Triterpenoid toosendanin (TSN) exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK). Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation. Melia toosendan Sieb. et Zucc. is a medicinal plant with analgesic, insecticidal and anti-inflammatory characteristics, which mainly grows in China and India in vitro and in vivoin vitro by Hoechst 33258 staining, DNA fragmentation analysis and flow cytometric analysis Natural products have become important molecular probes to study different cellular processes by virtue of their ability to bind to specific protein targets and interfere with their cellular functions. Therefore, large efforts have been directed at the identification of biologically active targets of natural products. Notable examples include the identification of prohibitin 1 as the target of marine natural product aurilide In this study, we chose TSN as a molecular probe to capture its target protein in HL-60 cells by serial affinity chromatography. A specific binding protein was identified as deoxycytidine kinase (dCK). To establish the relationship between dCK and TSN-induced apoptosis in HL-60 cells, we have provided three lines of evidence: a) recombinant dCK was directly activated by TSN; b) LP-503392, a dCK inhibitor, significantly inhibited TSN-induced apoptosis; and c) mutagenesis studies indicated that dCK activation was most likely caused by conformational change instead of auto-phosphorylation. Collectively, our results suggested that TSN binds dCK to induce conformational change and subsequently activates dCK to partially contribute to the pro-apoptotic effects in HL-60 cells.TSN-S was synthesized following the protocol reported previously  [17]. Th2CO3 (3×30 mL), washed with 50 mL ethyl acetate and 50 mL ether, respectively. The aqueous phase was adjusted to pH 1–2 with 1 N HCl, extracted with 100 mL ether, dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain crude product, which was recrystallized to give 28-hemisuccinyl-TSN (TSN-S) as white crystals . The structures was confirmed by comparison of its MS, NMR and IR spectral data with those in the literature 34H42O14; SIMS m/z 673(M-1); 1H NMR  δ: 4.30 ,1.90  2.78 ,5.25 ,2.68 ,1.73  2.08 ,3.67 ,4.60 ,5.25 ,3.76 ,1.90  2.23 ,2.97 ,1.31 ,4.30 ,7.13 ,6.15 ,7.33 ,0.82 ,5.82 ,1.15 ,2.10 ,1.97 ,2.66–2.70 ; 13C NMR  δ: 35.2, 73.3, 39.0, 28.0, 25.1, 69.3, 41.2, 48.3, 45.5, 207.7, 78.3, 45.5, 72.1, 58.4, 33.4, 35.2, 14.4, 64.8, 122.8, 142.3, 111.6, 140.7, 21.5, 95.1, 19.5, 171.6, 171.3, 174.3 (OCOCH2CH2COOH), 170.7 (OCOCH2CH2COOH), 28.8 (OCOCH2CH2COOH), 28.2 (OCOCH2CH2COOH); IR ): 3466 , 1730 .A mixture of TSN , succinic anhydride  and anhydrous sodium acetate (150 mg) in dry acetone (20 mL) was refluxed in 60°C water-bath for 10 h. The reaction mixture was filtered and the filtrate concentrated; the residue was then dissolved in 30 mL ethyl acetate. The solution was extracted with 3% NaTSN-S affinity matrices were designed and prepared based on previous report  [17]. TS2 atmosphere at 37°C.Human promyelocytic leukemia cell line (HL-60) was obtained from Type Culture Collection of Chinese Academy of Sciences . HL-60 cells were cultured in RPMI 1640 medium  supplemented with 10% (v/v) heat inactivated fetal bovine serum , 100 units/mL streptomycin and 100 µg/mL penicillin in a humidified 5% CO7) were washed twice with ice-cold PBS and extracted in 1 mL lysis buffer A composed of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5 mM MgCl2, 1% Nonidet P-40 (Sunshine Bio), 0.5 mM PMSF (Biosharp), 5 µg/mL Aprotinin (Bio Basic), 5 µg/mL Leupetin (Amresco), 5 µg/ml Pepstain (Bio Basic), shaken in ice-bath for 1.5 h. After centrifugation at 16000 rpm (20000 × g), 4°C for 15 min, the supernatants were used immediately for subsequent binding experiments.Identification of target proteins using serial affinity chromatography was performed as described previously 2, 1% Nonidet P-40, then shaken gently with lysate from HL-60 cells in ice-bath for 40 min and precipitated by microcentrifuge at 4°C for 1 min. The resulting supernatant was mixed with another 100 µL of TSN-S affinity matrices in ice-bath, again for 40 min. The resulting matrices was washed five times with 1.0 mL of lysate buffer B, resuspended in 30 µL of SDS sample buffer containing 4% (w/v) SDS, 20% (v/v) glycerol, 0.01% (w/v) bromophenol blue, 10% (v/v) 2-mercaptoethanol, and 0.125 M Tris–HCl (pH 6.8), heated in boiling water for 5 min, and then centrifuged for 1 min. The supernatant was subjected to SDS-PAGE. The resulting bands were stained with silver staining and then comparatively analyzed to identify specific binding proteins. For reproducibility, the above procedures were carried out for multiple times.TSN-S affinity matrices (100 µL) was washed four times with 1.0 mL of lysis buffer B containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5 mM MgClProtein band on the SDS-PAGE gel was excised and directly digested with trypsin. Tryptic peptides were separated by nano-flow microcapillary reversed-phase HPLC and eluted directly into the LTQ- Ion trap mass spectrometer  using a spray voltage of 3.0 KV at a capillary temperature of 170°C in positive ion mode. A full scan from m/z 400–1800 was performed in the ion trap at a resolution of 60,000. The top ten most intense ions were selected for MS/MS in the LTQ, with a normalized collision energy of 35%.The raw data were filtered by TurboSEQUEST algorithm. Protein identification was acquired with Turbo SEQUEST (Thermo Electron) algorithm in the BioWorks 3.1 Software against the International Protein Index (IPI) human protein database (Version 3.36). Turbo SEQUEST evaluated proteins by finding candidate peptides whose masses match the m/z values in spectra within a mass error tolerance. Turbo SEQUEST used a cross-correlation scoring. Identified peptides were filtered by the Xcorr in accordance with the charge state , DelCN≥0.1. The reverse database was used for false positive rate estimation to be <0.05.Modeling study was performed using Accelrys Discovery Studio 2.5 (DS 2.5). The coordinates for dCK containing 2′-deoxycytidine (dC) and ADP as cocrystalized ligands were obtained from the Protein Data Bank (PDB code: 1P60). The structure of TSN was generated using ChemDraw. Docking of TSN was accomplished using CDocker E. coli BL21 (DE3) strain.Wild-type dCK and seven mutants  were constructed using overlap extension PCR method and wild-type human dCK cDNA as the template E. coli BL21 (DE3) cells harboring the pET28 (a+) expression plasmids were grown in Luria-Bertani (LB) medium at 37°C until the A600 reached 0.4–0.6, then induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) about 12 h at 15°C and harvested by centrifugation 2, 0.5 mM DTT for 30 min at room temperature. An equal volume of Kinase-Glo® reagent was added and incubated for 10 min at room temperature. The luminescence was recorded on a LUMIstar OPTIMA reader.The activity of dCK was measured with Kinase-Glo® Luminescent Kinase Assay  as previously described +/PI− cells were considered as apoptotic cells.HL-60 cells were treated with 70 nM TSN or 0.1–10 µM LP-503392  alone or exposed to their binary combinations for 36 h. Then, they were harvested, washed and resuspended with PBS. Apoptotic cells were determined with an FITC Annexin V Apoptosis Detection Kit  according to the manufacturer’s protocol. Annexin Vt test value of P<0.05 was considered statistically significant.All experiments were repeated at least three times and the data were expressed as means ± standard deviations. Student Affinity matrices bearing bioactive compounds have been a useful tool in the identification of target proteins. A modified affinity chromatography, termed serial affinity chromatography, has been used to capture various target proteins. For example, the specific binding proteins of ginsenoside Rg1, FK506, benzenesulfonamide and methotrexate were successfully captured and identified by this method In the present study, HL-60 cells, the most sensitive cell line to TSN To explore the interaction between TSN and dCK, recombinant dCK was overexpressed and purified. The dCK activity was measured using Kinase-Glo® Luminescent Kinase Assay after incubation of recombinant dCK in the presence of TSN. Different concentrations ranging from 6.25 to 250 µM TSN were tested to examine the effects to dCK activity. As depicted in To further confirm whether dCK was directly activated by TSN, a commercially available dCK inhibitor LP-503392 Phosphorylation and conformational change of dCK have been suggested to lead dCK activation After the phosphorylation site and interaction sites were replaced by site-directed mutagenesis, the mutant proteins were expressed and purified . To explNaturally occurring small molecules are important tools for cell biology and are lead molecules for drug discovery. Identification of the molecular targets of natural products has had profound influence on studies of complex cellular machinery dCK (EC 2.7.1.74) is a rate-limiting enzyme in the salvage pathway of DNA synthesis. The enzyme phosphorylates deoxyadenosine, deoxyguanosine and deoxycytidine, with UTP or ATP as phosphate donor It is known that Ser74 residue is the only phosphorylation site that influences dCK activity In the present study, the effective concentrations of TSN towards recombinant dCK and HL-60 cells were significantly different. This difference indicates that dCK activation only plays certain and limited roles in the HL-60 cell apoptosis, and TSN, a known cytotoxic compound, acts on multiple targets and pathways to induce the complex apoptotic process. In addition, the relatively weak activation effects by TSN on dCK activity may amplify to interfere the biological process and to interact with other proteins and signaling systems for the final apoptotic effects in HL-60 cells. Therefore, combination of the present study and previous findings Since TSN possesses a variety of biological activities, these different activities should be a result of targeting multiple proteins or signaling pathways. From the apoptosis point of view, the interference on the complex network might be responsible for the final outcomes. The present findings indicate that TSN can bind dCK and activate the enzyme through conformational changes, which at least explains, in part, a new apoptotic route for TSN. The exact network or signaling systems by which TSN interacts with different proteins requires further analysis.Figure S1The synthetic route of TSN-S affinity matrices.(TIF)Click here for additional data file.Figure S213% SDS-PAGE of purified dCK mutants. Bands 1–8 represent dCK mutants of S35E, S35A, S35Q, S74E, S74Q, R128E, R128A and WT dCK respectively. M indicates molecular weight markers.(TIF)Click here for additional data file.Table S1Theoretical and detected molecular masses of amino acid sequences of human dCK.(DOC)Click here for additional data file.


