquitinated and associates with mitotic structures. Oct1Oct1 phosphorylation has been investigated previously within the context of the cell cycle. Mitotic phosphorylation was described at a different residue, S385 pS335 is also ubiquitinated, including by non-canonical K11-Ub late in mitosis. Ubiquitinated proteins have been previously associated with the spindle pole bodies and midbody pS335 is formed throughout mitosis, but is degraded rapidly prior to telophase, at which time degradation slows or stops. A model for Oct1 phosphorylation and ubiquitination through the cell cycle is shown in Oct1pS335 acquires new functions. In Xenopus egg extracts, Oct1 co-purifies with the spindle matrix, which helps maintain spindle shape. Oct1 also localizes with lamin B at the midbody. Lamin B1 and Oct1 are mutually required to localize each other to the midbody, suggesting that they form a complex. Aside from the specific localization to mitotic structures, results from both Oct1 loss- and gain-of-function experiments implicate Oct1 as a mitotic regulator, at least in some cell types. For example, the organization of the mitotic spindle is disrupted upon Oct1 siRNA knockdown in HeLa cells. In XEE, Oct1 depletion causes defects in spindle morphogenesis, implicating Oct1 in mitosis-specific functions. Based on the observed Oct1 localization patterns, it is likely that the mitotic irregularities caused by changes in Oct1 levels are directly associated with mitotic regulation. Furthermore, an intact serine at position 335 is important for the mitotic phenotype of over-expressed Oct1, implicating this residue in mitotic functions.Although a simple model is that Oct1 is phosphorylated at S335 and becomes non-functional, several lines of evidence suggest that Oct1We found that Oct1 is not required for the completion of mitosis in HeLa cells but rather appears to play a regulatory role. In other cell types, such as murine fibroblasts and A549 cells, the effect of Oct1 is more mild than in HeLa cells. Oct1 deficient embryos survive past gastrulation 2. HeLa cells were transiently transfected using polyethyleneimine  and pCG-FLAG-Oct1 Oct1 deficient MEFs have been described previously 335FKNMC. The antibody was purified in two steps, first by blocking with unphosphorylated peptide to remove non-phospho-specific antibodies, then by affinity purification using the phosphorylated epitope. Mouse antibodies against α-tubulin, γ-tubulin, CLASP-1, lamin B, and goat anti-Pit-1, were obtained from Santa Cruz. Mouse anti-pS10-H3 was obtained from Cell Signaling, and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Chemicon. Rabbit anti-APC1 and mouse anti-Cdh1 antibodies were purchased from Abcam. Mouse anti-FLAG and rabbit anti-Nek6 antibodies were from Sigma. For pan-Oct1, a rabbit antibody (Bethyl) was used with the exception of A commercial rabbit phospho-specific antibody (Bethyl) was raised against the peptide EALNLSXenopus egg extracts using AuroraA-conjugated beads was conducted as described Assembly of spindles from Coverslips were coated with Poly-L-lysine (Sigma) for 30 min and placed into 6 well dishes where counted cells were plated. Cells were washed with phosphate buffered saline (PBS) two times prior to fixation. Methanol fixation was performed as described SmaI restriction site. Expressed proteins in BL21 Codon-plus (Stratagene) Escherichia coli transformed with each construct were purified using glutathione-sepharose resin . Sequences were S335: 5′GCGATTTGAAGCCTTGAACCTCAGCTTTAAGAACATGTGCAAGTGA3′, 5′TCACTTGCACATGTTCTTAAAGCTGAGGTTCAAGGCTTCAAATCGC3′; A335: 5′GCGATTTGAAGCCTTGAACCTCGCCTTTAAGAACATGTGCAAGTGA3′, 5′TCACTTGCACATGTTCTTAAAGGCGAGGTTCAAGGCTTCAAATCGC3′. The GST-fused peptide  were incubated with recombinant Nek6 and CAK complex . Kinase activities were assayed with the purified substrate according to a manufacturer's protocol.Two sets of complimentary oligo DNA (S335 and A335) were anealed and ligated into pGEX-4T1 (Promega) using a Fzr1) and control transfections used siRNA pools (Dharmacon). Cells were cultured for 48 hr prior to analysis.siRNA pools targeting three different regions of Oct1 or Nek6 (Santa Cruz) were mixed with lipofectamine RNAi max (Invitrogen) and transiently transfected according to manufacturer's protocol. Control siRNAs were used in parallel and also purchased from Santa Cruz. Cells were cultured for 3 days prior to analysis. siRNA knockdown of CDH1 , and phosphatase inhibitors . 