The role of AMPK in EPA-induced eNOS phosphorylation was investigated in bovine aortic endothelial cells (BAEC), in mice deficient of either AMPKα1 or AMPKα2, in eNOS knockout (KO) mice, or in Apo-E/AMPKα1 dual KO mice. EPA-treatment of BAEC increased both AMPK-Thr172 phosphorylation and AMPK activity, which was accompanied by increased eNOS phosphorylation, NO release, and upregulation of mitochondrial uncoupling protein-2 (UCP-2). Pharmacologic or genetic inhibition of AMPK abolished EPA-enhanced NO release and eNOS phosphorylation in HUVEC. This effect of EPA was absent in the aortas isolated from either eNOS KO mice or AMPKα1 KO mice fed a high-fat, high-cholesterol (HFHC) diet. EPA via upregulation of UCP-2 activates AMPKα1 resulting in increased eNOS phosphorylation and consequent improvement of endothelial function  Eicosapentaenoic acid ; ω-3: EPA) is an ω-3 polyunsaturated fatty acid (PUFA), which is abundant in fish oils. Epidemiological and clinical trials have shown that ω-3 fatty acids, in particular EPA, reduce cardiovascular deaths Nitric oxide (NO) is essential for endothelial function. Decreased NO bioactivity is involved in the pathogenesis of many cardiovascular disorders such as hypertension, atherosclerosis, venous bypass graft disease, diabetic vascular disease 2+/calmodulin-dependent protein kinase kinase (CaMKK) The AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of α, β, and γ subunits. The α subunit imparts catalytic activity, while the β subunit contains a glycogen-binding domain (GBD) that also regulates the activity and the γ subunit forms the broad base of the protein and is required for AMP binding To investigate whether EPA activates AMPK in endothelial cells, confluent BAEC or HUVEC were treated with varying concentrations of EPA for 2 to 24 h. AMPK activation was indirectly assessed by western blot analysis of AMPK phosphorylation at Thr172, which is essential for AMPK activity We next examined the dose-dependent effects of EPA on AMPK-Thr172 and ACC-Ser79 phosphorylation. EPA did not affect phosphorylation of AMPK or ACC at a concentration of 5 µM . In contet al.We had previously demonstrated that AMPK phosphorylates and activates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells Both Akt and AMPK are capable of phosphorylating eNOS at Ser1179 L-NAME, a non-selective inhibitor of NOS . This increase was of a slightly greater magnitude to that seen with EPA (P<0.05). Treatment of BAEC with GW9662, an antagonist of PPARγ, partially abrogated the stimulatory effect of EPA on UCP-2 protein expression  agonists, have been shown to increase expression of UCP-2 in several tissues with EPA . The othpression , while tTo further verify that EPA increases endothelial UCP-2 expression and AMPK activation through PPARγ-dependent signaling, HUVEC were transfected with PPARγ-specific siRNA and assayed for PPARγ expression, UCP-2 expression, and AMPK phosphorylation in the presence of EPA. Western blotting verified that PPARγ expression was selectively and significantly reduced by the cognate PPARγ siRNA duplex compared with scrambled siRNA . ImportaTo determine whether EPA activates AMPK in vasculature, C57BL/6J mice were given EPA (500 mg/kg/d in drinking water) for 4 months, and aortic levels of AMPK, ACC, and eNOS phosphorylation and UCP-2 protein expression were determined. As shown in We also investigated if AMPK mediates the effects of EPA in an isoform-specific manner. As demonstrated in ex-vivo conditions. Acetylcholine (Ach) induced concentration-dependent arterial vasodilatation in all groups . It should be noted that, following treatment with AICAR or EPA, Ach-induced vasodilatation in aortas from Apo-E KO mice was significantly improved. In contrast, inhibition of AMPK with compound C abolished EPA-stimulated increases in Ach-induced relaxation in this group, where the maximal relaxation was 76.4±5.5% for EPA alone and 52.9±7.3% in the presence of compound C , while only modest increases in UCP-2 expression were observed with the PPARα agonist (Wy14643). The PPARγ antagonist (GW9662) but not the PPARα antagonist (MK-886) significantly blocked EPA-induced upregulation of UCP-2. These results could be further substantiated using specific PPARγ siRNA. Together, these data suggest that EPA induction of UCP-2 expression was mediated partly by PPARγ.de novo synthesis of diacylglycerol Another important observation of the present study is that chronic EPA supplementation significantly increased eNOS Ser1177 phosphorylation, NO release, and Ach-induced endothelium-dependent relaxation. Further, we have provided evidence that AMPK was required for increased NO bioactivity. Finally, we have shown that AMPK might have direct vaso-protective effects as EPA supplementation improves endothelial function without altering lipids in serum. Our results strongly imply that a direct activation of AMPK in endothelial cells might have broad physiological effects, leading to improvement of endothelial function. In addition to activating eNOS, activated AMPK increases fatty acid oxidation by phosphorylating and inhibiting ACC, which serves to decrease the concentrations of malonyl-CoA In summary, we have uncovered a novel pathway by which the ω-3 fatty acid EPA activates AMPK in endothelial cells. This pathway, which relies on UCP-2 as a mediator of AMPK activation, stimulates NO production through eNOS phosphorylation. Thus, AMPK activation may help account for the beneficial effects of fish oil on endothelial function and atherosclerosis.Bovine aortic endothelial cells (BAEC) and cell culture media were obtained from Clonetics Inc. . Human umbilical vein endothelial cells (HUVEC) and cell culture media were purchased from Cascade Biologics . FFA-free bovine serum albumin (BSA), palmitic acid, oleic acid, GW9662, and MK-886 were obtained from Sigma . Wy14643 and rosiglitazone were obtained from Cayman Chemical Co. , and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) was purchased from Toronto Research Chemicals, Inc . Antibodies against phospho-acetyl-CoA carboxylase (ACC) (Ser79), phospho-AMPK (Thr172), AMPK, and phospho-eNOS (Ser1177) were purchased from Cell Signaling Inc. . The antibodies against ACC were obtained from Alpha Diagnostic International, Inc. . All other chemicals and organic solvents were of the highest grade and were obtained from Sigma.To generate Apo-E/AMPK dual knockout (KO) mice, Apo-E KO mice obtained from Jackson Labs were crossbred for at least 5 generation with the mice deficient of AMPK α1. Deficiency of both Apo-E and AMPK α1 was confirmed by both RT-PCR and western blots by using the specific antibody against AMPK α1. As both AMPK KO mice and Apo-E KO had extensively been backcrossed to the C57BL6 background, C57BL6 mice aged 8-months were used as wild type control (WT) in the study. All animal protocols were approved by the Institutional Animal Care and Use Committee at University of Oklahoma Health Sciences Center.2/95% air at 37°C. The fatty acids were added to the cell cultures coupled to fatty acid-free BSA in the ratio of 2 mol of fatty acid to 1 mol of albumin BAEC were grown in EBM supplemented with 2% fetal bovine serum (FBS). HUVEC were maintained in Medium 200 supplemented with a low serum growth supplement (LSGS) before use. All culture media were supplemented with both penicillin (100 Units/ml) and streptomycin (100 µg/ml). Cells between passages 5 and 10 were used for all experiments. All cells were incubated in a humidified atmosphere of 5% COHUVEC (passages 3–5) were grown in antibiotic free-EGM-2 medium containing 2% FBS until 70% confluence and transfected with human-specific PPARγ siRNA, UCP2 siRNA, or corresponding scrambled siRNA for 48 h using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. The final concentration of siRNA was 200 nM.For NO detection, BAEC grown in 24-well plates were incubated for 30 min in the presence of 15 µM 4,5-diaminofluorescein diacetate (DAF-2 DA) in PBS or in PBS alone (control) in the dark at 37°C. Cells were then washed with PBS to remove excessive DAF-2 DA, and the change in fluorescence was recorded for 15 min at room temperature using a microplate reader  with the excitation wavelength set at 485 nm and the emission wavelength set at 530 nm. Changes in fluorescence were also visualized with a fluorescence microscope (Olympus IX71), and images were captured for analysis HUVEC were cultured in 6-well plates with control siRNA or UCP2 siRNA for 48 h, treated with EPA or vehicle for 24 h, washed with PBS, and scraped in 0.3 ml of PBS. ATP and ADP were then measured in quadruplicate by a luminometric method as described elsewhere BAEC or HUVEC and thawed mouse aortas were lysed in cold RIPA buffer. Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay system . Proteins were subjected to Western blots using ECL-Plus, as described previously AMPK activity was assayed using the SAMS peptide, as previously described Aortic rings (3–4 mm in length) extracted from C57BL6 aged 8-weeks, 20-weeks, 4-months or 8-months , Apo-E KO aged 8-months, Apo-E/AMPK dual KO mice (8-month old) or eNOS KO (8-week old) were further incubated with EPA (25 µM) or AICAR (2 mM) in endothelial cell basal media (EBM) for 24 h. After that, aortic rings were pre-constricted with phenylephrine in organ chambers . Endothelium-dependent and independent vasodilation responses were determined in the presence of acetylcholine (0.01 to 100 µM) and SNP (0.0001 to 1 µM), respectively. When indicated, compound C (20 µM) were added 30 min prior to addition of EPA.Statistical comparisons of vasodilation were performed using a two-way ANOVA. Intergroup differences were analyzed using Bonferroni's post test. Analysis of time-course studies was performed with repeated measures ANOVA. All other results were analyzed with one-way ANOVA. Values are expressed as mean ± SD. P values less than 0.05 were considered as significant.


This study aimed to analyze the relationship between clinical status and work characteristics of firefighters and other public officers who engaged on collection duties in the site of the hydrogen fluoride spill that occurred on September 27, 2012, in Gumi City, South Korea.We investigated the clinical status, personal history, and work characteristics of the study subjects and performed physical examination and several clinical examinations, including chest radiography, echocardiography, pulmonary function test, and blood testing in 348 firefighters, police officers, volunteer firefighters, and special warfare reserved force who worked at the hydrogen fluoride spill area.The subjects who worked near the accident site more frequently experienced eye symptoms (p = 0.026), cough (p = 0.017), and headache (p = 0.003) than the subjects who worked farther from the accident site. The longer the working hours at the accident area, the more frequently the subjects experienced pulmonary (p = 0.027), sputum (p = 0.043), and vomiting symptoms (p = 0.003). The subjects who did not wear respiratory protective devices more frequently experienced dyspnea than those who wore respiratory protective devices (p = 0.013). In the pulmonary function test, the subjects who worked near the accident site had a higher decease in forced vital capacity than the subjects who worked farther from the site (p = 0.019); however, no statistical association was found between serum calcium/phosphate level, echocardiography result, chest radiographic result, and probation work characteristics.The subjects who worked near the site of the hydrogen fluoride spill, worked for an extended period, or worked without wearing respiratory protective devices more frequently experienced upper/lower respiratory, gastrointestinal, and neurological symptoms. Further follow-up examination is needed for the workers who were exposed to hydrogen fluoride during their collection duties in the chemical plant in Gumi City. Hydrogen fluoride is a colorless, potent respiratory irritant with an unpleasant odor. Exposure to hydrogen fluoride causes strong irritation in the eyes, nose, and throat and may result in tears, eye redness, rhinorrhea, sore throat, cough, headache, dermatalgia, and other symptoms. Even in cases in which no symptoms are present for 1–2 days after exposure, fever, cough, dyspnea, cyanosis, and pulmonary edema may occur later. If hydrogen fluoride or its aqueous solution, hydrofluoric acid, comes in contact with the skin, it causes serious tissue damage. Absorbing a considerable amount percutaneously or through the airway causes hypocalcemia and hyperkalemia, which leads to arrhythmia and may result in death. Chronic repeated exposure to hydrogen fluoride increases bone density, which is also known as fluorosis of the bones . HydrogeAt approximately 3:40 pm on September 27, 2012, a spill accident occurred at the National Industrial Complex chemical plant, where only hydrofluoric acid is produced. Approximately 8 tons of 100% hydrogen fluoride (anhydrous hydrofluoric acid) was spilled from inside the tank lorry. The accident caused the death of 5 workers who were working at the accident site. The total number of people who visited medical institutions and clinics as a result of the accident in the period until October 21, 2012, was 12,243, demonstrating that the accident had an extensive influence on the neighborhood residents [Firefighters not only fulfill fire-extinguishing duties but also provide rescue and first-aid services during emergencies. In the process of performing such duties, they may be exposed to carbon monoxide, formaldehyde, hydrogen cyanide, hydrogen fluoride, and other combustion by-products -10. FireThe literature on the health impact of hydrogen fluoride includes studies on acute and chronic health impact after a local community spill ,7,13,14,This study aimed to investigate the work history and clinical symptoms of the people who worked at the accident site as collectors of the spilled hydrogen fluoride and to determine the relationship between the distance from the accident site, working time, and use of protective devices, and the detrimental effects of hydrogen fluoride. The study will provide baseline information about the health management of personnel who participated in the hydrogen fluoride collection.The subjects of this study were firefighters, police officers, volunteer firefighters, and special warfare reserved forces who were admitted to the general hospital in Gumi City for evaluation of the health impact of the hydrogen fluoride spill after performing hydrogen fluoride collection after the accident that happened on September 27, 2012, in the chemical plant in Gumi City. The total number of personnel who visited hospital in the period between October 1 and October 19, 2012, was 348 people, of whom 245 (70.4%) were firefighters, 46 (13.2%) were police officers, 34 (9.8%) were volunteer firefighters, and 23 (6.6%) were special warfare reserved forces.A preliminary self-administrated survey and medical examination by interview were performed to determine the work history such as distance from the accident site, working time, and use of protective devices, as well as symptoms that appeared after performing duties at the accident site, physical examination, and personal history. When completing the survey, prior consent was obtained using a consent form for the collection and use of personal information. During medical examination by interview with the permission of the patient, serum calcium and serum phosphate analyses, pulmonary function test, chest radiography, and echocardiography were performed. To determine the individual level of exposure to hydrogen fluoride, the distance from the accident site was classified into less than 100 m, from 100 m to 1 km, and more than 1 km . The worA chi-square test for trend was performed on the general sociodemographic characteristics, symptoms according to body part, and complaints about separate symptoms related to the distance from the accident site, working time, and the use of respiratory protective devices. To compare the results of the clinical tests with the distance to the accident site, an analysis of covariance (ANCOVA) was used, compensating for age, sex, smoking, hospitalization duration, working time, and use of protective devices. The SPSS for Windows version 14.0  was used in the statistical analysis.The subjects were divided into those who stayed closer than 100 m, between 100 m and 1 km, and farther than 1 km from the accident site, and their characteristics were compared. Among the various age groups, 58.3% and 51.8% of those younger than 30 years and those 30–39 years old, respectively, worked closer than 100 m to the spill side, and 56.8% and 56.3% of those aged 40–49 years and those older than 50 years, respectively, worked between 100 m and 1 km from the site. With regard to sex, 42.1% of the men and 10.0% of the women worked closer than 100 m to the site; therefore, the men were more likely to work closer to the spill site than women. Of the firefighters, 47.3% worked closer than 100 m to the accident site, whereas most  of the police officers, volunteer firefighters, and special warfare reserved forces worked within 100 m to 1 km from the site. The results of the medical examination by interview revealed that the subjects had abnormal eye symptoms in 30% of the cases if they worked within 100 m from the accident site, in 17.3% of cases if they worked between 100 m and 1 km, and in 20.0% of the cases if they worked farther than 1 km from the spill site. Therefore, the subjects who worked the closest to the spill site had the most number of symptoms related to the eyes (p < 0.05). The other symptoms had no statistical differences depending on the distance from the accident site . No statistically significant differences were observed for other body parts . Of the police officers and special warfare reserved forces, 47.8% and 87.0%, respectively, used a disposable or cotton mask. Meanwhile, 61.8% of the special warfare forces used no respiratory protective device (p < 0.05). No other statistically significant differences were found in the symptoms by body part and the use of respiratory protective devices . Other symptoms had no statistically significant differences depending on distance from site . Other symptoms had no statistically significant difference depending on working time  who did not use any protective device; however, it was not observed in those who used a cotton mask, a dust respirator, or a SCBA. Therefore, the proportion of the subjects who did not use a protective device was higher than that of the subjects who used protection (p < 0.05). Other symptoms did not have any statistically significant difference in terms of the use of respiratory protective devices (table not provided).Serum calcium level, serum phosphorus concentration, and pulmonary function test results were analyzed according to the distance from the accident site. The mean serum calcium level was 9.41 mg/dL . None of the subjects were calcium deficient, that is, a calcium level lower than 8.4 mg/dL. The results of pulmonary function test revealed that forced vital capacity (FVC) and FVC (%) were respectively 4.50 ± 0.67 L and 90.57 ± 10.01% for less than 100 m, 4.36 ± 0.75 L and 90.68 ± 11.35% for 100 m to 1 km, and 4.59 ± 0.86 L and 95.66 ± 11.43% for more than 1 km from the accident site, the latter being the highest values (p < 0.05). No other statistically significant difference was found based on the pulmonary function test results  and chest radiography , no statistically significant differences were found as well depending on the distance to the accident site, working time, and the use of protective devices (table not provided).When performing their duties, firefighters may be exposed to various chemical substances that are the result of combustion. In particular, in case of accidents at industrial sites, chemical substances that are produced and used at the plant may have direct and indirect effects on the body in addition to the combustion gas that is released during the fire ,22. The Hydrogen fluoride causes very strong irritations, including tears, throat tingling, nose irritation, cough, and headache ,3,7,21. The subjects who performed work in the accident site for more than 10 hours exhibited symptoms in the lungs, including sputum. When high-concentration hydrogen fluoride is inhaled, it may immediately cause upper airway irritation, such as throat tingling and cough, and lower airway irritation, such as chest pain, dyspnea, sputum, pneumonia, and pulmonary edema. However, when low-concentration hydrogen fluoride is inhaled, it also first causes upper airway irritation and lower airway irritation may naturally follow ,21. In tFirefighters use SCBA at fire-extinguishing and rescue sites to protect their respiratory organs. This equipment supplies the members of the fire brigade with oxygen and prevents them from inhaling smoke and combustion gas; it is approximately 200 times more effective for protecting the respiratory organs than a dust respirator that has an appropriate purification cartridge ,26,27. TAccording to the pulmonary function test results in this study, the personnel who worked in close proximity to the accident site had significantly decreased FVC. According to one experimental study, when healthy male workers were exposed to low hydrogen fluoride levels while performing professional duties, exposure time increased with the decrease in FVC, consistent with the result of our study. Decreased FVC may lead to air trapping due to slight obstruction of the small airways in the lungs . However+ ATPase pump cells, which may result in cell death and cellular energy failure [If hydrogen fluoride is absorbed in the body, blood fluoride increases and bonds with body calcium and magnesium-forming insoluble salts, and may cause hypocalcemia, hypomagnesemia, and hyperkalemia ,16,32. H failure . HoweverIn this study, it was impossible to use equipment for testing hydrogen fluoride levels; therefore, hydrogen fluoride levels in the air or urine fluoride levels, the biological exposure marker, were not measured. Instead, markers that indirectly reflected exposure level, such as distance to the accident site , the timThe duties of a firefighter are characterized with the possibility of acquiring posttraumatic stress disorder (PTSD) due to exposure to fire in sites of disaster, death, inhalation of harmful gases, and exposure to infectious diseases. Secondary consequences of the firefighter’s work are decreased job performance, depression, alcoholism-related problems, and other mental health problems ,34. The This study investigated and analyzed the clinical characteristics and working history of firefighters who performed collection work at the accident site of the hydrogen fluoride spill that occurred on September 27, 2012, in a chemical plant in the National Industrial Complex in Gumi City. When the workers who performed collection duties at the accident site worked in close proximity to the accident site, performed the work for a long time, or had no respiratory protection, they experienced upper and lower airway, gastrointestinal tract, and neurological symptoms. It is necessary to continue follow-up observation among these workers to evaluate for stress-related diseases and physical symptoms.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.The authors declare that they have no competing interests.All authors had access to the data and played a role in writing this manuscript. SYC conceived and designed the study. SYY and JSK were involved in writing the manuscript. JYN and JHY performed the data collection. SYC and KHW performed the statistical analysis, the interpretation of data. YBK had critically revised the manuscript. All authors read and approved the final manuscript.