500 µg of extract was incubated with 4 µg of antibody in IP buffer  overnight at 4°C. Protein-antibody complexes were precipitated using magnetic beads (Activmotif) and washed three times with IP buffer.Figure S1Further characterization of the phospho-specific antibody. (A) The phospho-specific antibody immunoprecipitates ubiquitinated Oct1 under denaturing conditions. IP was performed as described in the materials and B) Immunofluorescence images are shown. Left and right panels show early and late mitosis, respectively. NT = no treatment. METHODS: samples were prepared as described in the materials and (JPG)Click here for additional data file.Figure S2Localization of total Oct1 in Mitotic HeLa Cells. (A) Total Oct1 concentrates at spindle pole bodies and the midbody, and is excluded from mitotic chromosomes. Immunofluorescence images are shown. HeLa cells were fixed and stained with antibodies against α-tubulin and pan-Oct1. Arrow indicates position of additional puncta not recognized by phospho-Oct1 antibodies. (B) Pan-Oct1 antibodies recognizing different epitopes stain different mitotic structures. Cells were fixed and stained as in (A). Arrows indicate additional metaphase puncta of unknown etiology not recognized by phospho-Oct1 antibodies. (C) Side-by-side images of normal interphase and mitotic HeLa cells stained with phospho-Oct1 or pan-Oct1 antibodies. Cells were fixed as in (A).(JPG)Click here for additional data file.Figure S3Localization of Oct1 Phosphorylated at S335 to Mitotic A549 Lung Adenocarcinoma Cells. (A) Immunofluorescence images are shown. Specific mitotic cell is highlighted with an arrow. The cells were co-stained with antibodies to alpha-tubulin and with DAPI. (B) Similar images using anti-gamma-tubulin antibodies. Protocols were identical to those summarized in the (JPG)Click here for additional data file.Figure S4p335 and Anti-Histone H3S10 Antibodies.Comparison of the Properties of Anti-Oct1 (A) Low-magnification (200×) immunofluorescence images of HeLa cells stained with antibodies against phospho-histone H3 and phospho-Oct1335. Arrows indicate mitotic cells. White arrows show cells in earlier mitotic stages that co-stain with both antibodies. Yellow arrows show examples of cells staining only with the phospho-335 and not phospho-histone antibody. (B) Immunofluorescence images of mitotic HeLa cells stained with anti-Oct1p335 and anti-histone H3S10 antibodies. Scale bar: 20 µM.(JPG)Click here for additional data file.Figure S5Effect of Oct1 siRNA knockdown on HeLa cells stained with alpha- and gamma-tubulin antibodies. (A) Additional examples of mitotic abnormalities in HeLa cells treated with Oct1-specific siRNAs. Reference (JPG)Click here for additional data file.Figure S6Phospho-S335 IF Staining Pattern and Cell-cycle Phenotype of Primary MEFs. (A) Immunofluorescence images of mitotic stages from wild-type primary early-passage MEFs. (B) Mitotic examples of −/−Oct1 MEFs. These examples could not be easily staged due to abnormalities. Note the reduced pS335 staining. (C) Alignment of human Oct1 and mouse Oct1, Oct2 and Pit-1. Alignment was generated using a Clustal W-based algorithm within the Vector NTI software package. Lower panel shows a Western blot of HeLa cells, wild-type and Oct1 deficient fibroblasts. The Pit-1 antibody was obtained from Santa Cruz. (D) Cell cycle profile of primary early passage Oct1 deficient MEFs and wild-type littermate controls. Inset shows expanded view of cells with super-4N DNA content.(JPG)Click here for additional data file.Figure S7pS335 in HeLa cells.Effect of WT and S335A Oct1 overexpression on Oct1 IF images are shown of interphase and HeLa cells transiently transfected with wild-type or S335A mutant pCG-Oct1.(JPG)Click here for additional data file.Figure S8Ectopic Oct1 expression in HeLa cells increases alpha-tubulin levels, and induces formation of puncta containing alpha-tubulin but lacking DNA. IF images are shown of interphase HeLa cells transiently transfected as in (JPG)Click here for additional data file.Figure S9Additional evidence that Nek6 phosphorylates Oct1. (A) Nek6 was knocked down in HeLa cells using siRNAs as in B) Pan-Oct1 and phospho-Oct1 channel signal intensity was analyzed following Nek6 knockdown using ImageJ software (NIH). 