Axons form many presynaptic terminals, which are dynamic structures sharing presynaptic material along the axonal shaft. We discuss how internal  and external factors  may affect the formation and plasticity of inhibitory synapses.Synaptic connections in our brains change continuously and throughout our lifetime. Despite ongoing synaptic changes, a healthy balance between excitation and inhibition is maintained by various forms of homeostatic and activity-dependent adaptations, ensuring stable functioning of neuronal networks. In this review we summarize experimental evidence for activity-dependent changes occurring in inhibitory axons, in cultures as well as  Our brain is a complex organ with tremendous self-organizing abilities. Its computational power is based in the adjustable synaptic connections between neurons. When new experiences and memories are established, specific synapses in specific brain regions are changed, both in strength and in number. To ensure proper global functioning despite changes in local connectivity, these synaptic changes must be coordinated within neurons, as well as within neuronal circuits. An important aspect is the coordination between changes in excitatory and inhibitory synapses to regulate and maintain an overall balance between excitation and inhibition. When this balance is disturbed, neurological diseases such as autism or schizophrenia can develop .Homeostatic plasticity is a term that is used for plasticity mechanisms which ensure that overall neuronal spiking activity is maintained within neuronal networks. Many forms of homeostatic plasticity have been described in excitatory and inhibitory neurons . In neurin vitro  and in vivo studies.When prolonged changes occur in network activity, homeostatic mechanisms come into play which adjust excitatory and inhibitory synapses to compensate and restore the activity level in the network . GeneralActivity manipulations in cultures of dissociated hippocampal or neocortical neurons generally affect excitatory and inhibitory synapses in opposite directions. After a prolonged period of activity blockade, excitatory synapses get strengthened and inhibitory synapses are weakened, and synaptic changes are in opposite directions when activity is enhanced . Therefoin vitro homeostatic rules for scaling inhibitory synapses get complicated in more complex networks. In addition, other factors such as different cell (glia) types or the extracellular environment in more intact tissue potentially influence homeostatic plasticity compared to dissociated cultured cells.In contrast to dissociated cultures neurons in more intact tissue, such as acute slices or organotypic cultures, make more specific connections and form structured networks. This network configuration makes the interpretation of synaptic changes more complex. In slices that were submitted to activity manipulations, changes in inhibition have been observed opposite to  as well in vivo, sensory deprivation is used as experimental paradigm to lower activity levels in the primary sensory cortex . While in vitro activity manipulations by pharmacological means affect the activity of all neurons in equal amounts, sensory deprivation in vivo will affect different neurons in the circuitry differentially. Therefore, in vivo responses of inhibitory synapses to changes in activity vary widely and strongly depend on the specific cell types, cortical layer, and specific circuitry  along its shaft and contacts many different postsynaptic neurons. Presynaptic boutons along an axon show a large variety in their volumes, in the number of synaptic vesicles and in the presence or absence of mitochondria . It is nThe exchange of presynaptic proteins means that the exact composition of presynaptic terminals is continuously changing. These changes can occur in a correlated fashion with the postsynaptic site in some synapses, but can be uncoordinated in others . ReleaseSynaptic vesicles are kept at the presynaptic terminal by interacting with a scaffolding meshwork of actin, β-catenin, synapsin, and many other proteins . SynaptiLive imaging of axons have shown that transient and mobile release sites exist  and thatSynapse assembly is a complicated process involving interactions of multiple proteins. It does not necessarily need to be a linear process, where one component necessarily recruits the next, but some of the interactions could occur in parallel and the sequence of protein recruitment may vary. Rapid self-assembly of presynaptic components may be an important element during synaptogenesis. This would mean that the formation of a presynaptic terminal merely needs an initial trigger to ascertain a specific axonal location or postsynaptic partner, but then the new presynaptic terminal ``unfolds'' automatically by spontaneous clustering of its components. It is likely that multiple triggers can induce self-assembly. Indeed, it was recently reported that synaptic material is actively prevented from aggregating and assembling new synapses during transport , supportFigure 1). For instance, enhancement of synaptic strength by increasing vesicle capture or anchoring at presynaptic terminals would also result in lower amounts of ``free'' vesicles in the axonal shaft thereby reducing the chance that new synapses are formed at nascent sites . This illustrates that presynaptic plasticity and synapse formation should be considered mutually dependent processes when neighboring presynaptic terminals are sharing synaptic proteins and vesicles.A dynamic control of the strength and number of presynaptic terminals in axons implies that control of transport, capture, and release of synaptic material are essential processes regulating the formation, maintenance, and strength of presynaptic terminals. In a dynamic axon with competing presynaptic terminals, a general change in synaptic strength is expected to also have an effect on ongoing synapse formation within the same axon and vice versa  and we summarize current knowledge of cell adhesion molecules that are specific for inhibitory synapses.Cell adhesion molecules are transmembrane proteins, which play a role in recognition of synaptic partners during the initial contact and provide specificity of synaptic connections . In addiFigure 2A). Indeed, for a number of cell adhesion molecules, activity-dependent changes in expression level have been observed .To have their effect specifically at synapses, cell adhesion molecules should be enriched at synaptic membranes. There is experimental evidence that the distribution of cell adhesion molecules over the cellular surface can be regulated . For instivation . FurtherFigure 2C).Synaptic adhesion molecules execute their function by binding to a trans-synaptic partner at their extracellular domain. In some cases, the extracellular domain can be cleaved, with strong effects on local synaptic adhesion. For instance, activity-dependent cleavage of agrin was shown to mediate the formation of dendritic filopodia  and cleaFigure 2D).For some cell adhesion molecules activity can regulate the binding properties of the proteins. For instance, interactions between cadherins are affected by extracellular calcium concentrations  and inteUpon binding to other cell adhesion molecules, cell adhesion molecules cluster at the cell membrane and signal through interactions with many intracellular proteins, whose levels may be regulated in an activity-dependent manner. Ultimately, signaling through synaptic adhesion molecules in the presynaptic terminal result in direct or indirect alterations of the actin cytoskeleton and vesicle recycling, affecting the number, function, and/or stability of synapses . It willThe list of known synaptic adhesion molecules is rapidly growing, but our knowledge on the precise function of most of these proteins remains incomplete. Interestingly, several synaptic cell-adhesion molecules have been reported to be specifically involved in inhibitory, and not excitatory, synapses. These include sema4D , slitrk3Postsynaptic neuroligins and their presynaptic partners neurexins are transmembrane cell adhesion molecules that have been established as important synaptic regulators . When exLeucine-rich repeat (LRR) proteins have received considerable research attention recently. The members of the subfamily of Slitrk (Slit and Trk-like) proteins are involved in synapse formation and has been linked to several neurological disorders . Slitrk3Members of the closely related subfamily of leucine-rich transmembrane proteins (LRRTMs) have also been implicated in excitatory synapse formation and plasticity , but so in vitro model for epilepsy (Semaphorins are well-known as (repulsive) axon guidance molecules acting through rearrangements of the cytoskeleton in the growth cone. They play an important role in the early development of the brain . Some seepilepsy . The effepilepsy . It was epilepsy , but theepilepsy . It was epilepsy .There are many other cell adhesion molecule proteins and with continued research on inhibitory synapses, it is expected that more of them will be found to play a role at inhibitory synapses. Here we just mention a few that have been reported at inhibitory synapses.ephrin family, ephrinA5 and EphA3, can affect inhibitory synapses and they require NCAM for their action  in an experience-dependent manner and developmental downregulation of NCAM-PSA was shown to coordinate maturation of perisomatic inhibitory synapses in the visual cortex  has been reported to be important for the maturation of perisomatic inhibitory synapses in the cortex . NCAM acr action . In vivol cortex .Several components of the dystrophin-associated glycoprotein complex (DGC), such as dystroglycan, dystrophin, and dystrobrevin, are also specifically located at a subset of inhibitory synapses , but theIntegrins are receptors for extracellular matrix proteins, soluble factors, and counter-receptors on adjacent cells and they have an intracellular link to actin filaments via adaptor proteins . IntegriFinally, the cell adhesion molecule neurofascin has been shown to regulate the formation of a specific subset of inhibitory synapses on the axon initial segment of principal neurons .Above we have described how cell adhesion molecules may provide signals to inhibitory axons from direct cell–cell contacts. However, inhibitory synapses may also be affected by signals from more distal origin. Nearby dendrites or surrounding cells can secrete trophic (or anti-trophic) factors, which may affect inhibitory synapse function and/or formation. Indeed, retrograde signals from the postsynaptic dendrite, such as endocannabinoids, nitric oxide (NO) or brain-derived neurotrophic factor (BDNF), or glutamate spillover from nearby excitatory synapses are known to regulate synaptic release at inhibitory synapses during many forms of short-term and long-term plasticity . Here weBrain-derived neurotrophic factor (BDNF) is a secreted neurotrophin that has been shown in many different preparations to promote the formation and maturation of inhibitory synapses by presynaptic modifications . Only exA receptors  in neuregulin1 have been linked to schizophrenia . The maieceptors . This suFibroblast growth factors (FGFs) are secreted signaling glycoproteins, which exert their effect by binding to FGF receptor tyrosine kinases (FGFR). In the brain, FGF signaling is important for several developmental processes, including patterning of different brain structures and neurogenesis . In addiStudies with neuronal and astrocyte co-cultures and astrocyte-conditioned medium have shown that astrocyte-released factors are crucial for synapse formation and plasticity . For insA and GABAB receptors have been implicated in mediating this regulation . In this review we have painted a picture of the inhibitory axon as a dynamic structure that can quickly adjust to a changing environment, by responding to local signals from postsynaptic cells via adhesion molecules and to global signals from the local neuronal network. A highly dynamic inhibitory system might serve to quickly respond to changes to allow circuit rearrangements by excitatory connections. For a healthy brain changes at inhibitory and excitatory synapses need to be well-coordinated at all times as subtle defects in this coordination can cause defects in circuitry and may underlie psychiatric disorders. This means that the interplay between plasticity of excitatory and inhibitory synapses is an important factor for the stability of neuronal circuits. The precise response of the inhibitory axon will be determined by the combination of internal and external factors, such as the availability of synaptic proteins within the axon, or the combination of the extracellular factors and cell adhesion molecules that are present at the membrane. It will be an important challenge for future research to unravel the precise molecular and cellular mechanisms and to further uncover pathways that affect synapse formation and plasticity of inhibitory synapses.Research on activity-dependent adaptations in inhibitory axons continues to generate novel insight in the cellular processes of synapse formation and plasticity. Many open questions remain to be answered in the future and we listed a few of these in a small scheme (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.


Emerging strategies that center upon the mammalian target of rapamycin (mTOR) signaling for neurodegenerative disorders may bring effective treatment for a number of difficult disease entities. Here we show that erythropoietin (EPO), a novel agent for nervous system disorders, prevents apoptotic SH-SY5Y cell injury in an oxidative stress model of oxygen-glucose deprivation through phosphatidylinositol-3-kinase (PI 3-K)/protein kinase B (Akt) dependent activation of mTOR signaling and phosphorylation of the downstream pathways of p70 ribosomal S6 kinase (p70S6K), eukaryotic initiation factor 4E-binding protein 1 (4EBP1), and proline rich Akt substrate 40 kDa (PRAS40). PRAS40 is an important regulatory component either alone or in conjunction with EPO signal transduction that can determine cell survival through apoptotic caspase 3 activation. EPO and the PI 3-K/Akt pathways control cell survival and mTOR activity through the inhibitory post-translational phosphorylation of PRAS40 that leads to subcellular binding of PRAS40 to the cytoplasmic docking protein 14-3-3. However, modulation and phosphorylation of PRAS40 is independent of other protective pathways of EPO that involve extracellular signal related kinase (ERK 1/2) and signal transducer and activator of transcription (STAT5). Our studies highlight EPO and PRAS40 signaling in the mTOR pathway as potential therapeutic strategies for development against degenerative disorders that lead to cell demise. Neurodegenerative disease leads to either severe disability or death for a significant proportion of the world's population. For example, in regards to cognitive disease, it is estimated that greater than twenty-four million people are afflicted with Alzheimer's disease, pre-senile dementia, and associated disease that involve memory loss Given that effective treatments for the majority of neurodegenerative disorders do not exist, new strategies that can offer protection in the nervous system during oxidative stress are of great interest In a number of scenarios, mTOR activation may be necessary to prevent apoptotic neuronal cell death during oxidative stress. Cell death following exposure to oxidative stress in dopaminergic neurons can be prevented during application of agents that increase mTOR activity 2. Cell suspension was prepared at a density of 3–4×104 (24 well plate) or 1–1.5×105 (35 mm2 Petri dish). When confluent at 50–60%, cells were differentiated by MEM growth medium containing 10 μM all-trans retinoic acid (RA)  for 48 h. Experiments were initiated until cells grew to 60% – 70% confluence between passages 4–10 after differentiation.Per our prior protocols 2 dishes with cells of 60–70% confluence with glucose-free Hank's balanced salt solution (HBSS) containing 116 mmol/l NaCl, 5.4 mmol/l KCl, 0.8 mmol/l MgSO4, 1 mmol/l NaH2PO4, 0.9 mmol/l CaCl2, and 10 mg/l phenol red (pH 7.4). SH-SY5Y cultures were then placed into a Bactron II anaerobic glove box  and were maintained in an anoxic environment (95% N2 and 5% CO2) at 37°C for 6 hours. Following this period, the cultures were removed from the anoxic chamber and the glucose-free HBSS was replaced with media containing Dulbecco's modified Eagle medium (DMEM) , supplemented with 10% heat-inactivated fetal bovine serum, 1 mM pyruvate, 1.5 g/L sodium bicarbonate, 100 IU/ml penicillin, and 100 µg/ml streptomycin and maintained at 37°C in 95%/5% (v/v) mixture of humidified atmospheric air and CO2. For treatments applied prior to OGD, erythropoietin , the phosphatidylinositol-3-kinase (PI 3-K) inhibitors wortmannin  and LY294002 , extracellular signal-regulated kinase (ERK) inhibitor 3-(2-aminoethyl)-5-((4-ethoxyphenyl) methylene)-2,4-thiazolidinedione (ERK-1) (100 μM) , HCl (ERK-I) , and signal transducer and activator of transcription- 5 (STAT5) inhibitor N′-((4-Oxo-4H-chromen-3-yl)methylene) nicotinohydrazide (STAT5-I) (100 μM)  were each administered directly to the cultures 1 hour prior to OGD and treatments were continuous.Per our prior experimental protocols, oxygen-glucose deprivation (OGD) in SH-SY5Y cells was performed by replacing the media of the cultures in 35 mm2 dishes were used, and for each dish, the mean survival was determined by counting eight randomly selected non-overlapping fields with each containing approximately 20 cells (viable + non-viable). Each experiment was replicated 6 times with different cultures.SH-SY5Y cell injury was determined by bright field microscopy using a 0.4% trypan blue dye exclusion method 24 hours following treatment with OGD per our previous protocols Genomic DNA fragmentation was determined by the terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay 2448, 1∶1000), mTOR (1∶1000), p-p70S6K , p70S6K (1∶1000), p-Akt1 , Akt1 (1∶1000), p-STAT5 , STAT5 (1∶1000), p-4EBP1 , 4EBP1 (1∶1000), PRAS40 (1∶1000), p-PRAS40 , p-ERK 1/2 (Thr202/Tyr204), and cleaved caspase 3 (1∶1000). All antibodies were obtained from Cell Signaling, Beverly, MA. Following incubation, the membranes were incubated with a horseradish peroxidase (HRP) conjugated secondary antibody goat anti-rabbit IgG . The antibody-reactive bands were revealed by chemiluminescence  and band density was performed using the public domain NIH Image program .Cells were homogenized and following protein determination, each sample (25–50 μg/lane) was then subjected to 7.5%  or 12.5%  SDS-polyacrylamide gel electrophoresis separation. After blocking for 1 hour at room temperature with 5% skim milk, the membranes were incubated overnight at 4°C with a rabbit antibody against (p-  =  phosphorylated) p-mTOR (Ser 2448) or p-PRAS40 (Thr246) .Recombinant human mTOR  or PRAS40 protein  1 μg was incubated with 10 ng/ml EPO for 30 min at 30°C under continuous agitation in 30 μl kinase buffer containing 200 μM ATP . Samples were analyzed by Western blot analysis using SDS-polyacrylamide gel and rabbit antibody against p-mTOR  and siRNA pool for PRAS40  were used respectively. Transfection of siRNA duplexes was performed with Lipofectamine 2000 reagent according to manufacturer guidelines . Experimental assays were performed 72 hours post-transfection. For each siRNA assay, scrambled siRNA was used as control.Cells were plated into 35 mm dishes or 24-well plates. To silence Cell lysates of total protein (200 μg) were incubated with primary antibody against protein 14-3-3  or mTOR  overnight at 4°C. The complexes were collected with protein A/G-agarose beads, centrifuged, and then prepared for 14-3-3, mTOR, PRAS40, and p-PRAS40 western analysis.For each experiment, the mean and standard deviation (SD) was determined. Statistical differences among groups were assessed by means of analysis of variance (ANOVA) with the post-hoc Dunnett's test. Statistical significance was considered at P<0.05.EPO (10 ng/ml) was administered to cell cultures 1 hour prior to a 6 hour period of OGD and cell injury was determined 24 hours later through trypan blue dye exclusion method and TUNEL assay. This concentration of EPO was chosen since it previously was shown to provide significant cytoprotection in neuronal cells and vascular cells Application of rapamycin (50 nM) or transfection with mTOR siRNA during EPO (10 ng/ml) exposure increased cell injury and DNA fragmentation following OGD when compared to OGD exposure alone . Protect24482448 is an important target for both Akt and p70S6K 389 that serves as a marker of mTOR activity 65 and threonine70Given that inhibition of mTOR can block cytoprotection by EPO during OGD, we investigated whether EPO can activate mTOR and control the activity of its downstream targets p70S6K and 4EBP1. The carboxy-terminal  kinase domain of mTOR consists of a conserved sequence with homology to the catalytic domain of phosphoinositide 3 –kinase (PI 3-K) with phosphorylation sites of mTOR for its activation 2448, active form) and phosphorylated forms of its downstream targets p-p70S6K and p-4EBP1  during OGD exposure. In We assessed the expression of phosphorylated mTOR  given 1 hour prior to OGD also significantly increased the expression of phosphorylated p-mTOR, p-p70S6K, and p-4EBP1 3 hours following OGD exposure, illustrating that EPO activates mTOR and p70S6K through phosphorylation but inhibits 4EBP1 activation through phosphorylation. The phosphorylation of mTOR, p70S6K, and 4EBP1 during either exposure to OGD alone or exposure to EPO and OGD were prevented during rapamycin administration or the transfection with mTOR siRNA . During Under some conditions, pathways for cellular proliferation and survival require the involvement of mTOR and phosphoinositide 3 –kinase (PI 3-K)/Akt pathways PRAS40 during EPO (10 ng/ml) administration and OGD exposure improved cell survival and limited genomic DNA degradation to a greater extent than EPO alone  was performed at 1, 3, and 24 hours following OGD exposure. In Western blot analysis for phosphorylated p-PRAS40 (ThrWe next examined the role of the PI 3-K pathway for EPO to phosphorylate PRAS40. EPO (10 ng/ml) was administered without  and withAkt1 on PRAS40 phosphorylation during EPO administration alone and during EPO application with OGD exposure. In Akt1 eliminated the ability of EPO to phosphorylate PRAS40 without OGD exposure and during OGD exposure, illustrating that EPO also relies upon Akt1 to phosphorylate PRAS40  and phosphorylated p-STAT5 (Tyr694) (activate forms of ERK ½ and STAT5) were performed at 1, 3, and 24 hors following OGD exposure. As shown in Prior studies have demonstrated that EPO can activate extracellular signal related kinase (ERK 1/2) Neurodegenerative disease can lead to disability in multiple systems of the body mTOR during EPO application significantly prevented cellular protection by EPO, illustrating that EPO relies upon the activation of mTOR to protect neurons against oxidative stress.We show that treatment with EPO in SH-SY5Y cells prevents cellular injury and apoptotic DNA degradation during exposure to OGD. This cellular protection by EPO is dependent upon the activity of mTOR and its signaling pathways. Prior studies have shown that oxidative stress can block the activity of mTOR signaling pathways to alter cell metabolism and longevity L expression to block BAD activity that can result in apoptosis Consistent with prior studies that have demonstrated that EPO increases mTOR signaling in inflammatory cells We also show that phosphorylation of mTOR, p70S6K, and 4EBP1 by EPO are dependent upon the PI 3-K/Akt pathways. Our studies demonstrate that EPO cannot directly phosphorylate mTOR, but requires the PI 3-K/Akt pathways to activate mTOR and phosphorylate p70S6K, and 4EBP1. Prior work has shown that cellular growth and protection can require the involvement of mTOR and the PI 3-K/Akt pathways PRAS40 prevents both cellular injury and neuronal apoptosis during oxidative stress. Furthermore, gene silencing of PRAS40 during EPO administration and OGD exposure enhanced cell survival and further reduced genomic DNA degradation to a greater extent than EPO alone, suggesting that EPO relies upon PRAS40 inhibition for cellular protection. This increased degree of protection by EPO during the loss of PRAS40 appears to be tied to the promotion of mTOR signaling for EPO, since gene silencing of PRAS40 increased the phosphorylation of p-mTOR, p-p70S6K, and p-4EBP1 during EPO treatment. EPO also maintained the phosphorylation and inhibition of PRAS40 over a 24 hour course following OGD exposure to a significantly greater extent that during exposure to OGD alone. In other cell systems, phosphorylation of PRAS40 has resulted in decreased apoptotic cell death Given that mTOR signaling through p70S6K and 4EBP1 forms an important component for EPO neuronal protection during oxidative stress, we examined the role of PRAS40 that blocks mTOR activity and prevents the binding of p70S6K and 4EBP1 to Raptor in vitro incubation of EPO with PRAS40 does not lead to phosphorylation of PRAS40. In contrast, co-administration of the PI 3-K inhibitors wortmannin or LY294002 or gene silencing of Akt1 eliminated the ability of EPO to phosphorylate PRAS40, suggesting that EPO was dependent upon the PI 3-K/Akt pathways to phosphorylate PRAS40. Akt can phosphorylate threonine246 on PRAS40. This results in the dissociation of PRAS40 from mTORC1 EPO controls PRAS40 activity through the post-translational phosphorylation of PRAS40 and the subcellular binding of PRAS40 to protein 14-3-3. However, EPO does not appear to directly control the post-translational phosphorylation of PRAS40, but requires activation of the PI 3-K/Akt pathway similar to the regulation of mTOR by EPO. We show that Control of neuronal apoptosis by EPO involves suppression of caspase 3 activation through PRAS40 but not through ERK 1/2 or STAT5 pathways. Both the early and late phases of apoptotic cell injury can be the result of caspase 3 activation Our studies offer new insight into the neuroprotective pathways for EPO during OGD that can lead to oxidative stress. EPO prevents cell injury through activation of mTOR signaling and is dependent upon the inhibition of PRAS40 as vital regulatory pathway. EPO controls the mTOR signaling pathways of p70S6K, 4EBP1, and PRAS40 through PI 3-K and Akt. PRAS40 either alone or as a component of EPO signal transduction governs cell survival through caspase 3 activation that is independent of other cytoprotective pathways of EPO that involve ERK 1/2 and STAT5. Future studies that can further elucidate the role of PRAS40 and the ability of EPO to regulate this pathway may offer novel approaches for the treatment of a variety of multi-system disorders, such as those that involve neurodegeneration.