6 control and 7 Nek6 siRNA mitotic events were averaged in the case of pan-Oct1, and 5 control and 5 Nek6 siRNA mitotic events were averaged in the case of phopsho-Oct1. (C) GST-fused to the substrate peptide was tested as an in vitro kinase target as in 32P-ATP. (D) Full-length, His6-tagged recombinant Oct1 purified from E. coli was used. Right panel shows a Coomassie-stained gel of the same material. “Δ Oct1” indicates presumptive Oct1 deletion products that are also phosphorylated species. METHODS: Recombinant C-terminal His6-tag Oct1 was expressed and purified as described :1981–6).(JPG)Click here for additional data file.Figure S10Additional evidence of mutual localization mediated by lamin B1 and Oct1. (A) HeLa cells were transiently transfected with siRNAs against lamin B1 (Santa Cruz), incubated for 72 hr and Western blotted with antibodies against lamin B1 or GAPDH as a loading control. (B) IF images are shown of mitotic HeLa cells transiently transfected with control or lamin B1 siRNAs, and stained with antibodies against lamin B (B1+B2) and Oct1pS335 antibodies. Arrows highlight elongated/split spindle poles.(JPG)Click here for additional data file.Figure S11APC is responsible for the accumulation of K11-Ub signal at sites of Oct1 phosphorylation upon proteasome inhibition. HeLa cells were transiently transfected with control siRNAs or siRNAs directed against the APC component CDH1 , incubated for 48 hr. After 42 hr, cells were treated with MG132 as described in the materials section. Cells were then fixed and processed for IF. For quantification, mitotic events were scored +/− based on concordance between Oct1pS335 and K11-Ub staining.(JPG)Click here for additional data file.


Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism.Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection.We demonstrate that a novel member of the mitochondrial transporter protein family, Plasmodium infection.We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to  Plasmodium parasites and results in more than half a million deaths every year, mainly of African children Plasmodium is transmitted by anopheline mosquitoes, and the establishment of this protozoan parasite in the insect vector can be greatly hindered by mosquito antiplasmodial responses Plasmodium parasites have to traverse the midgut epithelium and avoid destruction by the mosquito immune system. Plasmodium invasion causes irreversible damage that leads to apoptosis of invaded midgut cells Plasmodium ookinete invasion by inducing expression of nitric oxide synthase (NOS) and heme peroxidase 2 (HPX2), two enzymes that mediate nitration Malaria is a deadly disease caused by Anopheles gambiae strain refractory to Plasmodium infection PlasmodiumPrevious studies have shown that reactive oxygen species (ROS) levels are elevated in an +) into the mitochondrial intermembrane space; giving rise to a membrane potential between the mitochondrial membranes. Mitochondrial ATP production is achieved through oxidative phosphorylation, a process where the F1F0-ATP synthase complex, uses the energy of the electrochemical gradient to phosphorylate ADP to ATP. Under normal circumstances, a small portion of electrons leak from the electron transport system, giving rise to ROS m), so that small changes in this parameter can drastically affect ROS generation Mitochondria are the main cellular energy-transducing site and also a significant source of ROS To assess mitochondrial function, cellular oxygen consumption rates can be measured and modulated by experimental addition of substrates and/or inhibitors Toxoplasma gondii infection through a mechanism that involves increased ROS generation A. gambiae, the mitochondrial carrier 1 (AgMC1), which affects mitochondrial coupling, ROS generation and susceptibility to Plasmodium infection.The uncoupling proteins (UCP) and the adenine nucleotide translocators (ANT) are members of the solute carrier family 25 (SLC25) Public Health Service Animal Welfare Assurance #A4149 01 guidelines were followed according to the National