Changes at the grounding line of ice streams have consequences for inland ice dynamics and hence sea level. Despite substantial evidence documenting upstream propagation of frontal change, the mechanisms by which these changes are transmitted inland are not well understood. In this vein, the frequency response of an idealized ice stream to periodic forcing in the downstream strain rate is examined for basally and laterally resisted ice streams using a one-dimensional, linearized membrane stress approximation. This reveals two distinct behavioural branches, which we find to correspond to different mechanisms of upstream velocity and thickness propagation, depending on the forcing frequency. At low frequencies , slope and thickness covary hundreds of kilometres inland, and the shallow-ice approximation is sufficient to explain upstream propagation, which occurs through changes in grounding-line flow and geometry. At high frequencies , penetration distances are tens of kilometres; while velocity adjusts rapidly to such forcing, thickness varies little and upstream propagation occurs through the direct transmission of membrane stresses. Propagation properties vary significantly between 29 Antarctic ice streams considered. A square-wave function in frontal stress is explored by summing frequency solutions, simulating some aspects of the dynamical response to sudden ice-shelf change. Nevertheless, it remains to be conclusively demonstrated that the acceleration of PIG is due The observational data thus highlight that upstream propagation of forcing at the ice front can occur through two mechanisms. One is the direct transmission of membrane stresses, acting along the body of the ice stream in the horizontal plane. The other process is through increased flow at the grounding line inducing changes in the geometry of the ice stream, notably steepening, that lead to increases in the gravitational driving stress and velocity. The aim of this paper was to understand the conditions under which both of the two mechanisms operate, and whether a clear distinction can be made between the two.frequency response means quantification of the relationship between spatial scales of response and frequency of forcing. A simple analogy is the way that temperature forcing propagates into a solid, e.g. snow. A forcing with a particular period induces a typical decay length and wavelength in the temperature field. Our fundamental objective is to determine when membrane stresses need to be incorporated into ice-stream models to accurately reflect observations, and how the time scale of the forcing at the ice front affects upstream propagation of velocity and thickness changes.Our approach is inspired by Shallow-ice models respond to forcing through geometric coupling, and are a well-researched topic in ice dynamics . These sIn this study, we use a simplified, vertically integrated, one-dimensional flow-line model of a basally or laterally resisted ice stream, described in §2. This model includes membrane stresses based on  and we uUsing model parameters from 29 Antarctic ice streams, we characterize the upstream propagation for a range of different forcing frequencies (§4). For each ice stream, we find two distinct types of behaviour dependent on the forcing frequency. Many ice streams have laterally resisted sections abutting basally resisted sections. In §5, a solution is presented for an ice stream that changes from lateral to basal resistance, broadening this methodology by allowing stacking of differently resisted ice-stream portions to better represent real ice-stream conditions. Our linearized model formulation allows the summation of different frequency forcings to create arbitrary forcings close to the ice front. This is demonstrated in §6 through the construction of a square-wave function for the strain rate just upstream of the grounding line, which may provide an approximation of the effects of ice-shelf thinning and thickening at the grounding line on the inland velocity and thickness. The work concludes with a discussion in §7.2.X*], such that −[X*]<x*<0 represents the horizontal position. Thickness is given by H*=s*−b*(x*), where s* represents the ice surface and b*(x*) the ice base. Time is denoted t*. The forcing is prescribed at x*=0, which is considered to be a small distance upstream of the grounding line. Grounding-line movement is not modelled explicitly, but we allow thickness changes at x*=0 to implicitly describe such motion. Hence, our focus in this study was on propagation and not on instability. Dimensional quantities are denoted with an asterisk (*) and non-dimensional quantities without.The physical basis of our model is similar to that of B* is the ice-stiffness parameter, u* is ice velocity, ρ* is the density of ice and g* is the gravitational acceleration. Here Bn*−=A*v, where A*v is the rate factor in the viscous relationship e*=A*vτn* relating the strain rate e* to the deviator stress invariant τ*. Depth-averaged viscosity for the one-dimensional case was used to obtain the force balance equation A* is a coefficient representing either the rate of sliding or the rate of internal deformation, in which case A*=2A*v/(n+2). n is a rheological index and m is a constant which is either n+2  or n+1 (for sliding according to a Weertman-type law). The problem is scaled usingε is defined as the aspect ratio of the ice stream and length is scaled with the distance from the divide to the grounding line, [X*]. A* and B* are scaled with [A*] and [B*], respectively. The velocity scale [u*] is prescribed and used to calculate the sliding coefficient [A*]. This gives the non-dimensional momentum equation  and first-order components (denoted with subscript 1) depend upon x and t. In this study, we are predominantly interested in the length scales associated with the upstream propagation of frontal effects occurring near the grounding line. For this reason, we choose the thickness, velocity and strain-rate scales at x=0 and define the zeroth-order solution from these parameters. We assume that the negative surface slope at the grounding line is the aspect ratio of ice thickness to ice-stream length, ε . The scaled sliding factor and ice stiffness are not perturbed and are set as constants, A=B=1 .For a basally resisted ice stream equation  as3.3whb=0 and H1=s1. At first order equations , and kx is a complex spatial wavenumber. This transformation provides the perturbations in terms of frequency responses. Any of the thickness, velocity or strain rate can be chosen as the forcing, with specified amplitude, and the other two perturbation amplitudes are then found by solving the model. We choose to use the strain rate as the leading forcing by setting kx,ω, solving this cubic for kx provides estimates of spatial wavelengthDL is the length upstream at which only e−1 of the perturbation remains, and the speed of upstream propagation of the resulting perturbation in velocity or thickness (phase speed) is given byConsider an ice stream such that quations , 3.5) a ab=0 andquations  simplify(b)B*=0 and kx as a function of ω,Ω tends to zero. Thus, the SIA is the low Ω limit of the MSA, corresponding to very weak coupling in the membrane term.equation  to giveequation  along wiequation  for the (c)kx=Re(kx)+i Im(kx). Analysing the balance of the terms for ω≫1 gives equations for the real and imaginary parts of the equation separately asDL for the MSA and SIA (for which Γ=0: see equation (L* is defined as the membrane coupling length (MCL) from DL tends to zero for high frequencies. For the MSA in the high-frequency limit, the wave speed diverges  as3.23equation , where Lequation ).DL is the maximum decay length in this case. The corresponding limits of the wave speed (equation (kx) as In the low-frequency limit, the spatial wavenumber and decay number can be calculated in the same manner as for the high-frequency limit  in the Equation  indicate(d)W* can be solved approximately by using the flow law and writingy*=W*, the lateral margin, and the velocity is v*. Integrating the second of these equations over the semi-width of the ice stream and setting v*=0 at y*=W* gives the centre-line velocity u*(x*)=v* asA* is the viscous rate coefficient given by A*=Bn*−, and the traction can be written asW* in equation . This gives the same phasing relationship . The deequation  and the equation  and scaltionship  and the tionship  as for t4.kx(ω). Perturbations decay towards the ice divide only for Im(kx)≤0. In the case of equation . The Glen index n=3 is used, and for the basally resisted case m=4 . We set B*=106 Pa yr1/3, corresponding to a temperature of around −30°C. The dimensionless viscosity parameter Ω is small for all streams considered: 0.0066≤Ω≤0.037. An example of the relation between forcing frequency and wavenumber, in terms of decay number Im(kx) and spatial wavenumber Re(kx), is shown in t*]=162 years, and the period Tp=2π/ω is varied between approximately half a day  and 16 000 years .We solve for the complex wavenumber for 29 Antarctic ice streams using drainage basin area and grounding-line velocity and thickness data taken from ω. It is best to view ω as the independent variable, and then plot the relationship between the decay number and wavenumber. The main axes are dimensionless but a dimensional scale for the decay length  and for the colour scale in terms of the dimensional forcing period kx)=−1.38) and a fast, high-frequency forcing with a shorter minimum decay length of D*L=61.9 km (Im(kx)=−6.54). The spatial wavenumber increases with frequency on the upper branch, and decreases as the frequency becomes very high on the lower branch. This dual branch behaviour is a generic feature of the solution, and is seen for all Antarctic ice streams considered in this study. Figure 3a shows that the difference between the decay lengths for the slow and fast branches  varies between the ice streams owing to the different geometries and properties of each stream .Figurekx)=Re(kx) ). Complex wavenumbers to the left of this line have wavelength λ greater than decay length DL. It can be seen that for the MSA this can be expressed as λ>DL; in other words, we do not expect to be able to observe the upstream sinusoidal variations except at sufficiently low frequencies, when the spatial wavelength is not significantly greater than the decay length.The black dashed line in V1 is the change in perturbed volume between t=0 and some time t=t1 and the amplitude of this volume change over a full period gives the maximum perturbed volume change at some point t1=tmax, 0≤tmax≤Tp, where Tp is the dimensionless period. However, because the forcing is periodic, V1=0 when t=0 and t1=Tp; thus, there is no net change in perturbed volume over one full period. ω is small), the magnitude of the flux perturbation is small whereas the maximum perturbed ‘volume’ amplitude is large, presumably because the small flux perturbation acts over a long time period for these slow forcings. On the high-frequency branch (when ω is large), the flux perturbation becomes independent of frequency and ω≈2.6 (T*p≈388 years).H/dx) . At Θ=0.25/0.75 variables vary completely out of phase. When the frequency ω is low, figure 6a shows that these changes are in almost perfect anti-phase , leading to the maximum flux at ω≈2.6. For high-frequency forcings, ω≫1, the magnitude of the thickness perturbation becomes very small but the magnitude of the perturbed velocity tends to a limiting value   and the H/dx) , all as equation , thus ).equation ). Thus, ω , both the perturbed drag and the driving stress are larger than the membrane stress perturbation and appear to be approximately in balance. On the branch of high frequencies , the perturbed driving stresses are very small, owing to very little thickness or slope change , and here the perturbed drag and membrane stress terms approximately balance.In shown in , these ae change b,c, and D*L=62 km, Im(kx)=−6.54), the SIA shows monotone behaviour: as ω increases and the forcing becomes rapid, the decay length becomes very small and perturbations decay rapidly as they travel upstream  in T*sp and record in T*sp=32.5 years), but varies widely between different ice streams. For example, for PIG T*sp=15.3 years (ω=0.025) but for the MacAyeal ice stream T*sp=117 years, indicating that a forcing of, for example, 50 years may be on the slow branch for PIG but on the fast branch for the MacAyeal ice stream. Similar results are found for laterally resisted ice streams, with slightly smaller T*sp values . Finally, Ω, and for small Ω (shown as sideways triangles and stars) the propagations are heavily damped. This can be understood by noting that, as Ω limit of the MSA.For very low frequencies, MSA and SIA predictions of decay length are very similar but not identical (see equations  and 3.2 and figuequation ). Additiequation : the SIA5.x=XC, along with the upstream boundary condition that the perturbations tend to zero towards the ice divide . To preserve scales for the matching at x=XC, all parameters and ω on both sides of the divide are kept the same; the only difference between the basal and lateral cases is the value of m . Because m is not involved in the high-temporal-frequency limit , only lAn example of an ice stream with parameters estimated for PIG  is shown6.x=0, just upstream from the grounding line, with amplitude Tp,ω=2π(2i−1)/Tp, i=1,2…,j. The step change in strain rate may represent some aspects of upstream changes caused by sudden ice-shelf loss due to, for example, calving events or ungrounding from a pinning point. An example of this square-wave forcing is shown in T*p=50 years. A step decrease in strain rate leads to a rapid drop in velocity and a slower increase in thickness. The relatively high frequency of the forcing accounts for the small change in thickness and the difference in response speed of thickness and velocity (velocity and thickness are out of phase), as shown in §4 for sinusoidal forcings. Although the periodic square wave is an idealized forcing, the approximate 5–10% thinning and the 50 per cent acceleration of PIG over a period of 25 years shown in Owing to the linear nature of the perturbation model, multiple solutions can be summed to build an arbitrary periodic forcing close to the ice front. As a simple example, we construct a square-wave function  in strai7.We have developed an analytical theory for the upstream propagation of a periodic forcing in the strain rate just upstream of the grounding line. Physically, this change in strain rate could be a response to changes in the back-pressure at the grounding line, perhaps due to ice-shelf thinning or thickening. The fundamental result of this theory is that there are two styles of upstream propagation of velocity perturbations, each associated with different periods of forcings. For low-frequency forcing, the propagation does not depend on membrane stresses while for high-frequency forcing it does.The model was employed to estimate response characteristics for 29 Antarctic ice streams using grounding-line parameters from For all ice streams considered, as the forcing frequency varies, two distinct response styles emerge: a slow, low-frequency branch  with spatial wavenumber increasing with frequency, and a fast, high-frequency branch  with spatial wavenumber decreasing with frequency. On the low-frequency branch, the response can propagate many hundreds of kilometres upstream. On this branch, velocity is approximately in phase with negative slope and in anti-phase with thickness, and drag and driving stress are approximately in balance and both are much larger than the contribution from membrane stresses. Thus, the SIA is sufficient to explain and model the dynamics of the low-frequency branch. The mechanism for propagation on this branch is due to changes in grounding-line flow and geometry rather than direct propagation of membrane stresses. On the high-frequency branch for the MSA, velocity responds very rapidly to forcing, but thickness and driving stress vary little. On this branch, changes in membrane stresses are balanced by changes in drag and the effects are directly propagated tens of kilometres upstream. Thus, we find that there is a clear distinction, based on the frequency of the frontal forcing, between the two mechanisms for upstream propagation. Furthermore, this distinction, in terms of the period at the demarcation between the two branches, varies significantly between ice streams. A surprising feature of the results was the propagation of high-frequency velocity effects beyond the boundary layer lengths proposed by n=3 is used (higher n might explain the magnitude). This highlights the difficulty in attributing any observed changes in thickness or velocity either to a frontal forcing or to spatial anomalies in basal slipperiness or changes at the lateral margins; moreover, it is clear that the frequency of forcing is an important parameter when attempting to make this distinction.Inland acceleration and thinning have been observed in Greenland and the Antarctic and various theories have been proposed to explain the mechanisms behind these changes. Both While our results are presented in terms of upstream propagation from grounding lines, we expect the same pattern to hold for variability generated internally in ice sheets . An example would be penetration of melt to the bed in the ablation zones of thick ice sheets that results in rapid speed-up. This mechanism, long known from Alpine glaciers , was obsAlthough we find that long-distance short-period upstream propagations are not associated with significant changes in thickness, further investigation is required to assess the real-world implications for rapid but non-periodic forcing, and for a nonlinear system. Within a decade, we will have a good forcing and thinning record for most ice streams. Our results clearly imply that each ice stream requires a separate analysis to discriminate between high-frequency and low-frequency forcing, because we find that the upstream response to a forcing is not a simple function of the temporal nature of forcing. The forcing time scale separating the fast and slow branches for each ice stream (Ω limit of the MSA and captures upstream propagation for sufficiently slow forcings, we caution that this does not imply that the SIA is appropriate for modelling ice streams. In particular, it performs poorly at modelling the propagation of decadal and sub-decadal components of the forcing. In addition, we prescribed the strain rate at the grounding-line, but Although the SIA represents a low


Trypanosoma brucei (TbDYRK2), a member of the excavate supergroup and separated from Drosophila by ∼850 million years. Bioinformatics showed the DYRKs clustering into five known subfamilies, class 1, class 2, Yaks, HIPKs and Prp4s. Only class 2 DYRKs were present in all four supergroups. These diverse class 2 DYRKs also exhibited conservation of N-terminal NAPA regions located outside of the kinase domain, and were shown to have an essential role in activation loop autophosphorylation of Drosophila DmDYRK2. Class 2 TbDYRK2 required the activation loop tyrosine conserved in other DYRKs, the NAPA regions were critical for this autophosphorylation event, and the NAPA-regions of Trypanosoma and human DYRK2 complemented autophosphorylation by the kinase domain of DmDYRK2 in trans. Finally, sequential deletion analysis was used to further define the minimal region required for trans-complementation. Our analysis provides strong evidence that class 2 DYRKs were present in the primordial or root eukaryote, and suggest this subgroup may be the oldest, founding member of the DYRK family. The conservation of activation loop autophosphorylation demonstrates that kinase self-activation mechanisms are also primitive.DYRK-family kinases employ an intramolecular mechanism to autophosphorylate a critical tyrosine residue in the activation loop. Once phosphorylated, DYRKs lose tyrosine kinase activity and function as serine/threonine kinases. DYRKs have been characterized in organisms from yeast to human; however, all entities belong to the Unikont supergroup, only one of five eukaryotic supergroups. To assess the evolutionary age and conservation of the DYRK intramolecular kinase-activation mechanism, we surveyed 21 genomes representing four of the five eukaryotic supergroups for the presence of DYRKs. We also analyzed the activation mechanism of the sole DYRK (class 2 DYRK) present in  Phosphorylation of activation loop residues is a wide-spread and important mechanism for regulating the catalytic activity of a protein kinase. Activation loop phosphorylation may be carried out by a separate protein kinase as part of a signaling cascade, or a kinase can autophosphorylate using an intermolecular or intramolecular mechanism Saccharomyces cerevisiae by acting as a functional antagonist to the Ras/PKA and target of rapamycin (TOR) pathways Dictyostelium, the homologous Yak1p regulates a starvation-induced growth arrest initiating a transition from vegetative growth to a developmental phase Drosophila and mice, respectively S. pombe provides positional information for both cell growth and cell division 2-M transition Caenorhabditis elegans, class 2 DYRK, MBK-2, promotes the oocyte-to embryo transition, and modulates asymmetrical cell division and the patterning of the embryonic axis The DYRK family can be further subdivided into five