Institutes of Health (NIH) Office of Animal Care and Use (OACU). This study was approved by the NIH Animal Care and User Committee (ACUC), under the NIH animal study protocol (ASP) ID ASP LMVR5.A. gambiae (G3 and L3-5 strain) and Aedes aegypti (Red eyes strain) A. gambiae females were dissected in isolation buffer consisting of 250 mM sucrose, 5 mM Tris-HCl, 2 mM EGTA, 1% (w/v) fatty acid-free bovine serum albumin (BSA), pH 7.4 and placed on an oxygraph chamber  with respiration buffer  supplemented with 0.0025% digitonin. After 1 min of equilibration, both NAD+-linked substrate (10 mM pyruvate +10 mM proline) and FAD+-linked substrate (10 mM succinate) were added to the chamber. Subsequently, 1 mM ADP was added to induce the state 3 respiratory state. State 4-like (oligomycin-stimulated) and uncoupled maximal respiratory rates  were measured upon addition of 7 µg of oligomycin and up to 7 µM FCCP, respectively. The RCR was calculated by dividing the state 3 respiratory rates to those obtained by the oligomycin-induced state 4 respiratory rates (2 min after oligomycin addition in the case of Anopheles gambiae and 5–6 minutes in the case of Aedes aegypti). Oxygen (O2) consumption was recorded in an oxygraph fitted with a Clark-type electrode in a water-jacketed chamber  for Anopheles gambiae and in a high-resolution oxygraph  for Aedes aegypti. For all experiments, temperature was maintained at 27.5°C. The respiratory inhibitors antimycin A, and cyanide were all capable of completely inhibiting the midgut O2 consumption. All mosquitoes used for the respiration assay were 4–7 days old. All mitochondrial respiration results represent the averaged oxygen consumption of at least six different midgut preparations analyzed in three independent experiments.Respiration assays were done following the procedures first described by Chance and Williams, 1955 P. berghei (GFP-CON transgenic 259cl2 strain) in vitro exflagellation assay, as described previously Aedes aegypti females (Red Eye strain) were fed with P. gallinaceum-infected chickens (∼5% parasitemia). 7 days after infection midguts were dissected and oocysts were counted under a light microscope after 2% mercurochrome staining. The distribution of the number of oocysts in individual mosquitoes were compared using the non-parametric Kolmogorov-Smirnov (KS) test, the median levels of infection were compared using the Mann-Whitney test and the prevalence of infection using the Chi-square test.Mosquitoes were infected with A solution of penicillin (100 units/ml) and streptomycin (0.1 mg/ml)  in 10% sugar solution was orally administered to mosquitoes on the first day post emergence. At the following day, they were injected with dsRNA from the gene of interest and kept with antibiotic solution in water (a sugar cube was provided separately) until 2 days post injection when the experiments were performed.TCTATTTTCTACTGC and R-CGAAGTGGAGGAACTGCTACTAA and cloned with TOPO TA cloning kit  following standard procedures. Primers were designed based on the cDNA sequence predicted by the bioinformatics annotation of the AgMC1 gene in the A. gambiae genome sequence (GenBank accession No. AGAP001297-PA).RNA was extracted from midguts of sugar-fed mosquitoes with Trizol  according to the manufacture’s protocol. First-strand cDNA was synthesized using QuantiTect Reverse Transcriptase . The full-length AgMC1 sequence (1498 bp) was amplified using the following primers: F-TGCACTCGT GGGCAGGAAACAGCTATGAC. The PCR product was used as a template to synthesize dsRNA in vitro using a MEGAscript RNAi kit . dsRNA was loaded to an RNA purification column supplied by the kit, eluted with water and concentrated to 3 µg/µl using a Microcon YM-100 filter . A similar cloning strategy was used for the AgMC1. The cDNA from AgMC1 was used to generate dsRNA templates of 698 bp using the following primers: AgMC1 (5′ to 3′) F-TAATACGACTCACTATAGGGAGCAAGCGTCCCCTACACT and R-TAATACGACTCACTATAGGGCGTTTTGACCACGTCGAAC. For A. aegypti MC1 dsRNA synthesis a nested PCR was performed using the external primers (5′ to 3′) F- GCCTTGCCCACTACAGTGAT and R-gggtccgaactgaactcatc and the internal primers fused with the T7 promoters F- TAATACGACTCACTATAGGGAACGATTGTGAATCCGTTGG and R- TAATACGACTCACTATAGGGTATAAGTGCGCCCTGATCCT (GenBank accession No. AAEL001329-RA).A 218-bp fragment of the lacZ gene was amplified using the primers (5′ to 3′) F-GAGTCAGTGAGCGAGGAAGC and R-TATCCGCTCACAATTCCACA and cloned into the pCRII-TOPO vector. T7 promoters were incorporated onto this fragment by amplifying the cloned insert using the following primers: M13F-GTAAAACGACGGCCAGT and M13R-CTCGAGTAATACGACTCACTATA A. gambiae and A. aegypti female mosquitoes were injected with 69 nl and 138 nl, respectively, of a 3-µg/µl solution of dsRNA from the gene of interest at 1–2 days post emergence. Control mosquitoes were injected with dsLacZ. Two days later, females from both groups  were used. The distributions of the number of oocysts in individual mosquitoes were compared using the non-parametric Kolmogorov-Smirnov (KS) test, the median levels of infection were compared using the Mann-Whitney test and the prevalence of infection with the Chi-square test. The effect of AgMC1 and AsMC1 silencing on P. berghei and P. gallinaceum, respectively, were each confirmed in two independent experiments that were merged.A. gambiae ribosomal protein S7 as an internal standard and analyzed by the 2-ΔΔCt method TGAAATCAAACCCAGCA and R-CCACGTCCAGTGGAGT CATA. S7, F-GGCGATCAT CATCTACGTGC and R-GTAGCTGCTGCAAACTTCGG. AaMC1, F- TTCATGACCCCGCTAGATGT and R- CCCTCGTGGTGACTGATTTT. RP-49, F-GCTATGACAAGCTTGCCCCCA and R-TCATCAGCACCTCCAGCT. AgDuox, F-CAAGGACAAGGACGGAAGAA and R-TCGCACATGTCGAAAATGAT. AgNox5, F-TGTCTTCACGCAGAAGGATG and R-ACCCACGGGATAGTTCACTG.RNA extraction and cDNA synthesis were performed as described above. Gene expression was assessed by SYBR green quantitative real-time PCR (qPCR)  in a Chromo4 system . PCR involved an initial denaturation at 95°C for 15 min, followed by 44 cycles of 10 s at 94°C, 20 s at 56°C, and 30 s at 72°C. Fluorescence readings were taken at 72°C after each cycle. A final extension at 72°C for 5 min was completed before deriving a melting curve (70°C–95°C) to confirm the identity of the PCR product. qRT-PCR measurements were made in triplicate. Relative quantitation results were normalized with 2O2 production was assessed by monitoring resorufin fluorescence due to the oxidation of 5 mM Amplex Red (Invitrogen) in the presence of 1.0 unit/ml of commercial horseradish peroxidase (HRP) (Sigma). Five midguts, from antibiotic treated mosquitoes, were dissected in 10 mg/ml 3-amino-1,2,4-triazole in PBS (AT)  and incubated at room temperature and dim light in AT solution with Amplex Red and HRP for 30 min. Amino-triazole was used to inhibit H2O2 consumption by midgut catalase. Fluorescence intensity was measured in the supernatant in a spectrofluorometer plate reader  operating at excitation and emission wavelengths of 530 nm and 590 nm, respectively. Background fluorescence generated as unspecific Amplex Red oxidation by the midgut in the absence of HRP was subtracted. After each experiment, a standard curve of reagent grade H2O2  was performed.HMidguts of sugar-fed mosquitoes were dissected in PBS and incubated at room temperature in dim light in RPMI supplemented with 10% FBS containing 5 µM of the intracellular ROS-sensitive dye DHE  for 20 min Females were fed with a solution containing PBS + MitoTracker Red  (20 mM) + ATP (0.5 mM), pH 7. One hour after feeding, the midguts were dissected in PBS, fixed with paraformaldehyde (4%) for 40–60 minutes in the dark, washed twice with PBS, permeabilized with Triton X-100 (0.01%) for 3–5 min, and washed again. F-actin was stained with Phalloidin-Alexa 488 (Molecular Probes) (165 nM) diluted in PBS + BSA 1% at room temperature for 20 min followed by two washings with PBS. Finally, the midguts were transferred to glass slides and mounted with VectaShield with DAPI (Vector Laboratories). Images were acquired on a SP2 confocal microscope (Leica). The staining pattern was confirmed in three independent experiments.Assessment of early oocyst numbers in the midgut was done 48 h post-infection, using immunofluorescence staining with anti-Pbs21 antibodies to detect the parasites. Midguts were dissected in ice-cold PBS, fixed for 30 s in 4% paraformaldehyde and returned to ice-cold PBS to stop the fixation. The midgut was opened longitudinally to remove the blood and the epithelia was carefully cleaned, fixed overnight with 4% paraformaldehyde at 4°C and washed twice with PBS. Midguts were permeabilized and blocked for 2 h at room temperature with PBT  under gentle shaking. Midguts were incubated with primary Ab  overnight at 4°C followed by three washing steps of 20 min with PBT at room temperature. The incubation with the secondary Ab  carried out for 2 h at room temperature. The samples were then washed as described above and mounted using VectaShield. The number of early oocysts was scored in a Leica fluorescence microscope under the 40x objective.http://www.sbg.bio.ic.ac. uk/∼phyre/), which identifies homologs with known structures using PSI-BLAST and generates a homology-based model using the three-dimensional coordinates and the amino acid sequence alignment information The sequence of AgMC1 was submitted for automated molecular modelling using the Phyre protein fold recognition server (Plasmodium-resistant A. gambiae (L3-5) refractory mosquito strain Plasmodium infection AgMC1 gene is located in Chr 2 division 7B, a chromosomal region in A. gambiae that has been associated with the refractory phenotype Aedes aegypti (87% homology) and Drosophila melanogaster (72%), and clusters with the mammalian SLC25 family members 39 (60%) and 40 (64%) and the yeast manganese trafficking protein 1 (MTM) (48%) Previous studies showed that expression of some genes related to the mitochondrial electron transport chain are induced in the A molecular model of the AgMC1 three-dimensional structure was constructed using the automated Phyre fold recognition server . The strAnopheles gambiae G3 susceptibility to Plasmodium berghei infection was investigated. AgMC1 expression was efficiently silenced and 2 days post-injection midgut mRNA levels decreased by 93% relative to the dsLacZ control group (P<0.001) (The effect of AgMC1-silencing on ol group . AgMC1 sP<0.001) . The effP<0.001) , indicatA. gambiae (L3-5 strain) is refractory (R) to infection with P. berghei and parasites are melanized as soon as they complete invasion and come in contact with mosquito hemolymph. AgMC1 silencing also increased the number of oocysts that developed in the R strain (P<0.01) (P<0.001) (Plasmodium in the mosquito midgut.The (P<0.01) , as wellP<0.001) . HoweverP<0.001) . BecauseP. berghei infection. The hypothesis that AgMC1 silencing enhanced infection by reducing mitochondrial ROS generation was investigated. AgMC1 silencing had no effect on ADP-stimulated mitochondrial respiration (state 3)  (p<0.005) (Aedes aegypti ortholog of AgMC1 (AaMC1) on midgut respiration was also evaluated. As in A. gambiae, silencing AaMC1 had no effect on state 3 respiration in A. aegypti midguts (P = 0.0199) in AaMC1-silenced midguts  and the prevalence of infection increased significantly from 63% to 80% .We investigated how AgMC1 silencing affected mitochondrial respiration and ROS generation. We have previously shown that increasing the level of ROS in the midgut by silencing genes involved in ROS detoxification such as catalase state 3) . However(P<0.05) . As a cop<0.005) , indicat midguts , while s midguts . The RCR midguts , indicatney Test); the difAs MC1 silencing increased proton permeability of the inner mitochondrial membrane, we investigate whether this affected mitochondrial membrane potential. Adult females were fed a solution containing MitoTracker Red, a fluorescent cationic dye that is selectively transported into energized mitochondria. MitoTracker Red uptake was greatly reduced in AgMC1 silenced midguts , indicatin vivo, as the products of intracellular DHE oxidation are highly fluorescent 2O2) production was measured directly using the Amplex Red detection system. Mosquitoes were pre-treated with antibiotics to eliminate the gut microbiota as a potential source of ROS and 3-amino-1,2,4-triazole was included in the reactions to inhibit catalase activity and prevent H2O2 degradation. AgMC1 was efficiently silenced (2O2 generation was significantly reduced in silenced midguts (P<0.0001)  to levels not significantly different from the AgMC1-silenced group. It is also noteworthy that FCCP had no significant effect on H2O2 production in midguts in which AgMC1 had been silenced, indicating that midgut mitochondria were already partially uncoupled. Taken together, these