subfamilies: class 1 (or DYRK1), class 2 (or DYRK2), Yaks, HIPKs (homeodomain-interacting protein kinase), and PRP4s (pre-messenger RNA [mRNA]-processing protein 4) S. cerevisiae, Yak1p is translocated to the nucleus in response to TOR signaling or glucose depletion and thereby gains access to relevant substrates C. elegans DYRK family member, MBK-2, is carefully regulated during the oocyte-to-embryo transition through a series of actions involving timely relocalization of the kinase thus providing access to relevant substrates Class 1 and 2 DYRKs are the best-characterized subfamilies, particularly with regard to understanding activation mechanisms or regulatory events controlling the kinase activity of these enzymes. Class 1 and 2 subfamily members share a conserved central kinase domain and an adjacent N-terminal DH-box (DYRK homology box) but have divergent extended N- and C-terminal regions Drosophila class 2 DYRK, DmDYRK2 . Although the NAPA region was located outside of the kinase domain, we demonstrated that it was essential for activation loop phosphorylation but not for the subsequent serine/threonine phosphorylation of substrates All class 1 and 2 DYRKs as well as YAKs characterized to date contain a YxY motif in the activation loop; but only the second tyrosine residue in this motif is phosphorylated, an autocatalytic event that is essential for full serine/threonine kinase activity of the protein Dictyostelium, C. elegans, Drosophila, and mammals . Here we show that, similar to other DYRKs, TbDYRK2 is phosphorylated on the conserved activation loop tyrosine, and phosphorylation of this residue is necessary for full serine/threonine kinase activity. In addition, deletion of the NAPA-1 region or point mutations within the NAPA-2 region eliminated tyrosine autophosphorylation. Finally, we demonstrate that the isolated NAPA-regions of TbDYRK2 and human DYRK2 were able to complement tyrosine autophosphorylation of Drosophila DmDYRK2 in trans providing evidence for the functional conservation of the NAPA-domain across species.In this report, we have taken advantage of the availability of completely sequenced genomes from highly diverse organisms to interrogate the evolutionary conservation and phylogenetic distribution of DYRK family members. We have also compared NAPA region function across evolutionary time, by cloning and analyzing the sole class 2 DYRK encoded by T. Our analysis demonstrates the evolutionary ancient nature of the DYRK family, and provides evidence that class 2 DYRKs were present in the primordial or root eukaryote. Our functional analysis of TbDYRK2 provides further evidence for the conservation of the mechanism of intramolecular phosphorylation of the DYRK activation loop and the potential role of the NAPA domain in this process.Anti-Flag M2-agarose and anti-Flag antibodies were purchased from Sigma . Anti-phosphotyrosine 4G10, was purchased from Millipore . Horseradish peroxidase conjugated with anti-mouse IgG, protein G sepharase, were obtained from GE Healthcare . The QuickChange site-directed mutagenesis kit was from Stratagene . Clean-blot IP detection reagent (HRP) was purchased from Thermo Scientific . The SuperSignal west pico chemiluminescent reagent for immunoblot detection was from Amersham Biosciences Inc. . All PCR and mutagenesis primers were synthesized by Integrated DNA Technologies .T. brucei was amplified from genomic DNA by PCR. The nucleotide sequence and conceptual translation are shown  open reading frame (encoding the first 149 amino acids of the protein). To the 5′-end of the coding sequences, we added an EcoR1 restriction site and consensus Kozak sequences; and in frame FLAG-epitope encoding sequence immediately following the initiating-Met ATG codon. At the 3′-end of the coding sequences, a stop codon followed by a second EcoR1 site was added. The EcoR1-EcoR1 fragment was then cloned into EcoR1 linearized pcDNA3 to generate HsNt-DYRK2.Constructs encoding the various proteins were then subcloned into the baculoviral shuttle vector pVL1393 (BD Biosciences). The various pVL1393 constructs were then used in conjunction with BaculoGold DNA (BD Biosciences) to generate recombinant baculovirus for expression in Sf9 (Spodoptera frugiperda 9) cells as described previously For most immunoprecipitations, Sf9 cell extracts (0.15 mg) were incubated with Protein G-Sepharose conjugated to anti-FLAG (Sigma) or anti-DmDYRK2 antibody for 1 h on a rotating platform at 4°C. Efficient immunoprecipitation of ΔN2-TbDYRK2 required longer incubation times with the anti-FLAG antibody (up to 10 h). Immunoprecipitates were pelleted, washed twice with 1 ml buffer A  and once with 1 ml of ice-cold TBS buffer . Immunoprecipitated proteins were separated by SDS–polyacrylamide gradient gel (4–20% NuPAGE gels) electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes, blocked with 3% bovine serum albumin in 20 mM tris-HCl (pH 7.6), 137 mM NaCl, containing 0.2% Tween-20 (TBS-T), and probed with a monoclonal anti-FLAG antibody (1∶5000 dilution) or anti-phosphotyrosine antibody (1∶2000 dilution). Immunoblots were then incubated with HRP horseradish peroxidase conjugate (1∶1000) dilution, and visualized by SuperSignal west pico chemiluminescent reagent detection according to the protocol of the manufacturer (Amersham Biosciences).2, 1 mM DTT), and incubated with kinase buffer containing ATP and 50 microM Woodtide as described previously For peptide kinase assays, WT and mutant forms of TbDYRK2 were immunoprecipitated in triplicate, washed three times in lysis (1% NP40 buffer) buffer, washed once in kinase buffer . TbDYRK2 is one of the shortest of the class 2 DYRKs containing 108 N-terminal amino acids, a central kinase domain of 300 residues, and a C-terminus of 47 amino acids . We also compared the WT protein to an activation loop mutant (Y269F) where we modified the analogous tyrosine residue shown to be phosphorylated in other DYRKs. To monitor expression of WT and mutant proteins, a Flag-epitope was fused to the N-terminus and the modified proteins were expressed using recombinant baculovirus-infected Sf9 cells. When approximately equal levels of the proteins were compared, WT-TbDYRK2 was found to be tyrosine phosphorylated whereas the kinase-inactive and activation loop mutants displayed little if any tyrosine phosphorylation .Drosophila and mammalian class 2 DYRKs with regard to activation loop tyrosine autophosphorylation and the importance of this phosphorylation event for full kinase activity.Next we compared the ability of the WT and mutant proteins to phosphorylate the DYRK substrate, Woodtide. For this assay, TbDYRK2 proteins were immunoprecipitated and the kinase activity of the immunocomplexes determined by incubation with the Woodtide peptide. The results paralleled the levels of tyrosine phosphorylation, the WT molecule was significantly more active than either the kinase-inactive or activation loop mutant . The actDrosophila DYRK2 (DmDYRK2) 103  from DmDYRK2 (ΔN2-DmDYRK2) essentially eliminated activation loop tyrosine autophosphorylation. However, autophosphorylation could be rescued in trans by co-expression of the isolated N-terminus of the same DmDYRK2 molecule (residues 1–198) Drosophila ΔN2-DmDYRK2 protein.Normally, the NAPA-1 region functions in trans-complementation studies extends to the first residue of the kinase domain. We cloned FLAG-epitope tagged versions of the analogous region from TbDYRK2 (residues 1–109) and from HsDYRK2 (residues 1–149). We then expressed the activation loop phosphorylation-defective ΔN2-DmDYRK2 protein alone or co-expressed it with the N-terminus of DmDYRK2, TbDYRK2 or HsDYRK2 in Sf9 cells. Protein levels were normalized , and a more extensive deletion ΔN1-DmDYRK2 missing residues 1–189 trans by co-expressing the isolated N-terminus of DmDYRK2. In contrast, co-expression of Nt-DmDYRK2 did not rescue tyrosine autophosphorylation by ΔN1-DmDYRK2  to rescue ΔN2-DmDYRK2 activation loop phosphorylation. Nt-DmDYRK2 contains 120 amino acids followed by the NAPA-1, DH-box, and NAPA-2 regions trans-complementation, we generated four additional constructs .Previously, we identified regions (termed NAPA-1 and 2) in the non-catalytic N-terminus of T. brucei is the causative agent of African trypanosomiasis or Sleeping Sickness and is one of three trypanosomatids that cause major human disease T. cruzi (Chagas Disease) and Leishmania major (leishmaniasis) also encode a single class 2 DYRK . The non-catalytic N-terminus of all three trypanosomatid DYRKs are relatively short, ranging in size from 82–112 amino acids. When aligned, homology between these three DYRKs begins with the first residue of the NAPA-1 region and ends with the final residue of the kinase domain . However, for all DYRKs thus far analyzed, it is only the second tyrosine in the YTY motif that is phosphorylated and essential for full serine/threonine kinase activity in vivo in two separate studies that analyzed the phospho-proteome of T. bruceiAs noted, TbDYRK2 contains an atypical DLG triplet at the beginning of the activation loop, a sequence shared with Drosophila ΔN2-DmDYRK2 mutant protein Click here for additional data file.Figure S2Phylogenetic tree of the DYRKs. DYRK family members identified in our search of 21 sequenced genomes clustered into five separate subfamilies: class 1, class 2, HIPK, PRP4, and Yak.(TIF)Click here for additional data file.Figure S3Multiple protein alignment of class 2 DYRKs.(TIF)Click here for additional data file.Figure S4WT and mutant forms of TbDYRK2 were expressed in Sf9 cells and immunoprecipitated with anti-FLAG antibody. TbDYRK2 proteins analyzed include full length (WT), kinase inactive (K138M), activation loop tyrosine to phenylalanine (Y269F), N-terminal -deletion lacking residues 1–78 (ΔN2), and the entire non-catalytic N-terminus (Nt) of the molecule. ΔN2 was expressed alone in Sf9 cells; or it was coexpressed with Nt as indicated. Immunoprecipitates were subjected to SDS/PAGE and immunoblot analysis. Levels of TbDYRK2 proteins were detected with anti-FLAG antibody (α-FLAG), and activation loop phosphorylation was monitored with anti-phosphotyrosine antibody (α-pTyr). Large (*) and small (**) IgG background bands are indicated. Experiments were performed at least three times.(TIF)Click here for additional data file.Figure S5L. major, T. brucei, and T. cruzi. Symbols * invariant, : conservative substitutions, and . semi-conserved substitutions.CLUSTAL W (1.83) multiple sequence alignment of class 2 DYRKs from the trypanosomatids (DOC)Click here for additional data file.Table S1Identifiers of individual DYRK subfamily members categorized in each eukaryotic supergroup.(DOCX)Click here for additional data file.