data indicate that AgMC1 silencing decreases mitochondrial uncoupling and ROS production in the mosquito midgut.Because even modest changes in mitochondrial membrane potential can greatly affect ROS generation silenced  and H2O2<0.0001) . Further<0.0001)  or NADPH<0.0001) , two oxiMitochondrial ROS generation is regulated by a number of factors such as the oxygen pressure, substrate availability, efficiency of the electron flow and mitochondrial membrane potential; and changes in any of these parameters can potentially modulate the host’s susceptibility to infection. Knockdown of the uncoupling protein 2 (UCP2) in mice increases mitochondrial membrane potential, increases ROS production and confers resistance to Toxoplasma infection A. gambiae Mn-SOD to be active, because reducing Mn-SOD activity would increase mitochondrial ROS levels and we found that AgMC1 silencing has the opposite effect, decreasing ROS generation.Although the substrate of AgMC1 is unknown, the binding region of the predicted protein structure  is linedThe reduced respiratory control ration (RCR) in AgMC1 and AaMC1-silenced midguts  are due Plasmodium, and ROS/RNS production has been shown to limit Plasmodium infection in anopheline mosquitoes 2O22O2 detoxification by silencing catalase enhances A. gambiae resistance to P. berghei infection. Conversely, decreasing ROS, by oral administration of antioxidants prevents melanization of PlasmodiumPlasmodium infection, indicates that mitochondria ROS can modulate antiplasmodial responses. Lower levels of H2O2 in the invaded cells are expected to reduce the rate of nitration, increasing the probability that ookinetes escape unharmed and evade detection by the mosquito complement system. Recently, mitochondrial ROS have also been proposed to be important modulators of immunity. For example, ROS generated by mitochondria of endothelial cells in response to hypoxia decreases TLR4 expression Reactive oxygen and nitrogen species (ROS and RNS) are important effectors of the immune system. The mosquito midgut is an important barrier against a variety of pathogens including bacteria and A. gambiae midgut and characterized a novel member of the mitochondrial carrier gene family, AgMC1, that modulates midgut mitochondrial membrane potential and ROS production. Our studies revealed a novel link between mitochondrial metabolism and mosquito epithelial responses to Plasmodium invasion.We explored mitochondrial function in the Figure S1Sequence alignment of members of the mitochondrial family from different species. AgMC1 (AGAP001297-PA) and the putative A. aegypti  and D. melanogaster  putative ortholog genes, human mitochondrial carriers HsSLC25A-39 (NP_057100) and SLC25A-40 (NP_061331), and yeast manganese trafficking factor for mitochondrial ; human uncoupling protein 5 , A. gambiae , A. aegypti  and D. melanogaster  uncoupling proteins; Human SLC25A6  and yeast  adenine nucleotide translocators, A. gambiae , A. aegypti  and D. melanogaster  putative adenine translocators; human SLC25A3  and yeast  phosphate carriers, and A. gambiae , A. aegypti  and D. melanogaster  putative phosphate carriers.(PDF)Click here for additional data file.Figure S2Effect of AaMC1 silencing on A. aegypti susceptibility to P. gallinaceum infection.Aedes aegypti females were fed P. gallinaceum infected blood (5% parasitemia) and the intensity of infection was determined 8 days after feeding using mercurochrome to stain the midguts. Medians are indicated by the red lines and distributions were compared using the Kolmogorov-Smirnov test.(TIFF)Click here for additional data file.Figure S3Effect of AgMC silencing by systemic injection of dsRNA on AgMC1, AgDuox and AgNox5 midgut mRNA levels. Significant differences relative to the dsLacZ control are indicated by the asterisks .(TIFF)Click here for additional data file.Figure S4Effect of AgMC1 silencing and co-silencing AgDuox or AgNox5 by systemic injection of dsRNA on AgMC1, AgDuox and AgNox5 midgut mRNA levels. Significant differences are indicated by the asterisks .(TIFF)Click here for additional data file.Table S1Amino acid sequence identity and homology of the predicted AgMC1 protein with other members of the solute carrier family.(PDF)Click here for additional data file.